By Cláudia S. F. Queiroga · iii Foreword The present thesis dissertation is the result of more than four years of research at the Animal Cell Technology Unit of Instituto de Tecnologia

By Cláudia S. F. Queiroga

First Edition: October 2012

Second Edition: December 2012

Front cover

By Cláudia S. F. Queiroga

ITQB-UNL/IBET Animal Cell Technology Unit

Instituto de Tecnologia Química e Biológica/

Instituto de Biologia Experimental e Tecnológica

Av. Da República EAN, 2780-157 Oeiras, Portugal

Phone: +351 21 446 91 00; Fax:+351 21 442 11 61

http://tca.itqb.unl.pt

http://www.itqb.unl.pt

http://www.ibet.pt

Copyright © 2012 by Cláudia S. F. Queiroga

All Rights Reserved

Printed in Portugal

ii

From left to right: Prof. Carlos C. Romão, Dr. Roberto Motterlini, Prof. Cláudia N. Santos, Cláudia S.F. Queiroga, Dr. Helena L.A. Vieira, Prof. Sílvia V. Conde and Dr. Paula M. Alves

Supervisors

Dr. Helena L.A. Vieira, Head of the Biology of Cytoprotection Laboratory, Chronic Diseases

Research Center (CEDOC), Faculdade de Ciências Médicas (FCM), Universidade Nova de

Lisboa (UNL) (supervisor).

Dr. Paula Marques Alves, Principal Investigator and Head of the Animal Cell Technology

Unit at Instituto de Tecnologia Química e Biológica (ITQB), UNL, and Executive Director of

Instituto de Biologia Experimental e Tecnológica (IBET), Oeiras, Portugal (co-supervisor).

Jury

Dr. Roberto Motterlini, INSERM U955, Faculty of Medicine, Université Paris Est (Paris XII),

France.

Professor Carlos B. Duarte, Head of Neuronal Cell Death and Neuroprotection Laboratory

and Associate Professor, Faculdade de Ciências e Tecnologia, Universidade de Coimbra,

Portugal.

Professor Sílvia V. Conde, Pharmacology Department, FCM-UNL, Portugal.

Dr. Cláudia N. Santos, PhD from Disease and Stress Biology Laboratory. IBET/ITQB-UNL,

Portugal.

http://www.linkedin.com/search?search=&currentCompany=C&company=Faculty+of+Medicine%2C+University+of+Paris+Est&sortCriteria=R&keepFacets=true

iii

Foreword

The present thesis dissertation is the result of more than four years of research at the

Animal Cell Technology Unit of Instituto de Tecnologia Química e Biológica –

Universidade Nova de Lisboa and Instituto de Biologia Experimental e Tecnológica

(Oeiras, Portugal) and at the Biology of Cytoprotection Laboratory at Centro de

Estudos de Doenças Crónicas – Faculdade de Ciências Médicas, Universidade

Nova de Lisboa (Oeiras, Portugal), under the supervision of Dr. Helena L.A. Vieira

and Dr. Paula M. Alves.

This thesis aims at contributing with novel knowledge on the cellular mechanisms

involved CO-induced cytoprotection in the pathological model of cerebral ischemia.

Several brain models were used, from subcellular to systemic level (in vitro and in

vivo models).

iv

v

Ao meu mano.

vi

Acknowledgements

I would like to express my sincere gratitude to all people that direct or indirectly

helped me during this thesis. I would also like to acknowledge the good conditions of

the institutes where I worked: ITQB, IBET and CEDOC.

To my supervisor, Dr. Helena Vieira, it is a pleasure doing science with you. You

taught me many different things without invading my space to grow. Also, in the

personal side, there are some special people that we have the pleasure to find few

times in our lives. In my life, you are one of them. Two feet give a better walk and the

result of such good friendship is so many serious, scary and laughing stories,

adventures, jokes, trips, cryings, scientific divagations almost philosophic, beers,

orange hair, all the mental spirals. The Talking Cricket only exists because Pinnochio

is in a search of courage, lealty and honesty. You don’t need to search it no more!

To my co-supervisor, Dr. Paula Alves, for the excellent example of hard-working and

for inspiring the others to do always more and better. Leading a team as Animal Cell

Technology Unit can only be achieved by a true leader.

To all Animal Cell Technology Unit team, especially to the friends I was lucky to find.

For sharing all the lab frustrations maintaining the sanity when no one else is seeing

it! Sofia L. for the inspiration for the life and the romantic side, a little star of light and

energy to live in a roller coaster; Sofia A. for all the “nothing” conversations, text

messages, work, existencial doubts, cientific discussions, shared papers, parties,

confessions, cryings and shakra calibrations; Tiago for all our moments as the

morning coffees; Rita for all the sharings; Cristina, for all the warm reception after we

moved to CEDOC; Pedro, for all the “engenheirices”, “almocinhos”, “brigadeiros”,

“bons bocados” e muppet music in the car; to the Italian committee, Marco and Zé,

for all the laughing.

The CO-team, which started really small (nano-clusters): Raquel, Rita, Sara, for all

the scientific discussions and for the good environment in the lab, so important for

our brain.

To CEDOC colleagues, in a final phase I still had the time to meet some people with

whom I started new routines and friendships. New lab mates from Cilia Regulation

and Disease, Rita, Petra, Bárbara and Susana; Maria, for all our moments in the

beginning and end of the day; Cristina E, continuing the good neighbor relations;

Marta for being a fountain of laugh. To all the others for different reasons.

vii

To our colaborators in differents parts of the work, to Simone, Alessandro, Marius,

Sílvia Conde, Carlos B. Duarte. Thanks to Alfama and in particular to Prof. Carlos

Romão for allowing the access to carbon monoxide gas.

To the financial support provided by Fundação para a Ciência e Tecnologia

(SFRH/BD/43387/2008).

To my FCUL friends, in particular Ana Maria and Pedro, some things never change.

To all my ITQB/InTerQB friends. To Neuza for all the conversations that started as

group works and presentations but evolved to conversations about personal life and

personal decisions. Lia, Licas, Damas, Gonçalo, Rui, Bárbara, Margarida and all the

others that I am not mentioning, our group is a great way to relax through laugh

therapy.

Ao João e Sérgio pela inquestionável amizade, suportando o tempo, a distância,

confusões, mudança de prioridades na vida e ajudando nas vicissitudes; Carlos e

família pela intimidade criada, ajudando nos momentos complicados; Judas, Tony,

Filipa, Cátia, Rita, Marta, Joana, Ana, Vítor e tantos outros; Daniela, Mário, Cuco,

Susana, Magda, Tina, Guedes, Joi, Nuno por me darem a juventude em semanas e

dias complicados e cansativos, são vocês a verdadeira fonte da minha jeunesse!

Ao Sérgio, pela boa surpresa numa altura que nada fazia prever. Os desígnios de

Deus são bons e inquestionáveis e acredito que Ele nos guarda muita coisa boa.

A toda a minha extensa família, que me leva até à Lixa do Alvão por ser um

verdadeiro interruptor “off” para descansar, e “on” para as verdadeiras prioridades;

Sónia, Manuel, Delfina e todos os outros pela ligação invisível mas contínua, pelas

boas histórias e gargalhadas. À minha madrinha, tio-padrinho, Fábio, Rui e Ricardo.

Pais, devo-vos tudo. À minha mãe por me fazer viver que a união é o pilar que nos

faz vencer na vida e nos dá a força para nos mantermos firmes mas não frios. Pai,

um teimoso não teima sozinho e se há coisa que herdei de ti foi a teimosia! Mano,

quando tu estás por perto tudo fica mais seguro, completo e simples. És, sem

dúvida, o que eu tenho de mais precioso e por isso te dedico esta tese.

viii

ABSTRACT

Perinatal complications are a serious clinical problem, in particular hypoxic-

ischemic (HI) episodes, which are caused by birth asphyxia or uterine and fetal blood

flow interruption. HI corresponds to 23% of all neonatal deaths worldwide, being one

of the top 20 leading causes of burden of disease in all age groups (source:

http://www.who.int/en/). Recent progress in neonatology has contributed to

reductions in mortality rates. Nevertheless, there is still high neurological morbidity

related to HI accounting for significant disability. Therefore, there is an urgent need

for investigation in this field.

Preconditioning (PC) is defined as stimulation below the threshold of injury that

activates endogenous protective mechanisms and prevents tissue and cellular

damage. Because PC is a physiological process, the organism fully adapts to low

doses of carbon monoxide (CO), an endogenously produced molecule that induces

the process and confers protection in several systems.

The aim of this PhD project was to evaluate the role of CO in the pathological

model of cerebral ischemia. Cellular mechanisms involved in CO-induced

cytoprotection were also studied using different cerebral models.

Chapter I introduces some general concepts of the brain field, constitution and

general functional pathways. Particular attention is given to the key role of

mitochondria in the variety of cell death mechanisms occurring after cerebral HI

episodes. Additionally, the advantages of PC induction as well as the potential

clinical importance of CO are addressed.

In Chapter II the ability of CO to prevent apoptosis in primary culture of

astrocytes via directly targeting mitochondria and preventing the release of pro-

apoptotic factors from these organelles into cytosol is demonstrated. For the first

time, it was shown that CO augments the mitochondrial content of oxidized

glutathione (GSSG) that modulates adenine nucleotide translocase (ANT)

glutathionylation. This reversible post-translational modification of ANT increases its

ADP/ATP translocation activity, preventing its pore-forming function through the inner

ix

membrane, which avoids mitochondrial membrane permeabilization (MMP) and,

therefore, apoptosis. These CO-induced processes are reverted by the presence of

beta-carotene, an antioxidant molecule, revealing the key signalling role of reactive

oxygen species (ROS) in CO’s biological functions.

The CO effect in mitochondria is not limited to MMP prevention. Chapter III

describes how CO improved cellular metabolism and reinforced oxidative

phosphorylation by: (i) increasing ATP generation, (ii) augmenting O2 consumption

and (iii) decreasing the ratio between glucose consumption and lactate production.

Moreover, CO increased cytochrome c oxidase (COX) specific activity and

mitochondrial biogenesis. Silencing Bcl-2 expression by siRNA transfection reverted

CO effects, showing that Bcl-2 cytoprotective property also involves cell metabolism

modulation.

In Chapter IV a model of primary co-culture of neurons and astrocytes (2D) is

used to explore the non-cell autonomous role of CO, namely in astrocyte-neuron

communication. It is demonstrated that CO-pre-treated astrocytes were more

efficient at reducing neuronal cell death than control astrocytes. CO stimulated

purinergic signalling from astrocytes to neurons, by releasing adenosine and/or ATP,

which activated neuronal A2A receptors and, in turn, transactivates TrkB receptors,

finally promoting neuronal survival.

After exploring CO biological functions in organelle and cellular models, a

systemic approach, using animal models, is reported in Chapter V. In a neonatal

brain HI rat model, CO was tested as a PC inducer for promoting cell death inhibition.

In the brains of rat pups exposed to 250 ppm of CO before the cerebral HI induction,

there was (i) apoptotic nuclei profiles reduction, (ii) caspase-3 activation decline, (iii)

Bcl-2 expression increment and (iv) reduction on cytochrome c release from

mitochondria.

Finally, a general and integrated discussion is presented in Chapter VI, for

positioning the work developed in the present thesis in the current scientific context

and for suggesting future investigation. In conclusion, this thesis has shed light into

x

CO’s cytoprotective role, by way of the induction of PC, for decreasing the

consequences of HI insult.

xi

RESUMO

Complicações perinatais são um problema clínico sério, em particular os

episódios de hipóxia-isquémia (HI), causados por asfixia durante o nascimento ou

por interrupção sanguínea no útero. HI corresponde a 23% do total mundial de

mortes neonatais, sendo uma das 20 causas de morbilidade em todas os grupos

etários (fonte: http://www.who.int/en/). Progressos recentes na neonatologia têm

contribuído para uma redução na taxa de mortalidade. No entanto, existe uma

elevada morbilidade neurológica relacionada com HI, aumentando a incapacidade

mental e física. Assim, há uma necessidade crescente de investigação nesta área.

O pré-condicionamento (PC) é definido como um estímulo abaixo do limite de

dano que activa os mecanismos endógeneos protectores prevenindo o dano

tecidular e celular. Como o PC é um processo fisiológico, o organismo adapta-se

totalmente a baixas concentrações de monóxido de carbono (CO), uma molécula

produzido endogenamente que inicia o processo protector e a protecção em

diferentes sistemas.

O objectivo deste projecto de doutoramento foi avaliar o papel do CO no modelo

patológico de isquémia cerebral. Mecanismos celulares envolvidos na citoprotecção

induzido pelo CO foram também estudados, usando diferentes modelos cerebrais.

O Capítulo I introduz alguns conceitos gerais no campo do cérebro, a sua

constituição e vias de funcionamento geral. Foi dada uma atenção particular ao

papel central da mitocôndria nos vários mecanismos de morte celular que ocorrem

após episódios de HI cerebrais. Em adição, as vantagens da indução do PC, assim

como a potencial relevância clínica do CO foram também contempladas.

No Capítulo II é demonstrada a habilidade do CO para a prevenção da apoptose

em culturas primárias de astrócitos por acção directa na mitocôndria e também a

inibição da libertação de factores pró-apoptóticos da mitocôndria para o citosol. Pela

primeira vez, foi demonstrado que o CO aumenta o conteúdo mitocondrial de

glutationo oxidado (GSSG) que modela a glutationilação do ANT (adenine nucleotide

translocase). Esta modificação pós-translacional do ANT é reversível e aumenta a

xii

sua actividade de translocação do ADP/ATP o que previne a formação de poro na

membrana interna, que, por sua vez, evita a permeabilização das membranas

mitocondriais (MMP) e a apoptose. Estes processos induzidos pelo CO são

revertidos na presença do beta-caroteno, uma molécula antioxidante, revelando o

papel fulcral das espécies reactivas de oxigénio (ROS) na sinalização das funções

biológicas do CO.

O efeito do CO na mitocôndria não é limitado à prevenção da MMP. O capítulo

III descreve como o CO melhora o metabolismo celular e reforça a fosforilação

oxidative: (i) aumenta a producção de ATP, (ii) aumenta o consumo de O2 e (iii)

diminui o rácio entre o consumo de glucose e produção de lactato. Em adição, o CO

aumenta a actividade específica da citocromo c oxidase (COX) e a biogénese

mitocondrial. O silenciamento da expressão de Bcl-2 por transfecção com siRNA

reverteu os efeitos do CO, mostrando que a propriedade citoprotectora da Bcl-2

também envolve modelação do metabolismo celular.

No Capítulo IV, um modelo 2D de culturas primárias de neurónios e astrócitos

foi usado para explorar o papel celular não-autónomo do CO, especialmente na

comunicação astrócito-neurónio. É demonstrado que os astrócitos pré-tratados com

CO foram mais eficientes a reduzir a morte neuronal do que os astrócitos controlo. O

CO estimula a sinalização purinérgica dos astrócitos para os neurónios, através da

libertação de adenosina e/ou ATP, que activam os receptores neuronais A2A que, por

sua vez, activam os receptores TrK B para culminar na sobrevivência neuronal.

Após a exploração as funções biológicas do CO em modelos de organelos e

celulares, passou-se para um modelo sistémico, usando modelos animais, como

reportado no Capítulo V. Num modelo de HI neonatal em rato, o CO foi testado

como inductor de PC para promover a inibição da morte celular. Nos cérebros dos

ratos recém-nascidos expostos a 250 ppm de CO, antes da indução da HI cerebral,

verificou-se (i) redução no perfil de núcleos apoptóticos, (ii) descréscimo na

activação de caspase-3, (iii) aumento na expressão de Bcl-2 e (iv) redução na

libertação de citocromo c da mitocôndria.

xiii

Finalmente, uma discussão geral e integrada é apresentada no Capítulo VI,

posicionando o trabalho desenvolvido durante a presente tese no contexto científico

currente e sugerindo algumas direcções de investigação futura. Em conclusão, esta

tese contribuiu para a compreensão do papel citoprotector do CO, por indução do

PC para diminuir as consequências do insulto de HI.

xiv

Thesis publications

1. Queiroga C.S.F., Almeida A.S., Martel C., Brenner-Jan C., Alves P.M. and

Vieira H.L.A., “Glutathionylation of adenine nucleotide translocase induced by

carbon monoxide prevents mitochondrial membrane permeabilization and

apoptosis”, Journal of Biological Chemistry 2010 May 28;285(22):17077-88.

2. Almeida A.S., Queiroga C.S.F., Sousa M.F., Alves P.M. and Vieira H.L.A.,

“Carbon monoxide modulates apoptosis by reinforcing oxidative metabolism in

astrocytes: role of Bcl-2”, Journal of Biological Chemistry 2012 Feb 13; 287(14):

10761-10770.

3. Queiroga C.S.F, Tomasi S., Widoroe M., Alves P.M., Vercelli A. and Vieira

H.L.A., “Preconditioning triggered by carbon monoxide (CO) provides neuronal

protection following perinatal hypoxia-ischemia” PLos One, 2012, 7(8):e42632.

xv

Abbreviations

Abbreviation Full form

2-VP 2-vinylpiridine

αβmeATP α,β-methyleneadenosine 5’-triphosphate lithium salt

AGA 18α-glycyrrhetinic acid

AIF Apoptosis-Inducing Factor

ANT Adenine Nucleotide Translocase

AK Adenylate Kinase

Ap5A P1, P5-diadenosine-5’-pentaphosphate

Apaf-1 Apoptotic Protease Activating Factor 1

ARL ARL67156

BBB Blood-Brain Barrier

Bcl-2 B-cell lymphoma-2

BCNU Carmustine

BCSFB Blood-Cerebrospinal Fluid Barrier

BDNF Brain-Derived Neurotrophic Factor

BME Basal Medium Eagle’s

CCA Common Carotid Artery

CL Contralateral

CNS Central Nervous System

CO Carbon Monoxide

CORM’s Carbon Monoxide Releasing Molecules

COX Cytochrome c Oxidase

CsA Cyclosporine A

CSF Cerebrospinal Fluid

CTL Cytotoxic T Lymphocytes

Cyp D Cyclophilin D

DiOC 3,3’-dihexyloxacarbocyanine iodide

http://atlasgeneticsoncology.org/Genes/APAF1ID422.html

xvi


DG Dentate Gyrus

DMEM Dulbecco’s Minimum Essential Medium

DPCPX 8-cyclopentyl-1,3-dipropylxanthine

DTNB 5, 5’-dithio-bis 2-nitrobenzoic acid

EA Ethacrynic Acid

EAA Excitatory Aminoacids

EPO Erythropoeitin

FBS Fetal Bovine Serum

GFAP Glial Fibrillary Acidic Protein

GSSG Oxidized Glutathione

GST Glutathione S-Transferase

HIF Hypoxia Inducing Factor

HIR Hypoxia-Ischemia and Reperfusion

HO Heme-Oxygenase

Hoe Hoechst 33342

IAP Inhibitor of Apoptotic Proteins

ICAD Inhibitor of Caspase Activated DNAse

IL Ipsilateral

JAK Janus Kinase

MMP Mitochondrial Membrane Permeabilization

NBTI S-(ρ-nitrobenzyl)-6-thioinosine

NDS Normal Donkey Serum

NO Nitric Oxide

NOS Nitric Oxidase Synthase

NSC Neural Stem Cells

ONOO- Peroxinitrite

P/S Penicillin/Streptomycin

xvii


PBS Phosphate Buffered Saline

PARP-1 Poly (ADP-ribose) polymerase-1

PC Preconditioning

PI Propidium Iodide

PNS Peripheral Nervous System

PTP Permeability Transition Pore

ROS Reactive Oxygen Species

RT Room Temperature

RT-qPCR Real Time-quantitative Polymerase Chain Reaction

SCH SCH58261

SD Standard Deviation

sGC Soluble Guanilyl Cyclase

SOD Superoxidase Dismutase

STAT Signal Transducer and Activator of Transcription

t-BHP tert-butyl hydroperoxide

TIAs Transient Ischaemic Attacks

TNB 5-thio-2-nitrobenzoate

TNF Tumour Necrosis Factor

tPA Tissue-Plasminogen Activator

VDAC Voltage Dependent Anion Channel

VEGF Vascular Endothelial Factor

xviii

LIST OF FIGURES

Figure Page Legend Title

1.1. 4 Eschematic representation of the (A) human and (B) rat brain.

1.2. 5 Brain barriers.

1.3. 6 Eschematic representation of a typical neuronal structure.

1.4. 7 Synapse types: (A) electrical and (B) chemical.

1.5. 8 Schematic representation of several brain cell populations.

1.6. 11 Schematic representation of a slice of the human brain.

1.7. 14 Apoptotic pathways.

1.8. 16 Regulated non-apoptotic cell death.

1.9. 22 Preconditioning and mitochondria.

1.10. 23 General preconditioning mechanism in the brain.

1.11. 24 Heme group metabolization by the enzyme heme-oxygenase (HO).

1.12. 31 Main questions and systems of this thesis.

2.1. 57 Carbon monoxide at 100 μM does not confer protection against astrocytic apoptosis but 50 μM of CO confers protection against apoptosis in delayed periods of time.

2.2. 58 Carbon monoxide confers protection against apoptosis.

2.3. 60 Relevance of ROS on carbon monoxide protective role.

2.4. 62 Carbon monoxide effect on the mitochondrial membrane depolarization, inner membrane permeabilisation, mitochondrial swelling and cytochrome c release.

2.5. 64 Influence of ROS on CO effect at mitochondrial level.

2.6. 65 Carbon monoxide effect on ADP/ATP translocase activity of ANT.

2.7. 66 Carbon monoxide effect on mitochondrial GSSG/GSH ratio.

2.8. 69 Role of ANT glutathionylation in MMP modulation.

2.9. 70 Effect of CO in ANT-GST interaction.

2.10. 72 Effect of mitochondrial GSSG in cell death, MMP and post-translational ANT modifications.

xix

Figure Page Legend Title

3.1. 94 Carbon monoxide confers protection against apoptosis.

3.2. 95 CO increases ATP generation in astrocytes.

3.3. 97 Effect of CO on ATP production and protection against cell death under glycolysis limiting conditions.

3.4. 99 Effect of CO on cytochrome c oxidase activity and mitochondria biogenesis.

3.5. 101 Role of CO in expression of Bcl-2 and Bcl-2-COX interaction.

3.6. 103 Role of Bcl-2 in CO-induced astrocytic metabolism modulation.

4.1. 123 Representation of experimental time-line.

4.2. 125 Effect of astroglial pre-treatment with carbon monoxide in neuronal apoptosis.

4.3. 126 CO influences ATP and adenosine content in co-culture supernatant.

4.4. 127 Adenosine and αβmeATP protect neurons against cell death.

4.5. 128 Effect of SCH58261 (SCH), suramin and8-cyclopentyl-1,3-dipropylxanthine (DPCPX)in the CO mechanism.

4.6. 129 Effect of 18α-glycyrrhetinic acid (AGA), S-(ρ-nitrobenzyl)-6-thioinosine (NBTI) and ARL67156 (ARL) in the CO mechanism.

4.7. 130 Effect of K252a in the CO mechanism.

5.1. 149 Experimental groups and time-points schematic representation.

5.2. 153 Stereological measurement of apoptosis.

5.3. 155 Effect of carbon monoxide treatment on neuronal apoptosis.

5.4. 157 Carbon monoxide effect in hippocampus after perinatal hypoxia-ischemia – apoptotic profiles.

5.5. 158 Carbon monoxide effect in hippocampus after perinatal hypoxia-ischemia – cleaved caspase 3 expression.

5.6. 160 Effect of carbon monoxide on apoptotic markers in hippocampal extracts after 6 and 24 h of HI – protein expression and sub-cellular localization.

6.1. 174 Main achievements of this PhD thesis.

xx

LIST OF TABLES

Table Page Legend Title

1.1. 18 Mitochondrial Membrane Permeabilization (MMP) consequences in the cellular processes.

1.2. 20 Mitochondrial quality control system levels.

1.3. 21 Molecules intervenients on the preconditioning pathways.

1.4. 26 Protective actions of HO-1 and HO-2.

1.5. 27 Protective actions of CO.

1.6. 29 Protective actions of CORMs.

3.1. 96 Metabolic hallmarks

4.1. 122 List of compounds used to modulate CO effect.

6.1. 179 This thesis contribution in carbon monoxide downstream molecular effectors, its modifications and time window of action.

xxi

TABLE OF CONTENTS

Chapter Page Title

I 1 Introduction

II 41 Carbon Monoxide Direct Effect On Non-Synaptic Mitochondria: mitochondrial membrane permeabilization prevention

III 81 Carbon Monoxide Direct Effect On Non-Synaptic Mitochondria: mitochondrial metabolism reinforcement

IV 113 Carbon Monoxide Non Cell-Autonomous Role: purinergic signalling

V 139 Carbon Monoxide Effect On Perinatal Hypoxia-Ischemia: apoptosis prevention

VI 169 Discussion and Conclusions

I INTRODUCTION

“A major part of brain function in decision-making is the testing of predictions against

reality — in essence all people are 'scientists'”

Chris Chambers, 2011

Chapter I

CONTENTS

1. Brain ....................................................................................................................... 3

1.1. Neuronal Cells ................................................................................................ 5

1.2. Glial Cells........................................................................................................ 7

1.3. Cell-to-cell communication .............................................................................. 8

2. Hypoxia-Ischemia and Reperfusion ........................................................................ 9

3. Cell Death ............................................................................................................. 12

3.1. Apoptosis ...................................................................................................... 12

3.2. Necrosis ........................................................................................................ 15

3.3. Regulated non-apoptotic cell death .............................................................. 15

4. Mitochondria ......................................................................................................... 17

4.1. Mitochondrial role in cell metabolism ............................................................ 17

4.2. Mitochondrial control of cell death ................................................................ 18

4.3. Mitochondrial quality control ......................................................................... 19

5. Preconditioning ..................................................................................................... 20

5.1. Preconditioning in the brain .......................................................................... 22

6. Carbon Monoxide ................................................................................................. 23

6.1. Heme-oxygenase 1 and the central nervous system .................................... 25

6.2. Carbon monoxide and the central nervous system ....................................... 27

6.3. Carbon monoxide as therapeutic molecule ................................................... 28

7. Aims and Scope of the Thesis .............................................................................. 29

8. References ........................................................................................................... 32

Cláudia S.F. Queiroga has written the whole chapter based on the referred bibliography.

INTRODUCTION

3

Ch

apte

r I

1. BRAIN

The interest in the brain is very ancient, with many theories involving the function

of this organ. In 1906, Camillo Golgi and Santiago Ramón y Cajal received the Nobel

Prize in Physiology and Medicine for their work in the brain. This prize is a landmark

of the beginning of a more systematic era in the field of Neurosciences.

The human brain is part of the central nervous system (CNS), together with the

spinal cord, and is an organ of extremes: extreme importance and extreme

complexity. It integrates and centralizes the information sensed all over the body and

initiates the signaling for the organism to react to it. The sophisticated network allows

a rapid response as a result of cooperation between several organs and muscles.

Due to the major importance of the brain in the body, its function is preserved by (i)

the thick bones of the skull, protecting against traumatic impacts (ii) the suspension

in the cerebrospinal fluid (CSF), which acts as a shock absorber and (iii) biological

barriers, as blood-brain barrier (BBB), decreasing the risk of infections (Purves et al.

2001). Despite all evolutionary strategies, the brain is still susceptible to several

types of damage and disease. Stroke is the first cause for brain injury, constituting

along with chronic neurodegeneratives diseases (as Parkinson, Alzheimer and

Hungtinton Disease) and the psychiatric disorders an enormous threat with the

increasing life expectancy, in particular because the nature of these pathologies is

not well understood.

The weight of a human brain is about 2% of the whole body weight (but

consumes up to 25% of oxygen), with a volume of 1130 cm3 in women and 1260 cm3

in men (Cosgrove et al. 2007). It is divided into left and right hemispheres that are

connected by corpus callosum, allowing the communication between them. Within

the brain there are four chambers interconnected and filled with CSF, called

ventricules.

Chapter I

4

The cerebral areas are represented in Figure 1.1. In order to attribute functions

to each brain region electrophysiological and behavioral studies can be performed.

However, each area is responsible for more than a single function being its action the

integrative communication between several areas. Despite all the knowledge already

achieved, the exact correlation is still undisclosed.

Figure 1.1. Schematic representation of the (A) human and (B) rat brain. The organization of the

areas in the brain vary according to the species.

There are two barriers, which are key players in the communication between its

periphery and the brain (Figure 1.2.). The most described is the BBB (Figure 1.2. –

A), which is constituted by the endothelial cells of the blood capillaries together with

associated astrocytic end-feet processes and perivascular neurons. As cellular

junctions are tight this physical barrier avoids the entrance of toxic factors into the

brain and restricts the exchanges between blood and brain, maintaining its normal

function. The second barrier is the blood-cerebrospinal fluid barrier (BCSFB, Figure

1.2. – B), constituted by the epithelial cells of the choroid plexus that separate the

blood from the CSF (Siegel et al. 1999; Francis et al. 2003).

Choroid plexus is a vascular tissue presented in all the cerebral ventricules and

the major site for the secretion of the CSF, holding four important functions:

synthesis and secretion of carrier proteins (as thyroid hormones), detoxification and

excretion (as amyloid β-peptide), signaling in response to peripheral inflammation

and a role in the regulation of the iron metabolism (Siegel et al. 1999).

(B)

(A)

INTRODUCTION

5

Ch

apte

r I

The cellular components in the brain comprise neuronal and glial cells.

Figure 1.2. Brain barriers. (A) The blood-brain barrier (BBB). (Adapted from (Francis et al. 2003))

(B) Blood—Cerebrospinal Fluid Barrier (BCSFB) (Adapted from (Siegel et al. 1999))

1.1. Neuronal Cells

Neurons are polarized cells, highly specialized in the transmission of

information. They can be classified according to their function, location, the

synthesized and released neurotransmitter, shape and number of extensions

from the cell body. The basic structure of a neuron (Figure 1.3.) includes (i)

dendrites that receive the information, (ii) the cell body, where the cellular

processes occurs and (iii) the axon, which transmits the information to the

following element in the network and is coated by myelin to facilitate the action

potential conduction velocity. However, neurons are not standardized. The size

of the axon together with the ramification and size of the dendrites are co-

related with the brain region and function (Purves et al. 2001). Bridging

information in the brain is a very specialized neuronal function, and, therefore

neurons have lost other cellular abilities, namely presenting limited energy

reservoirs and are not able to divide.

Adult neurogenesis is the daily generation of new neurons from neural stem

cells (NSC), occurring in specific brain regions called neural stem cells niches –

dentate gyrus in the hippocampus and subventricular zone. At least five steps

seem to be involved in adult neurogenesis: (i) the proliferation of stem cells and

the expansion of progenitor cells, (ii) the migration of newborn neurons

(neuroblasts) to the appropriate area, (iii) differentiation, (iv) integration into

Chapter I

6

circuits and (v) survival (reviewed in Vieira et al. 2011). Nevertheless, neuronal

renewal is limited, hence the advance on scientific knowledge on

neurodegeneration is of extreme relevance.

Figure 1.3. Eschematic representation

of a typical neuronal structure. Adapted

from (Morris 1988). Typically, the dendrites

receive the information, signaling it to the

cell body and the signaling continues

through the axon until the following

element in the network.

Synapse is the process of interneuronal communication being described as

signal transmission (electrical and chemical – Figure 1.4.) between two

neurons, a pre-synaptic from which the signal is transmitted and the post -

synaptic one that receives the information. The space between them is called

synaptic cleft. Nevertheless, nowadays the definition of synapse also includes

astrocytes – tripartite synapse (Mirko Santello 2012). These astroglial cells

remove the excess of neurotransmitters and recycle some of these factors,

avoiding the over-stimulation of neuronal cells, which assures their normal

function.

Electrical synapses are more associated to fast responses, such as

defensive reflexes. The signal transmitted through gap junctions is the same or

smaller in the post-synaptic neuron than that of the originating neuron (Figure

1.4. – A). While, in chemical synapses, vesicles are released from the pre-

synaptic neuron into the synaptic cleft (Figure 1.4. – B). Once in this space, the

neurotransmitter can bind to its receptor on the membrane of the post-synaptic

neuron, initiating an intracellular cascade. Neurotransmitters can be divided in

INTRODUCTION

7

Ch

apte

r I

amines, excitatory aminoacids, inhibitory aminoacids, purines, gases, lipids and

polypeptides (Purves et al. 2001).

Figure 1.4. Synapse types: (A) electrical and (B) chemical. Adapted from (Purves et al. 2001).

1.2. Glial Cells

Glial cells can be divided in macroglia and microglia. Macroglial cells

include astrocytes, oligodendrocytes and Schwann cells. Astrocytes (Figure

1.5.) are very important cells in the central nervous system, since they provide

the structural and metabolic support essential for neuronal maintenance.

Oligodendrocytes (Figure 1.5.) are the myelin-producing cells, for the

myelinization of the axon, which is of great importance to assure a correct and

functional signal transmission. Microglia are defined as specialized

macrophages of the CNS. Schwann cells belong to the peripheral nervous

system (PNS) and are responsible for (i) cleaning up PNS debris, (ii)

myelinization of the nerve axons and (iii) guide the regrowth of PNS axons

(Purves et al. 2001).

Chapter I

8

Astrocytes (from astro, star shaped, and cyte, cell, Figure 1.5.) are the main

neuronal suppliers of extracellular matrix proteins, growth factors and recyclers of

neurostransmitters in the CNS, establishing an important metabolic cooperation with

neurons (there is 1.4 astrocytes per neuron in the human brain (Theodosis et al.

2008)). The extension of numerous processes form highly organized anatomical

domains and with little overlap between adjacent cells, in close contact with

synapses or blood vessels. The domains communicate through gap junctions,

structuring a functional network.

The unique astrocytic phenotype, differential receptors and ion channels

expression become these cells as central in the regulation of morphology,

proliferation, differentiation, homeostasis and survival of different neuronal

subpopulations (Matyash et al. 2010).

Whereas neurons communicate using electric or chemical transmission, astrocytes

use gap junction channels that are regulated by extra- and intracellular signals

allowing exchange of information (Giaume 2010).

Figure 1.5. Schematic

representation of several

brain cell populations.

1.3. Cell-to-cell communication

Communication between neurons and astrocytes is bidirectional, tightly regulated

and any dysfunction can affect both cell populations. There are two important

astrocyte-neuron cycles. In the glutamine-glutamate cycle, the neurotransmitter

INTRODUCTION

9

Ch

apte

r I

glutamate is released from neurons and rapidly removed from the synaptic space by

astrocytes. Astrocytic glutamine synthase converts glutamate into glutamine, which

is released to synaptic space and uptaken by the neurons. Secondly, astrocytes

uptake glucose more efficiently than neurons and after its convertion into lactate, this

substrate is released to the synaptic space to be used as an energy source by

neurons (astrocyte-neuron lactate shuttle). In addition, astrocytic function also

modulates extracellular ionic homeostasis, neurotransmission, glutathione

metabolism and aminoacid recycling (Theodosis et al. 2008).

Astrocyte-neuron network is very complex (a single astrocyte enwraps multiple

neurons and one neuron interacts with 4-8 astrocytes). The revealing of cell-to-cell

communications can only add benefits, in particular in pathologic conditions. More

and more astroglial cells arise with an important biological role in neuroinflammation

and neuroprotection, namely in Alzheimer Disease, Amyotrophic Lateral Disease and

Cerebral Ischemia. Alexander Disease is caused by a mutation in glial fibrillary acidic

protein (GFAP) an astrocytic specific gene and the patients present symptoms also

related to dysfunction on other brain cells (neurodegeneration and abnormal

myelination) (Allaman et al. 2011).

2. HYPOXIA-ISCHEMIA AND REPERFUSION

Cerebral ischemia and reperfusion (HIR) is the main cause of brain damage

leading to worldwide mortality and morbidity (Vannucci and Hagberg, 2004; Dirnagl

et al., 2009; Dirnagl and Schwab, 2009). The absence of nutrients reservoirs in the

neurons develops into a strong dependence on blood flow, thus the abrupt

interruption on the circulation (ischemic stroke), decreasing the access to oxygen

(hypoxia) and nutrients (ischemia) can cause severe brain damage. The hemorrhagic

stroke is another type of cerebral incident, which occurs when a blood vessel bursts

spilling blood in the spaces surrounding the brain cells. The damage on the human

brain begins from the moment the stroke happens and, depending on the time of

blood flow interruption, the subsequent damage can have different levels of severity,

even being irreversible (Hossain 2008). To date, administration of thrombolytic

Chapter I

10

tissue-type plasminogen activator (tPA) is the one approved protocol for the

treatment of cerebral HIR. Still, there are limitations: the short time window and the

risk of haemorrhage (Yepes et al. 2008; Yepes et al. 2009). Thus, there is a crucial

and urgent need for novel therapies.

When blood flow is re-established (reperfusion), mitochondria can overproduce

reactive oxygen species (ROS) exacerbating cell injury by oxidative stress, which

can lead to cell death and/or to an increase on the BBB permeability. Likewise, the

interruption of oxygen supply favours anaerobic metabolism and lactate production,

leading to acidosis. Moreover, the Ca2+ intracellular concentration augments inducing

damaging cellular effects: (i) interferes with oxidative phosphorylation, causing

mitochondria failure and ATP production decrease; (ii) activates calpain, which

degrades the cellular matrix and (iii) activates phospholipase A2, which acts on

phospholipids, changing the membrane structure. Modifications in the membrane

permeability induce the release of excitatory amino acids (EAA), overstimulating the

excitatory receptors, which can induce glutamate excitotoxicity (Guo et al. 2011;

Yenari et al. 2012).

In the core of ischemic insult, cells die rapidly by necrosis. While, around the

core, a penumbra region is formed where cells die mostly by apoptosis, which occurs

during hours or days after the insult, establishing a time frame to limit the tissue

lesion by anti-apoptotic therapies (Figure 1.6.; (Buchan et al. 2007))

INTRODUCTION

11

Ch

apte

r I

Figure 1.6. Schematic representation of a slice of the human brain. In an hypoxic-ischemic and

reperfusion episode, one may consider the formation of distinct brain regions, core, where the more

severe consequences occur and penumbra, where cell death is more controlled but with a broader time

window.

In newborns cerebral HIR can be due to perinatal complications and is a common

cause of damage in the immature brain (1-6 of every 1000 live term birth) (reviewed

in Hossain 2008). In addition to the time of blood flow interruption, the lesion in

perinatal brain depends on the developmental stage. Although there are still some

aspects to unravel, it is accepted that the main molecular players, consequences and

protective mechanisms differ between the mature and immature brain (Hossain

2008). The lesion affects determined areas and not the whole brain, however can

cause cognitive, sensory and motor disabilities, which increases the importance on

the finding of new treatments (Sanders et al. 2010). Hypothermia is an approved

protocol for treatment of neonatal ischemia, however there is still an uncertainty on

the involved cellular mechanisms.

Affected area

Cellular consequences Type of cell

death Time of cell

death

Core Rapid impairment of cellular

functions Necrosis Minutes

Penumbra

Loss of synaptic activity; excitotoxicity; energy failure;

mitochondrial impairment; acidosis and inflammation

Apoptosis Hours or days

Chapter I

12

It is accepted that, at least, three types of cell death can occur in HIR episodes:

apoptosis, necrosis and/or autophagy, which can be interconnected by several

proteins (Carloni et al. 2008). Due to the up-regulation of pro-apoptotic factors in

early maturation ages, apoptosis has a major importance in developmental brain.

3. CELL DEATH

Nowadays, the definition of the different cell death types is under a strong

scientific debate. One can assume three types of cell death: apoptosis, necrosis and

regulated non-apoptotic cell death (autophagy, necroptosis and PARP-1 mediated

cell death).

3.1. Apoptosis

Apoptosis is a programmed and energy-dependent form of cell death. The term

apoptosis (from Greek, apo – from, off, without, -ptosis – falling) was first described

in 1842, but only published in 1972 by Kerr, Wyllie and Currie (Kerr et al. 1972).

Sidney Bremer, Robert Horvitz and John E. Sulston received the Nobel Prize in 2002

for their work in apoptosis.

During this tidy regulated process there are several morphological modifications:

cell shrinkage, chromatin condensation (pyknosis), DNA and nuclei fragmentation

(karyorhexis). Apoptotic bodies are formed (budding), which are packed organelles

with the integrity preserved, are engulfed by macrophages, parenchymal or

neoplastic cells and degraded within phagolysosomes. Thus, it is a way of removing

non-needed, harmed, old or mutated cells without interfering with the remaining

ones, with no inflammatory response or damage perpetuation. For the phagocytic

cells to recognize the cell fragments, the apoptotic bodies expose phosphatidylserine

in the outer leaflet of the plasma membrane – “eat me” signal (Tsujimoto et al. 2006;

Elmore 2007; Galluzzi et al. 2007; Wu et al. 2007).

During development and aging, apoptosis has an essential role in maintaining

cell homeostasis, as significant as mitosis. Therefore apoptosis regulation assumes a

INTRODUCTION

13

Ch

apte

r I

great importance to avoid serious defects. The excess of apoptosis can cause

atrophy, elimination of healthy cells or difficulty in wound healing. On the other hand,

the absence of apoptosis leads to tumour formation, autoimmune, inflammatory and

viral diseases (Elmore 2007).

One can distinguish three apoptotic pathways (Figure 1.7.): intrinsic, extrinsic

and perforin/granzyme pathways, which differ in the initiator stimuli, however

converging in the execution phase (Elmore 2007).

In intrinsic pathway, mitochondrial dysfunction is the initiator cellular signal. The

mitochondrial damage results in mitochondrial membrane permeabilization (MMP)

with not only the interruption on mitochondrial functions, but also with the release of

pro-apoptotic factors from mitochondria into cytosol. One example is cytochrome c,

which completes the apoptosome formation with Apaf-1 and caspase-9, being

responsible for the activation of the execution phase (Galluzzi et al. 2009). The

extrinsic pathway is initiated by the interaction between death ligand and death

receptor. This pathway involves members of the tumour necrosis factor (TNF)

receptor gene superfamily, which are transmembranar proteins, containing a

cytosolic “death domain”, important for death signal transmission leading to caspase-

8 activation (Locksley et al. 2001). Cytotoxic T lymphocytes (CTLs) are able to kill

target cells by a third pathway, by secreting perforin that allows the release of

cytoplasmic granules (that transport serine proteases as granzyme A and B) through

the pore and into the target cells (Pardo et al. 2004) – perforin/granzyme pathway.

Chapter I

14

Figure 1.7. Apoptotic pathways. Adapted from (Elmore 2007).

The already described pathways (Figure 1.7.) converge in caspase-3 activation,

initiating the execution phase of apoptosis. The executioner caspases (3, 6 and 7)

can be activated by any initiator caspase (caspase 2, 4, 8, 9, 10 or 12). Caspase 1, 5

and 11 are considered to be inflammatory ones (Ow et al. 2008).

Caspase-3 has different downstream targets, namely ICAD (Inhibitor of Caspase

Activated DNAse) whose cleavage releases its ligand CAD that, after the

translocation to the nucleus, degradates chromosomal DNA and causes chromatin

condensation. Furthermore, caspase-3 assumes a key role in apoptosis since it is

also involved in the cytomorphological changes and in the disintegration of the cell

into the apoptotic bodies (Elmore 2007).

During the execution phase, other important proteins, which are located in the

mitochondrial intermembranar space, are released after mitochondrial membrane

permeabilization (MMP) and participate in the caspase-independent mechanisms,

such as: Smac/DIABLO and Apoptosis Inducing Factor (AIF). Smac/DIABLO, along

with the serine protease HtrA2/Omi, inhibit an inhibitor of apoptotic proteins (IAP),

favouring the occurrence of apoptosis. AIF is cleaved by a cytosolic calpain and

translocated to the nucleus for degradating DNA (Modjtahedi et al. 2006).

INTRODUCTION

15

Ch

apte

r I

3.2. Necrosis

Necrosis (from Greek, “dead”) involves plasmatic membrane disruption with the

leakage of cytoplasmatic content leading to inflammation and enlarging the lesion

(Morse et al. 2009). It is associated with a bioenergetic catastrophe resulting from

ATP depletion, with calpains activation, resulting from infection, toxins or trauma

(Rami et al. 2008). Calpains are cytosolic proteases Ca2+-dependents required for

normal cell function. In models of acute neurodegeneration such as ischemia,

traumatic brain injury or epilepsy, calpains are pathologically activated, cleaving

neural proteins (Vosler et al. 2008).

3.3. Regulated non-apoptotic cell death

Regulated non-apoptotic cell death types (Figure 1.8.) includes autophagy or

type II, necroptosis and PARP1-mediated necrotic cell death (Degterev et al. 2008).

Autophagy (meaning “self-eating”) is a genetically controlled cell process for the

renewal of damaged intracellular organelles and proteins, where the target is

engulfed by multimembrane vesicles (autophagosomes) and degradated by

lysosomal fusion (Hotchkiss et al. 2009). When autophagy is initiated, microtubule-

associated protein light chain 3-I (LC3-I), a cytosolic protein, is modified, linked to a

phosphatidylethanolamine, becoming LC3-II, and integrates the autophagosome

membranes (Zhu et al. 2005); while beclin 1 is a component of phosphatidylinositol-

3-kinase (PI3K) complex, a cross-talk between apoptosis and autophagy (Carloni et

al. 2008). Autophagy was firstly recognized as a specific type of cell death, also

called type 2. However, it is now increasingly accepted that cells may die

coincidentally with autophagy rather than by autophagy. This pathway may function

as a power gateway during ischemia since energy and metabolites can be produced

– cytoprotective (Ginet et al. 2009). However, autophagy can also activate apoptosis

(Carloni et al. 2008) and the limit that divides noxious and protective role is still under

discussion. Autophagy can oppose apoptosis by engulfing mitochondria, which will

block the mitochondrial pathways of apoptosis and compromise the energy

Chapter I

16

production. However, autophagy can allow apoptosis and delay necrosis by providing

energy substrates to the cell or through molecular interconnections with apoptosis

(Ginet et al. 2009). Despite the fact that autophagy role and intensity is not well

classified, it is clear that it is a crucial process in homeostasis and in response to

injury.

Necroptosis is defined as having similar initiator stimuli as apoptosis, however

with necrotic morphological features (Fig. 1.8. b). Thirdly, Poly (ADP-ribose)

polymerase-1 (PARP1) is a nuclear enzyme responsible for maintaining genome

stability. Conversely, the excess of PARP1 activity can initiate caspase-independent

cell death pathways. One is driven for a rapid depletion of cytosolic NAD+, which will

lead to an energetic catastrophe, culminating in necrotic death. Secondly and

associated with acute neuronal injury, in excitatory neuronal cells, PARP1

translocates to the cytosol, and conducts AIF translocation to the nucleus, mediating

cell death.

Figure 1.8. Regulated non-apoptotic cell death. Adapted from (Ow et al. 2008)

Cell death pathways are regulated processes extremely important in tissue

function and homeostasis and may be blocked by survival factors. Furthermore, the

described pathways have several crosstalk players, indicating that in case of an

INTRODUCTION

17

Ch

apte

r I

insult (as cerebral HIR) the occurring mechanisms are variable, according to each

cellular state. Thus, a combined or adjustable therapy might provide maximal benefit.

4. MITOCHONDRIA

4.1. Mitochondrial role in cell metabolism

Mitochondria are dynamic organelles that have a key cellular role both in

metabolism (energy production) and in cell death (MMP).

Cellular energy is mostly produce in the inner mitochondrial membrane by

oxidative phosphorylation (80-90% of ATP). This is a very productive metabolic

pathway (26 of 30 ATP molecules per 1 glucose molecule) based on electron

transfer along an electron transport chain, from Complex I to Complex IV, through

oxidation-reduction reactions.

Glycolysis produces metabolites to be used in the tricarboxylic acid cycle,

producing compounds to supply the first electronic transporters in the oxidative

phosphorylation: NADH and succinate to complex I (NADH ubiquinone reductase)

and complex II (succinate dehydrogenase), respectively. The generated electrons

move to complex III (ubiquinol-cytochrome c oxidoreductase), and finally to complex

IV (cytochrome c oxidase - COX), via cytochrome c, in which O2 is the final electronic

acceptor, leading to H2O formation. While electrons are transported from one

transporter to the next, H+ ions are pumped to the intermembranar mitochondrial

space; leading to the formation of a gradient, maintaining a high transmembranar

potential (ΔΨm). ATP is synthesized in the final enzymatic complex, ATP synthase,

by driving back protons to the mitochondrial matrix (Nelson et al. 2004). In response

to injury, ΔΨm can decrease, the gradient of protons is unbalanced, disturbing ATP

production, ion homeostasis and protein transport into mitochondria, which has an

impact on cellular functions (Tait et al. 2008).

The oxidative phosphorylation has residual ROS production as by-product. 1 to

3% of the molecular oxygen is not converted to H2O, forming the very reactive O2·,

which can be rapidly converted by superoxide dismutase (SOD) to a less reactive

Chapter I

18

specie, H2O2. ROS formation can augment in stress conditions, activating others

signaling pathways, which can be damaging or protective, according to ROS

concentration (Guo et al. 2011). In ischemia-reperfusion episodes and, for preserving

ΔΨm, ATP synthase can revert its activity and hydrolises ATP; this is another source

of ROS overproduction (Christophe et al. 2006).

4.2. Mitochondrial control of cell death

Mitochondria are key organelles in the cell and their damage can lead to cell

death by two means: (i) the release of various pro-apoptotic proteins that are

confined to the intermembranar space, after MMP and (ii) due to disruption of the

respiratory chain (affecting energy cellular state associated pathways) (Kroemer et

al. 2007; Galluzzi et al. 2009). (Table 1.1.)

Table 1.1. Mitochondrial Membrane Permeabilization (MMP) consequences in the cellular

processes.

The mitochondrial membrane permeability is regulated via the interaction with pro

or anti-apoptotic proteins from Bcl-2 (B-cell lymphoma-2) family. Bcl-2 family

members contain Bcl-2 homology domains and can be divided in three classes:

apoptosis inhibitors, as Bcl-2 or Bcl-xL, apoptosis inducers, as Bax and Bak, and

BH3-only proteins that regulate anti-apoptotic bcl-2 proteins to promote apoptosis, as

Bad or Bik (Tait and Green 2008; Youle et al. 2008). Bcl-2 proteins are localized in

the endoplasmic reticulum, nucleus and in the outer membrane of mitochondria

(Chen et al. 2009; Galluzzi et al. 2009).

The exact mechanism for MMP is still under the focus of intense investigation,

with three potential models. One model is based on oligomerization of the pro-

Cell Death-related Cell Metabolism-related

ROS overproduction

Release of numerous pro-apoptotic factor (which will activate several proteases and nucleases)

Mitochondrial transmembrane potential dissipation

Respiratory chain uncoupling

ATP synthesis arrest

INTRODUCTION

19

Ch

apte

r I

apoptotic protein Bax; in response to death stimulus, translocates from the cytosol

into the outer mitochondrial membrane, where there is pore formation and membrane

permeabilization. A second model is based on VDAC (voltage dependent anion

channel), which is a physiological pore in the outer mitochondrial membrane allowing

the passage of solutes up to 5 kDa. Whenever VDAC interacts with Bax, the opening

of the pore enlarges with the leakage of factors superior than 5 kDa (such as

cytochrome c). Finally, the third model involves the permeabilization of the outer and

inner membranes, through the dynamic interaction between VDAC, cyclophilin D and

adenine nucleotide translocase (ANT). The complex of proteins is denominated

permeability transition pore (PTP) and allows the release of solutes up to 1500 Da.

PTP model appears to be the more relevant in ischemia-reperfusion injury (Vieira et

al. 2000; Belzacq et al. 2003; Mattson et al. 2003; Hausenloy et al. 2009).

As discussed previously, MMP is considered as the point of no return, since it is

the responsible for the activation of caspase dependent and independent apoptotic

pathways (section 3).

4.3. Mitochondrial quality control

Mitochondria population form a network within the cell to ensure cellular function

and survival. Thus, the correct function of each mitochondrion has to be ensured by

a quality control system (Table 1.2.). The first level of this hierarchical organization is

the intraorganellar quality control system. It has the aim of avoiding or detecting

misfolded proteins and to circumvent undesirable accumulation of dysfunctional

proteins that can be harmful (Tatsuta et al. 2008). When the first level fails, the

accumulation of misfolded or damaged proteins can have an effect on mitochondrial

integrity. In a second level of quality control, the mitochondrial fusion and fission are

modulated. In stress conditions, the fusion is advantageous to exchange genetic

material, avoiding the accumulation of defective content. While, fission facilitates the

degradation of defective mitochondria by autophagy (mitophagy). Nevertheless, if

neither of the levels is efficient or sufficient, cell undergoes apoptosis (Rugarli et al.

2012).

Chapter I

20

Table 1.2. Mitochondrial quality control system levels. Based in (Rugarli and Langer 2012).

5. PRECONDITIONING

In 1943, Noble was the first to propose that short periods of global hypoxia can

protect the entire mammalian organism and preserve brain energy metabolism

during longer periods of hypoxia. However, was only in 1964 that Janoff (Janoff

1964), introduced the term ‘preconditioning’.

The preconditioning (PC) phenomenon has been described as an insult that does

not cause damage but induces a cellular protective state (tolerance) against a

subsequent and more severe challenge (Kirino 2002; Alkan 2009). It can be an event

of early response (tolerance induced in minutes or hours), or late response within

days with de novo protein synthesis. PC induction involves (i) a stimuli, as ischemic

insult or chemical agent; (ii) the stimuli recognition by the cell, through

neurotransmitters, receptors, ion channels and redox-sensitive enzymes; (iii) the

transduction of the recognized stimuli with signalling molecules such as ROS,

adenosine, caspases and/or transcription factors and (iv) the integration of the

previous steps to effect the signal. The effectors are ubiquitous including variations

on enzymatic activities or gene transcription (Gidday 2006; Stenzel-Poore et al.

2007; Dirnagl et al. 2008) (Table 1.3). Because the protection results from an

endogenous boost, the knowledge of these cellular pathways is of extreme

importance for the design of new therapies to induce or reinforce cellular defences.

Intraorganellar Quality Control System

Organellar Quality Control System

Within mitochondria a combination of chaperones and proteases to assure:

(i) the correct folding of newly imported proteins; (ii) protection of the existing proteins from stress; (iii) degradation of irreversible damaged proteins

Equilibrium between mitochondrial fission and fusion to maintain mitochondrial population:

(i) stress conditions (nutrient deprivation, oxidative stress, impairment of cytosolic protein synthesis) – mitochondrial hyperfusion (ii) mitochondrial dysfunction – fusion inhibited, fission favoured to eliminated abnormal organelles

INTRODUCTION

21

Ch

apte

r I

Table 1.3. Molecules intervenients on the preconditioning pathways. Sensors, the molecules that

recognize the PC stimulus; transducers, that signal the induction of a protective state to effectors, in

order for the cell accomplish the preconditioning level. Adapted from (Dirnagl et al. 2003; Gidday 2006).

Sensors Transducers Effectors

Neurotransmitters Cytokins Toll-like receptors Ion channels Redox-sensitive enzymes

Depending on the PC stimulus ROS MAPKs Adenosine NF-kB PkB/Akt Caspases Transcription factors

Genes transcription Transient or protracted During minutes, hours or days

Another interesting aspect of PC is cross-tolerance. One PC inducer can promote

tolerance to another different type of damage. Moreover, the tolerance initiation in

one organ has demonstrated to cause protection in a distinct organ (Kirino 2002).

Additionally, patients suffering angina before a myocardial infarction have smaller

infarcts and longer survival – transient ischaemic attacks (TIAs, (Dirnagl et al. 2003)).

Furthermore, there are already several clinical trials to test the efficacy of PC

strategies. One of the most described is erythropoietin (EPO), already used in

patients with anemia. EPO is a kidney-derived glycoprotein hormone that acts as (i)

erythroid progenitor cell proliferation, (ii) cytoprotective, (iii) a gene target of HIF

(hypoxia inducible factor), which is activated in the vast majority of stress episodes

and (iv) PC induction increases EPO amounts. The reported protective cascade

includes the action of Janus Kinase 2 (JAK 2), Akt (or protein kinase B) and STAT

(signal transducer and activator of transcription) (Gidday 2006).

It is also described in heart that transient interruptions in blood flow during early

reperfusion (postconditioning), reduced ischemic injury to levels similar to that

achieved with PC (Vinten-Johansen et al. 2005) with promising results in clinical

trials (Gerczuk et al. 2011).

Mitochondria are considered as gatekeepers of life and death, and therefore are

proposed to assume a central role also in PC (Busija et al. 2008). Several evidences

co-relate mitochondria and PC (Figure 1.9.): (i) PC protects the integrity of

respiratory chain and membrane fluidity, prevents mitochondrial swelling, maintains

Chapter I

22

mitochondrial energy metabolism, which all together, increase ATP levels (Galeffi et

al. 2000; Dave et al. 2001; Zhang et al. 2003); (ii) in heart, PC cytoprotection

includes the inhibition of MMP (Javadov et al. 2000); (iii) PC-induced phophorylation

of mitochondrial proteins regulates MMP and apoptotic proteins (Maurer et al. 2006;

Zhao et al. 2006; Nishihara et al. 2007); and (iv) PC activates mitochondrial DNA

repair (Chen et al. 2003; Li et al. 2006). In summary, mitochondria play a key role in

PC regulation and is a possible therapeutic target.

Figure 1.9. Preconditioning and mitochondria. Adapted from (Dirnagl and Meisel 2008).

5.1. Preconditioning in the brain

Neuronal cells present higher energy demand than glial cells, since they conduct

excitatibility events. Nevertheless, they present lower energy reservoirs, which

position this cellular population as more dependent on mitochondria and on

astrocytes (Galluzzi et al. 2009). Thus, neurons are particularly susceptible to HIR;

however there are limited therapeutic options to this severe condition, being PC-

induction a potential therapy.

Several experimental evidences have emerged during the last years in order to

understand this endogenous pathway (Koch et al. 2012). For instance, in rats

INTRODUCTION

23

Ch

apte

r I

hypoxic preconditioning during the prenatal period is effective for reducing hypoxic-

ischaemic injury after birth (Gidday 2006). Although many aspects need more

scientific attention, the cerebral factors can be resumed in Figure 1. 10.

Figure 1.10. General mechanisms of preconditioning in the brain. Adapted from (Dirnagl et al.

2003; Gidday 2006).

There are increasing efforts to translate PC into clinical solutions, because it

takes advantage of endogenous cytoprotective mechanisms. For applying PC-based

strategies it is necessary at least, partially predictable pathological situations of

cerebral ischemia, such as pre-surgery period and in the case of perinatal

complications leading to asfixia.

Clinical trials are already approved, most of them based in ischemic PC

(clinicaltrials.gov). As examples, “The neuroprotective effect of remote ischemic

preconditioning on ischemic cerebral vascular disease” (NCT01321749) and

“Remote ischemic preconditioning in subarachnoid hemorrhage (RIPC-SAH)”

(NCT01158508).

6. CARBON MONOXIDE

Carbon monoxide (CO) is an endogenous molecule commonly known for being

toxic due to its high affinity to heme proteins, which can compromise oxygen delivery

in tissues (formation of carboxyhaemoglobin) and can limit oxidative phosphorylation

Chapter I

24

in cells (via cytochrome c oxidase inhibition) (Ahlstrom et al. 2009). Claude Bernard

first published an accurate description of the physiology of carbon monoxide

poisoning (Bernard 1857).

This gas is formed by heme-oxygenase (HO) activity after heme degradation,

along with free iron and biliverdin (a strong antioxidant molecule), rapidly converted

into bilirrubin, by biliverdin reductase (Figure 1.11.).

Figure 1.11. Heme group metabolization by the enzyme heme-oxygenase (HO), giving rise to free

iron, biliverdin (rapidly converted to bilirubin) and carbon monoxide.

In biological systems CO binds almost exclusively to transition metals, namely

iron, manganese, vanadium, cobalt, tungsten, copper, nickel and molybdenum,

which are present in structural and functional proteins (Boczkowski et al. 2006). The

number of molecules targeted by CO in mammals is very limited; the majority are

heme-containing proteins, whose function is regulated by the iron of this prosthetic

group. Iron is involved in the control of protein function by being part of heme

structure. In contrast to NO, that can bind to Fe3+ and Fe2+, CO is only able to accept

electrons from Fe2+, which promotes a selectivity of CO-targeted heme-proteins

(Roberts et al. 2004; Boczkowski et al. 2006). CO also activates soluble guanylate

cyclase (sGC) and nitric oxide synthase (NOS), but higher levels of CO are usually

required and its physiological role is not yet clarified (Queiroga et al. 2012). Finally,

the last mitochondrial electron transport chain complex, cytochrome c oxidase,

appears as another potential binding target for CO in vivo (Cooper et al. 2008).

INTRODUCTION

25

Ch

apte

r I

Despite the biological functions associated to CO in vivo and the existence of

several proteins capable of binding CO in vitro; it is still a matter of discussion the

actual physiological targets of CO.

6.1. Heme-oxygenase 1 and the central nervous system

HO has two main described isoforms: HO-1 or inducible and HO-2 or constitutive

and both respond to stress by the increase in expression or activity, respectively

(Ryter et al. 2006). HO-3 has been found only in the rat brain, but no activity on the

human brain (Zhu et al. 2011). HO activity plays an important role on the redox state

of the cell and it is described as crucial for cellular maintenance and survival in many

systems (listed in Table 1.4) such as brain (Dore 2002), heart (Piantadosi et al.

2008), intestine (Nakao et al. 2008), liver (Babu et al. 2007) and lung (Morse et al.

2009). Its activation reduces oxidative stress in cells and inhibits inflammation, both

due to removal of heme and because of the biological activity of HO-1 products

(Grochot-Przeczek et al. 2012).

Chapter I

26

Table 1.4. Protective actions of HO-1 and HO-2.

HO isoform Protective action Model Reference

HO-1

Prevents apoptosis in transplanted organs Transplanted heart Akamatsu et al.

2004

Reduces neuroinflammation Autoimmune

encephalomyelitis Chora et al. 2007

Prevents damage in cerebral ischemia Brain Atkins et al. 1999; Wang et al. 2011

Anti-inflammatory Gastrointestinal

Vascular Durante 2011; Zhu et al. 2011

Prevents development of ischemia-reperfusion injury Prevents graft rejection

Liver

Wang et al. 2011

Attenuates hypertension Lower blood pressure in established hypertension

Vascular Hosick et al. 2012

HO-2 HO-2 deficiency increases brain swelling and inflammation after intracellular hemorrhage

Brain Wang et al. 2008

The induction of HO-1 expression can be achieved by a myriad of inducers,

supporting the consideration of HO-1 as a therapeutic funnel, since its function is

required for the activity of several others therapeutic molecules (Otterbein et al.

2003). In addition, Soares and Bach (Soares et al. 2009) proposed HO-1 system

(HO-1, CO, Fe and biliverdin) to work as a funnel, albeit each molecule has different

mechanism, the global and protective effect is constructed together. HO-2 is

described to be a less likely target to main control of protection in diseases, since its

expression is not inducible (Soares and Bach 2009).

INTRODUCTION

27

Ch

apte

r I

6.2. Carbon monoxide and the central nervous system

Carbon monoxide beneficial role has been described in the literature, namely the

anti-proliferative in vascular smooth muscle cells (Stanford et al. 2003); anti-

inflammatory in lung (Ryter et al. 2007) and heart (Otterbein 2002); protective against

organ rejection (Musameh et al. 2007) and HIR injury in various models (Chatterjee

2007; Nakao et al. 2008). Some examples of CO anti-apoptotic effect are reported in

endothelial cells (Brouard et al. 2000), pulmonary cells (Otterbein et al. 1999) and

muscle (Harder et al. 2008). For review (Ryter et al. 2007) and Table 1.5. However,

the protective effect in the brain environment has been poorly described. We

previously described that in primary cultures of neuronal cells exogenous CO

prevents apoptosis by inducing a PC state (Vieira et al. 2008). Still, CO pathways

remain elusive and further studies must be carried to improve the possibility to use

this molecule as therapeutic agent.

Table 1.5. Protective actions of CO.

Protective action Model Reference

Prevents apoptosis Endothelial cells

Rat mitochondria

Brouard et al. 2000

Piantadosi et al. 2006

Reduces inflammatory responses and endoplasmatic reticulum stress

Metabolic disorders Joe et al. 2001

Reduces neuroinflammation Brain Chora et al. 2007

Energy maintenance Ischemic myocardium Ahlstrom et al. 2009

Reduces brain injury after transient middle cerebral artery occlusion

Brain Zeynalov et al. 2009

Preventing cerebral injury resulting from cardiac bypass procedures using deep hypothermic circulatory arrest

Brain Mahan et al. 2012

Chapter I

28

6.3. Carbon monoxide as therapeutic molecule

CO administered in combination with others strategies may extend the

therapeutic window. In particular, CO-induced PC would enhance endogenous

properties with clinical benefits, is not invasive and would not require very specialized

equipment. Others inhalation therapies have been introduced in clinics (Robinson et

al. 2009), for reducing toxicity resulting from metabolization of administered drugs,

since drug extraction occurs by exhalation. The therapeutic application of nitric oxide

(NO) as vasodilator in several disease models and also in injured lungs of premature

and newborn babies is now widely accepted (Bloch et al. 2007). NO is chemically

similar to CO; however, unlike CO, NO reacts rapidly with molecular oxygen, and

produces peroxynitrite (ONOO-), which is highly reactive. Likewise, noble gases have

also been studied for medical applications, especially xenon. Recently, Ryang and

co-workers (Ryang et al. 2011) described the efficacy of argon in protecting rat

brains in a model of transient middle cerebral artery occlusion. Taken all together,

potential CO-inhaled based therapy has the added value of integrating two critical

advantages: it is chemically inert compared to NO and is an endogenous molecule

compared to noble gases.

For overcoming a possible systemic CO toxic effect, the use of CO-releasing

molecules (CORM’s) has been proposed (Motterlini 2007). These small organic and

organometallic compounds are able to deliver CO in a timely and tissue-specific

manner, allowing a significant reduction in carboxyhaemoglobin formation and

toxicity, which opens novel windows of opportunity for clinical applications. The

biological activities of CORMs include, among others (see Table 1.6.):

cardioprotection in isolated perfused rat heart, protection in acute hepatic reperfusion

injury in rats, endothelial cells protection during cold preservation and injury

impairment in the case of HI injury during kidney transplantation, among others

(Motterlini et al. 2010). Furthermore, and very recently, CORM-3 was shown to

modulate the inflammatory response and to reduce brain damage in an adult rat

model of hemorrhagic stroke (Yabluchanskiy et al. 2012), indicating that CORM-3

can cross the blood brain barrier.

INTRODUCTION

29

Ch

apte

r I

At present there are two clinical trials phase II on CO gas inhalation-based

therapy: for treating patients with intestinal paralysis after colon surgery, for

prevention of post-operative ileus (NCT01050712) and for the improvement of

tolerability in patients receiving kidney transplants (NCT00531856).

Table 1.6. Protective actions of CORMs.

Protective action Model Reference

Alleviate vascular and immune-related dysfunctions Motterlini et al. 2002

Protects against HI and oxidative stress Cardiac cells

(CORM-3) Clark et al. 2003 Stein et al. 2012

Late PC induction Myocardial infarction

(CORM-3) Stein et al. 2005

Reduces inflammation Microglia

(CORM-3) Bani-Hani et al. 2006

Protects against seizure-induced neonatal vascular injury

Brain (CORM A1)

Zimmermann et al. 2007

Protects against myocardial infarction in hyperglycaemic rats

Heart (CORM-3)

Filippo et al. 2011

Regulation on mitochondrial respiration Heart

(CORM-3) Lo Iacono et al. 2011

7. AIMS AND SCOPE OF THE THESIS

This PhD thesis aims at clarifying the cellular pathways involved in the carbon

monoxide (CO) capacity to confer cytoprotection against apoptosis following hypoxia-

ischemia and reperfusion (HIR) in the brain. Three different model systems were

used to address this objective:

(i) isolated non-synaptic mitochondria – as mitochondria are the apoptosis

gatekeepers, central in cellular metabolism, the main subcellular source of ROS

and CO affinity to cytochrome c oxidase is documented, the direct CO effect on the

Chapter I

30

mitochondrial membrane permeabilization (chapter II), mitochondrial metabolism

(chapter III) and its upstream events were studied;

(ii) primary monocultures of astrocytes and primary co-cultures of neurons and

astrocytes (in vitro) – CO protective action in primary cultures of neurons was already

demonstrated (Vieira et al. 2008), thus CO capability to revert cell death was tested

in primary cultures of astrocytes (chapter II). Furthermore, approaching with a more

physiological model – co-cultures – the relevance of carbon monoxide pre-treatment

to cell-to-cell communication and its downstream events were investigated (chapter

IV);

(iii) perinatal model of hypoxia-ischemia (HI) in rats (in vivo) – complementing the

cellular and subcellular role of CO in vitro, the in vivo performance in order to validate

CO as neuroprotector and its influence in anti-apoptotic pathways was pursued. The

model used was a perinatal model of hypoxia-ischemia in rat pups (chapter V).

A schematic representation of the main goals proposed for this thesis as well as

the systems employed are summarized in Figure 1.12.

INTRODUCTION

31

Ch

apte

r I

Figure 1.12. Main questions and systems of this thesis.

Chapter I

32

8. REFERENCES

Ahlstrom, K., B. Biber, et al. (2009). "Metabolic responses in ischemic myocardium after

inhalation of carbon monoxide." Acta Anaesthesiol Scand 53(8): 1036-42.

Akamatsu, Y., M. Haga, et al. (2004). "Heme oxygenase-1-derived carbon monoxide protects hearts from transplant associated ischemia reperfusion injury." Faseb J 18(6): 771-2.

Alkan, T. (2009). "Neuroproctective effects of ischemic tolerance (preconditioning) and postconditioning." Turk Neurosurg 19(4): 406-12.

Allaman, I., M. Belanger, et al. (2011). "Astrocyte-neuron metabolic relationships: for better and for worse." Trends Neurosci 34(2): 76-87.

Atkins, C. M. and J. D. Sweatt (1999). "Reactive oxygen species mediate activity-dependent neuron-glia signaling in output fibers of the hippocampus." J Neurosci 19(17): 7241-8.

Babu, A. N., S. S. Damle, et al. (2007). "Hemoglobin-based oxygen carrier induces hepatic heme oxygenase 1 expression in Kupffer cells." Surgery 142(2): 289-94.

Bani-Hani, M. G., D. Greenstein, et al. (2006). "Modulation of thrombin-induced neuroinflammation in BV-2 microglia by carbon monoxide-releasing molecule 3." J Pharmacol Exp Ther 318(3): 1315-22.

Belzacq, A. S., H. L. Vieira, et al. (2003). "Bcl-2 and Bax modulate adenine nucleotide translocase activity." Cancer Res 63(2): 541-6.

Bernard, C. (1857). "Lecons sur les Effets des Substaces Toxiques et Medicamenteuses." Paris: J-B Bailliere et Fils.

Bloch, K. D., F. Ichinose, et al. (2007). "Inhaled NO as a therapeutic agent." Cardiovasc Res 75(2): 339-48.

Boczkowski, J., J. J. Poderoso, et al. (2006). "CO-metal interaction: Vital signaling from a lethal gas." Trends Biochem Sci 31(11): 614-21.

Brouard, S., L. E. Otterbein, et al. (2000). "Carbon monoxide generated by heme oxygenase 1 suppresses endothelial cell apoptosis." J Exp Med 192(7): 1015-26.

Buchan, A. M. and J. Kennedy (2007). "Strategies for therapy in acute ischemic stroke." Nat Clin Pract Neurol 3(1): 2-3.

Busija, D. W., T. Gaspar, et al. (2008). "Mitochondrial-mediated suppression of ROS production upon exposure of neurons to lethal stress: mitochondrial targeted preconditioning." Adv Drug Deliv Rev 60(13-14): 1471-7.

INTRODUCTION

33

Ch

apte

r I

Carloni, S., G. Buonocore, et al. (2008). "Protective role of autophagy in neonatal hypoxia-ischemia induced brain injury." Neurobiol Dis 32(3): 329-39.

Chatterjee, P. K. (2007). "Novel pharmacological approaches to the treatment of renal ischemia-reperfusion injury: a comprehensive review." Naunyn Schmiedebergs Arch Pharmacol 376(1-2): 1-43.

Chen, D., M. Minami, et al. (2003). "Upregulation of mitochondrial base-excision repair capability within rat brain after brief ischemia." J Cereb Blood Flow Metab 23(1): 88-98.

Chen, Z. X., R. Velaithan, et al. (2009). "mitoEnergetics and cancer cell fate." Biochim Biophys Acta 1787(5): 462-7.

Chora, A. A., P. Fontoura, et al. (2007). "Heme oxygenase-1 and carbon monoxide suppress autoimmune neuroinflammation." J Clin Invest 117(2): 438-47.

Christophe, M. and S. Nicolas (2006). "Mitochondria: a target for neuroprotective interventions in cerebral ischemia-reperfusion." Curr Pharm Des 12(6): 739-57.

Clark, J. E., P. Naughton, et al. (2003). "Cardioprotective actions by a water-soluble carbon monoxide-releasing molecule." Circ Res 93(2): e2-8.

Cooper, C. E. and G. C. Brown (2008). "The inhibition of mitochondrial cytochrome oxidase by the gases carbon monoxide, nitric oxide, hydrogen cyanide and hydrogen sulfide: chemical mechanism and physiological significance." J Bioenerg Biomembr 40(5): 533-9.

Cosgrove, K. P., C. M. Mazure, et al. (2007). "Evolving knowledge of sex differences in brain structure, function, and chemistry." Biol Psychiatry 62(8): 847-55.

Dave, K. R., I. Saul, et al. (2001). "Ischemic preconditioning preserves mitochondrial function after global cerebral ischemia in rat hippocampus." J Cereb Blood Flow Metab 21(12): 1401-10.

Degterev, A. and J. Yuan (2008). "Expansion and evolution of cell death programmes." Nat Rev Mol Cell Biol 9(5): 378-90.

Dirnagl, U. and A. Meisel (2008). "Endogenous neuroprotection: mitochondria as gateways to cerebral preconditioning?" Neuropharmacology 55(3): 334-44.

Dirnagl, U., R. P. Simon, et al. (2003). "Ischemic tolerance and endogenous neuroprotection." Trends Neurosci 26(5): 248-54.

Dore, S. (2002). "Decreased activity of the antioxidant heme oxygenase enzyme: implications in ischemia and in Alzheimer's disease." Free Radic Biol Med 32(12): 1276-82.

Durante, W. (2011). "Protective role of heme oxygenase-1 against inflammation in atherosclerosis." Front Biosci 17: 2372-88.

Chapter I

34

Elmore, S. (2007). "Apoptosis: a review of programmed cell death." Toxicol Pathol 35(4): 495-516.

Filippo, C. D., M. Perretti, et al. (2011). "Acute myocardial infarction in streptozotocin-induced hyperglycaemic rats: protection by a carbon monoxide-releasing molecule (CORM-3)." Naunyn Schmiedebergs Arch Pharmacol 385(2): 137-44.

Francis, K., J. Beek, et al. (2003). "The Blood-brain Barrier (BBB)." Expert Rev Mol Med 5: 1-19.

Galeffi, F., S. Sinnar, et al. (2000). "Diazepam promotes ATP recovery and prevents cytochrome c release in hippocampal slices after in vitro ischemia." J Neurochem 75(3): 1242-9.

Galluzzi, L., K. Blomgren, et al. (2009). "Mitochondrial membrane permeabilization in neuronal injury." Nat Rev Neurosci 10(7): 481-94.

Galluzzi, L., M. C. Maiuri, et al. (2007). "Cell death modalities: classification and pathophysiological implications." Cell Death Differ 14(7): 1237-43.

Gerczuk, P. Z. and R. A. Kloner (2011). "Protecting the heart from ischemia: an update on ischemic and pharmacologic conditioning." Hosp Pract (Minneap) 39(3): 35-43.

Giaume, C. (2010). "Astroglial Wiring is Adding Complexity to Neuroglial Networking." Front Neuroenergetics 2.

Gidday, J. M. (2006). "Cerebral preconditioning and ischaemic tolerance." Nat Rev Neurosci 7(6): 437-48.

Ginet, V., J. Puyal, et al. (2009). "Enhancement of Autophagic Flux after Neonatal Cerebral Hypoxia-Ischemia and Its Region-Specific Relationship to Apoptotic Mechanisms." Am J Pathol.

Grochot-Przeczek, A., J. Dulak, et al. (2012). "Haem oxygenase-1: non-canonical roles in physiology and pathology." Clin Sci (Lond) 122(3): 93-103.

Guo, M. F., J. Z. Yu, et al. (2011). "Mechanisms related to neuron injury and death in cerebral hypoxic ischaemia." Folia Neuropathol 49(2): 78-87.

Harder, Y., M. Amon, et al. (2008). "Ischemia-Induced Up-Regulation of Heme Oxygenase-1 Protects from Apoptotic Cell Death and Tissue Necrosis." J Surg Res.

Hausenloy, D. J., S. B. Ong, et al. (2009). "The mitochondrial permeability transition pore as a target for preconditioning and postconditioning." Basic Res Cardiol 104(2): 189-202.

Hosick, P. A. and D. E. Stec (2012). "Heme oxygenase, a novel target for the treatment of hypertension and obesity?" Am J Physiol Regul Integr Comp Physiol 302(2): R207-14.

INTRODUCTION

35

Ch

apte

r I

Hossain, M. A. (2008). "Hypoxic-ischemic injury in neonatal brain: involvement of a novel neuronal molecule in neuronal cell death and potential target for neuroprotection." Int J Dev Neurosci 26(1): 93-101.

Hotchkiss, R. S., A. Strasser, et al. (2009). "Cell death." N Engl J Med 361(16): 1570-83.

Janoff, A. (1964). "Alterations in Lysosomes (Intracellular Enzymes) During Shock; Effects of Preconditioning (Tolerance) and Protective Drugs." Int Anesthesiol Clin 2: 251-69.

Javadov, S. A., K. H. Lim, et al. (2000). "Protection of hearts from reperfusion injury by propofol is associated with inhibition of the mitochondrial permeability transition." Cardiovasc Res 45(2): 360-9.

Joe, Y., M. Zheng, et al. (2001). "The role of carbon monoxide in metabolic disease." Ann N Y Acad Sci 1229: 156-61.

Kerr, J. F., A. H. Wyllie, et al. (1972). "Apoptosis: a basic biological phenomenon with wide-ranging implications in tissue kinetics." Br J Cancer 26(4): 239-57.

Kirino, T. (2002). "Ischemic tolerance." J Cereb Blood Flow Metab 22(11): 1283-96.

Koch, S., R. L. Sacco, et al. (2012). "Preconditioning the Brain: Moving on to the Next Frontier of Neurotherapeutics." Stroke.

Kroemer, G., L. Galluzzi, et al. (2007). "Mitochondrial membrane permeabilization in cell death." Physiol Rev 87(1): 99-163.

Li, W., Y. Luo, et al. (2006). "Ischemic preconditioning in the rat brain enhances the repair of endogenous oxidative DNA damage by activating the base-excision repair pathway." J Cereb Blood Flow Metab 26(2): 181-98.

Lo Iacono, L., J. Boczkowski, et al. (2011). "A carbon monoxide-releasing molecule (CORM-3) uncouples mitochondrial respiration and modulates the production of reactive oxygen species." Free Radic Biol Med 50(11): 1556-64.

Locksley, R. M., N. Killeen, et al. (2001). "The TNF and TNF receptor superfamilies: integrating mammalian biology." Cell 104(4): 487-501.

Mahan, V. L., D. Zurakowski, et al. (2012). "Inhaled carbon monoxide provides cerebral cytoprotection in pigs." PLoS One 7(8): e41982.

Mattson, M. P. and G. Kroemer (2003). "Mitochondria in cell death: novel targets for neuroprotection and cardioprotection." Trends Mol Med 9(5): 196-205.

Matyash, V. and H. Kettenmann (2010). "Heterogeneity in astrocyte morphology and physiology." Brain Res Rev 63(1-2): 2-10.

Chapter I

36

Maurer, U., C. Charvet, et al. (2006). "Glycogen synthase kinase-3 regulates mitochondrial outer membrane permeabilization and apoptosis by destabilization of MCL-1." Mol Cell 21(6): 749-60.

Mirko Santello, C. C., Paola Bezzi (2012). Gliotransmission and the tripartite synapse. Synaptic Plasticity. A. i. E. M. a. Biology. 970: 307-331.

Modjtahedi, N., F. Giordanetto, et al. (2006). "Apoptosis-inducing factor: vital and lethal." Trends Cell Biol 16(5): 264-72.

Morris, C. G. (1988). Psychology: an introduction, Prentice Hall.

Morse, D., L. Lin, et al. (2009). "Heme oxygenase-1, a critical arbitrator of cell death pathways in lung injury and disease." Free Radic Biol Med 47(1): 1-12.

Motterlini, R. (2007). "Carbon monoxide-releasing molecules (CO-RMs): vasodilatory, anti-ischaemic and anti-inflammatory activities." Biochem Soc Trans 35(Pt 5): 1142-6.

Motterlini, R., J. E. Clark, et al. (2002). "Carbon monoxide-releasing molecules: characterization of biochemical and vascular activities." Circ Res 90(2): E17-24.

Motterlini, R. and L. E. Otterbein (2010). "The therapeutic potential of carbon monoxide." Nat Rev Drug Discov 9(9): 728-43.

Musameh, M. D., C. J. Green, et al. (2007). "Improved myocardial function after cold storage with preservation solution supplemented with a carbon monoxide-releasing molecule (CORM-3)." J Heart Lung Transplant 26(11): 1192-8.

Nakao, A., D. J. Kaczorowski, et al. (2008). "Application of heme oxygenase-1, carbon monoxide and biliverdin for the prevention of intestinal ischemia/reperfusion injury." J Clin Biochem Nutr 42(2): 78-88.

Nelson, D. L. and M. M. Cox (2004). Lehninger Principles of Biochemistry, W.H. Freeman.

Nishihara, M., T. Miura, et al. (2007). "Modulation of the mitochondrial permeability transition pore complex in GSK-3beta-mediated myocardial protection." J Mol Cell Cardiol 43(5): 564-70.

Otterbein, L. E. (2002). "Carbon monoxide: innovative anti-inflammatory properties of an age-old gas molecule." Antioxid Redox Signal 4(2): 309-19.

Otterbein, L. E., L. L. Mantell, et al. (1999). "Carbon monoxide provides protection against hyperoxic lung injury." Am J Physiol 276(4 Pt 1): L688-94.

Otterbein, L. E., M. P. Soares, et al. (2003). "Heme oxygenase-1: unleashing the protective properties of heme." Trends in Immunology 24(8): 449-455.

INTRODUCTION

37

Ch

apte

r I

Ow, Y. P., D. R. Green, et al. (2008). "Cytochrome c: functions beyond respiration." Nat Rev Mol Cell Biol 9(7): 532-42.

Pardo, J., A. Bosque, et al. (2004). "Apoptotic pathways are selectively activated by granzyme A and/or granzyme B in CTL-mediated target cell lysis." J Cell Biol 167(3): 457-68.

Piantadosi, C. A., M. S. Carraway, et al. (2008). "Heme oxygenase-1 regulates cardiac mitochondrial biogenesis via Nrf2-mediated transcriptional control of nuclear respiratory factor-1." Circ Res 103(11): 1232-40.

Piantadosi, C. A., M. S. Carraway, et al. (2006). "Carbon monoxide, oxidative stress, and mitochondrial permeability pore transition." Free Radic Biol Med 40(8): 1332-9.

Purves, D., G. J. Augustine, et al. (2001). Neuroscience, Sinauer Associates, Inc.

Queiroga, C. S., A. S. Almeida, et al. (2012). "Carbon monoxide targeting mitochondria." Biochem Res Int 2012: 749845.

Rami, A. and D. Kogel (2008). "Apoptosis meets autophagy-like cell death in the ischemic penumbra: Two sides of the same coin?" Autophagy 4(4): 422-6.

Roberts, G. P., H. Youn, et al. (2004). "CO-sensing mechanisms." Microbiol Mol Biol Rev 68(3): 453-73.

Robinson, B. R., K. P. Athota, et al. (2009). "Inhalational therapies for the ICU." Curr Opin Crit Care 15(1): 1-9.

Rugarli, E. I. and T. Langer (2012). "Mitochondrial quality control: a matter of life and death for neurons." Embo J 31(6): 1336-49.

Ryang, Y. M., A. V. Fahlenkamp, et al. (2011). "Neuroprotective effects of argon in an in vivo model of transient middle cerebral artery occlusion in rats." Crit Care Med 39(6): 1448-53.

Ryter, S. W., J. Alam, et al. (2006). "Heme oxygenase-1/carbon monoxide: from basic science to therapeutic applications." Physiol Rev 86(2): 583-650.

Ryter, S. W., H. P. Kim, et al. (2007). "Protective functions of heme oxygenase-1 and carbon monoxide in the respiratory system." Antioxid Redox Signal 9(12): 2157-73.

Sanders, R. D., H. J. Manning, et al. (2010). "Preconditioning and postinsult therapies for perinatal hypoxic-ischemic injury at term." Anesthesiology 113(1): 233-49.

Siegel, G. J., B. W. Agranoff, et al. (1999). Basic Neurochemistry: Molecular, Cellular and Medical Aspects, Lippincott-Raven.

Chapter I

38

Soares, M. P. and F. H. Bach (2009). "Heme oxygenase-1: from biology to therapeutic potential." Trends Mol Med 15(2): 50-8.

Stanford, S. J., M. J. Walters, et al. (2003). "Heme oxygenase is expressed in human pulmonary artery smooth muscle where carbon monoxide has an anti-proliferative role." Eur J Pharmacol 473(2-3): 135-41.

Stein, A. B., R. Bolli, et al. (2012). "Carbon monoxide induces a late preconditioning-mimetic cardioprotective and antiapoptotic milieu in the myocardium." J Mol Cell Cardiol 52(1): 228-36.

Stein, A. B., Y. Guo, et al. (2005). "Administration of a CO-releasing molecule induces late preconditioning against myocardial infarction." Journal of Molecular and Cellular Cardiology 38(1): 127-134.

Stenzel-Poore, M. P., S. L. Stevens, et al. (2007). "Preconditioning reprograms the response to ischemic injury and primes the emergence of unique endogenous neuroprotective phenotypes: a speculative synthesis." Stroke 38(2 Suppl): 680-5.

Tait, S. W. and D. R. Green (2008). "Mitochondria and cell death: outer membrane permeabilization and beyond." Nat Rev Mol Cell Biol 11(9): 621-32.

Tatsuta, T. and T. Langer (2008). "Quality control of mitochondria: protection against neurodegeneration and ageing." Embo J 27(2): 306-14.

Theodosis, D. T., D. A. Poulain, et al. (2008). "Activity-dependent structural and functional plasticity of astrocyte-neuron interactions." Physiol Rev 88(3): 983-1008.

Tsujimoto, Y., T. Nakagawa, et al. (2006). "Mitochondrial membrane permeability transition and cell death." Biochim Biophys Acta 1757(9-10): 1297-300.

Vieira, H. L., P. M. Alves, et al. (2011). "Modulation of neuronal stem cell differentiation by hypoxia and reactive oxygen species." Prog Neurobiol 93(3): 444-55.

Vieira, H. L., D. Haouzi, et al. (2000). "Permeabilization of the mitochondrial inner membrane during apoptosis: impact of the adenine nucleotide translocator." Cell Death Differ 7(12): 1146-54.

Vieira, H. L., C. S. Queiroga, et al. (2008). "Preconditioning induced by carbon monoxide provides neuronal protection against apoptosis." J Neurochem 107(2): 375-84.

Vinten-Johansen, J., Z. Q. Zhao, et al. (2005). "Postconditioning--A new link in nature's armor against myocardial ischemia-reperfusion injury." Basic Res Cardiol 100(4): 295-310.

Vosler, P. S., C. S. Brennan, et al. (2008). "Calpain-mediated signaling mechanisms in neuronal injury and neurodegeneration." Mol Neurobiol 38(1): 78-100.

INTRODUCTION

39

Ch

apte

r I

Wang, B., W. Cao, et al. (2011). "Carbon monoxide-activated Nrf2 pathway leads to protection against permanent focal cerebral ischemia." Stroke 42(9): 2605-10.

Wang, C. F., Z. Y. Wang, et al. (2011). "Dual protective role of HO-1 in transplanted liver grafts: a review of experimental and clinical studies." World J Gastroenterol 17(26): 3101-8.

Wang, J. and S. Dore (2008). "Heme oxygenase 2 deficiency increases brain swelling and inflammation after intracerebral hemorrhage." Neuroscience 155(4): 1133-41.

Wegener, S., B. Gottschalk, et al. (2004). "Transient ischemic attacks before ischemic stroke: preconditioning the human brain? A multicenter magnetic resonance imaging study." Stroke 35(3): 616-21.

Wu, J., J. D. Holstein, et al. (2007). "Purinergic receptor-stimulated IP3-mediated Ca2+ release enhances neuroprotection by increasing astrocyte mitochondrial metabolism during aging." J Neurosci 27(24): 6510-20.

Yabluchanskiy, A., P. Sawle, et al. (2012). "CORM-3, a carbon monoxide-releasing molecule, alters the inflammatory response and reduces brain damage in a rat model of hemorrhagic stroke*." Crit Care Med 40(2): 544-52.

Yenari, M. A. and H. S. Han (2012). "Neuroprotective mechanisms of hypothermia in brain ischaemia." Nat Rev Neurosci 13(4): 267-78.

Yepes, M., B. D. Roussel, et al. (2008). "Tissue-type plasminogen activator in the ischemic brain: more than a thrombolytic." Trends Neurosci.

Yepes, M., B. D. Roussel, et al. (2009). "Tissue-type plasminogen activator in the ischemic brain: more than a thrombolytic." Trends Neurosci 32(1): 48-55.

Youle, R. J. and A. Strasser (2008). "The BCL-2 protein family: opposing activities that mediate cell death." Nat Rev Mol Cell Biol 9(1): 47-59.

Zeynalov, E. and S. Dore (2009). "Low doses of carbon monoxide protect against experimental focal brain ischemia." Neurotox Res 15(2): 133-7.

Zhang, H. X., G. H. Du, et al. (2003). "Ischemic pre-conditioning preserves brain mitochondrial functions during the middle cerebral artery occlusion in rat." Neurol Res 25(5): 471-6.

Zhao, H., R. M. Sapolsky, et al. (2006). "Phosphoinositide-3-kinase/akt survival signal pathways are implicated in neuronal survival after stroke." Mol Neurobiol 34(3): 249-70.

Zhu, C., X. Wang, et al. (2005). "The influence of age on apoptotic and other mechanisms of cell death after cerebral hypoxia-ischemia." Cell Death Differ 12(2): 162-76.

Chapter I

40

Zhu, X., W. G. Fan, et al. (2011). "Heme oxygenase-1 system and gastrointestinal inflammation: a short review." World J Gastroenterol 17(38): 4283-8.

Zimmermann, A., C. W. Leffler, et al. (2007). "Cerebroprotective effects of the CO-releasing molecule CORM-A1 against seizure-induced neonatal vascular injury." Am J Physiol Heart Circ Physiol 293(4): H2501-7.

II CARBON MONOXIDE DIRECT EFFECT ON NON-SYNAPTIC MITOCHONDRIA:

mitochondrial membrane permeabilization prevention

This chapter is based on the following manuscript:

Glutathionylation of adenine nucleotide translocase induced by carbon monoxide prevents mitochondrial membrane permeabilization and apoptosis

Cláudia S.F. Queiroga, Ana S. Almeida, Cécile Martel, Catherine Brenner, Paula M.

Alves and Helena L.A. Vieira (2010) Journal of Biological Chemistry, 28;285(22):17077-88.

“Mitochondria are gatekeepers of life and death.” Brian O'Rourke

MITOCHONDRIAL MEMBRANE PERMEABILIZATION PREVENTION

43

Ch

apte

r II

ABSTRACT

The present work demonstrates the ability of carbon monoxide (CO) to prevent

apoptosis in primary culture of astrocytes. For the first time, the anti-apoptotic

behaviour can be clearly attributed to the inhibition of mitochondrial membrane

permeabilisation (MMP), a key event in the intrinsic apoptotic pathway. In isolated

non-synaptic mitochondria CO partially inhibits (i) loss of membrane potential, (ii) the

opening of a non specific pore through the inner membrane, (iii) swelling and (iv)

cytochrome c release, which are induced by calcium, diamide or atractyloside (a

ligand of adenine nucleotide translocase, ANT). CO directly modulates ANT function

by enhancing ADP/ATP exchange and prevents its pore-forming activity. Additionally,

CO induces reactive oxygen species (ROS) generation, and its prevention by β-

carotene, decreases CO cytoprotection in intact cells, as well as in isolated

mitochondria, revealing the key role of ROS. On the other hand, CO induces slight

increase in mitochondrial oxidized glutathione (GSSG), which is essential for

apoptosis modulation by (i) delaying astrocytic apoptosis, (ii) decreasing MMP and

(iii) enhancing ADP/ATP translocation activity of ANT. Moreover, CO and GSSG

trigger ANT glutathionylation, a post-translational process regulating protein function

in response to redox cellular changes. In conclusion, CO protects astrocytes from

apoptosis by preventing MMP, acting on ANT (glutathionylation and inhibition of its

pore activity) via a preconditioning-like process mediated by ROS and GSSG.

Chapter II

44

CONTENTS

1. Introduction ........................................................................................................... 46

2. Material and methods ........................................................................................... 49

2.1. Materials ....................................................................................................... 49

2.2. Cell culture .................................................................................................... 49

2.3. Isolation of non-synaptic mitochondria from cortex ....................................... 49

2.4. Preparation of CO solutions .......................................................................... 50

2.5. Apoptosis induction/prevention ..................................................................... 50

2.6. Assessment of apoptosis-associated parameters ........................................ 51

2.7. Measurement of ROS generation ................................................................. 51

2.8. Quantification of mitochondrial swelling ........................................................ 52

2.9. Cytochrome c release detection ................................................................... 52

2.10. Mitochondrial depolarisation detection ........................................................ 52

2.11. Inner membrane permeabilisation assay .................................................... 52

2.12. ADP/ATP translocase activity assessment ................................................. 53

2.13. Glutathione content quantification .............................................................. 53

2.14. Mitochondria isolation from primary culture of astrocytes ........................... 54

2.15. Immunoprecipitation ................................................................................... 54

2.16. Immunoblotting ........................................................................................... 55

2.17. Statistical analyses ..................................................................................... 55

3. Results ................................................................................................................. 56

3.1. Carbon monoxide prevents apoptosis in astrocytes ..................................... 56

3.2. ROS generation is crucial for CO-induced cytoprotection ............................ 59

3.3. CO inhibits mitochondrial membrane permeabilisation (MMP) in isolated non-

synaptic mitochondria .......................................................................................... 61

3.4. ROS are important molecules for CO prevention of MMP ............................ 63

3.5. CO facilitates ADP/ATP translocation function of ANT ................................. 64

3.6. CO augments GSSG/GSH ratio in isolated mitochondria ............................. 65


45

Ch

apte

r II

3.7. GSSG signalling and protein glutathionylation are involved in the modulation

of ANT activity and MMP by CO ........................................................................... 66

3.8. Role of glutathione S-transferase (GST) ....................................................... 70

3.9. Prevention of mitochondrial GSH recycling protects astrocytes from cell death

and inhibits MMP .................................................................................................. 70

4. Discussion ............................................................................................................. 72

5. Acknowledgments ................................................................................................. 76

6. References ............................................................................................................ 77

Cláudia S.F. Queiroga had carried out all the majority of the experimental part performed, as well as involved on the decisions on how to execute the experiments, as well as on the

discussion and interpretation of the results.

Chapter II

46

1. INTRODUCTION

Preconditioning (PC) is induced by potentially hazardous stimuli below their

threshold of injury, resulting in subsequent tissue protection, or tolerance, which is

defined as a condition of transiently increased resistance to injury. PC was first found

to be triggered by short episodes of ischemia, called ischemic preconditioning;

however, the stimuli can also be pharmacological or chemical (Kirino 2002).

Understanding the preconditioning phenomenon can be a tool to elucidate the

cellular endogenous protective mechanisms. Many factors are involved in the

signalling, transducing or executing the PC response. Reactive oxygen species

(ROS) are crucial signalling molecules during PC development and are mostly

generated in the mitochondria (Dirnagl et al. 2008). The four protein complexes

associated with the respiratory chain are the primary source of ROS by handling the

bulk of oxygen metabolism (Busija et al. 2008). Furthermore, respiratory chain

inhibition induces preconditioning and cytoprotection against focal cerebral ischemia

via ROS generation (Wiegand et al. 1999).

Apoptosis occurs via two distinct pathways: an extrinsic pathway (relying on cell

surface membrane receptors) and an intrinsic pathway, which is triggered by several

conditions of intracellular stress, leading to mitochondrial membrane permeabilisation

(MMP). In many models, MMP induces (i) mitochondrial transmembrane potential

dissipation, (ii) respiratory chain uncoupling, (iii) ROS overproduction, (iv) ATP

synthesis arrest and (v) the release of several death-regulating molecules (activating

proteases and nucleases), making the cell death process irreversible (Kroemer et al.

2007; Galluzzi et al. 2009). Depending on the cell type and apoptosis stimuli, MMP

can occur only in the outer membrane or in both mitochondrial membranes (inner

and outer membranes) via the mitochondrial permeability transition pore (PTP).

Permeability transition pore consists in a sudden increase in the inner membrane

permeabilisation to solutes up to 1500 Da; PTP is based at least on the dynamic

interaction between voltage dependent anion channel (VDAC), cyclophilin D (CypD)

and adenine nucleotide translocase (ANT) (Zoratti et al. 1995; Bernardi et al. 2001).


47

Ch

apte

r II

Apoptotic or necrotic cell death due to hypoxia-ischemia and reperfusion injury

clearly involves the process of mitochondrial permeability transition (Kroemer et al.

2007).

ANT is the most abundant inner membrane protein responsible for the vital

function of stoichiometric ADP/ATP exchange on inner membrane. However, ANT

can switch to a lethal function, corresponding to its pore-forming activity. ANT can

interact with different proteins depending on the cell type or in response to apoptotic

stimuli. Anti- or pro-apoptotic members of the Bcl-2 family, such as Bcl-2 or Bax, can

physically interact with ANT facilitating its antiporter or pore-forming activity,

respectively (Marzo et al. 1998; Belzacq et al. 2003). On the other side, glutathione-

S-transferase (GST) interacts with ANT in normal tissue, in colon carcinoma cells

and in vitro. This interaction is lost during apoptosis induction, suggesting that GST

behaves as an endogenous repressor of PTP and of ANT pore activity (Verrier et al.

2004). The ANT pore forming property is also modulated by oxidation of critical thiol

groups in cysteine residues (cysteine 56, 159 and 256) facing the matrix side

(Costantini et al. 1996; McStay et al. 2002). Several thiol-cross linking agents (such

as diamide, dithiodipyridine or phenylarsine oxide) enforce PTP opening and pore-

forming activity of ANT, which is not prevented by Bcl-2 (Costantini et al. 2000;

McStay et al. 2002).

Carbon monoxide (CO) is an endogenous product of heme degradation by

heme-oxygenase (HO), which also generates free iron and biliverdin (Ryter et al.

2006). HO plays an important role in cell redox state, acting as an antioxidant

enzyme, which can be especially important for tissues with weak endogenous

antioxidant defences, such as the myocardium and the nervous system (Dore 2002).

HO activity has been suggested to modulate and prevent cerebral cell death in

several models; this neuroprotective property has been mainly attributed to bilirubin

antioxidant activity (Boehning et al. 2003; Schipper 2004), however little data is

available for CO in the central nervous system.

Low concentrations of CO confer an increased resistance to apoptosis triggered

by several stimuli in different models, including endothelial cells, vascular smooth

Chapter II

48

muscle cells, liver or lung. CO also mediates other biological functions, such as anti-

inflammation, arrest of proliferation or vasodilatation (Ryter et al. 2006) and this

molecule presents a strong potential in therapeutic applications (Bannenberg et al.

2009). More recently, it has been shown that ROS, generated by mitochondria

(Sandouka et al. 2005; D'Amico et al. 2006), are imperative signalling molecules for

CO biological functions, such as anti-inflammation, cardioprotection, anti-proliferation

or anti-apoptosis in several systems (Bilban et al. 2008). Carbon monoxide is

described to bind to cytochrome c oxidase (complex IV), which slows down the rate

of electron transport enabling electron to accumulate, including at complex III. Thus,

the lifetime of the ubisemiquinone state of coenzyme Q is prolonged, increasing the

propensity to reduce O2 into superoxide (O2-), which is enzymatically converted to

other ROS (Chin et al. 2007; Zuckerbraun et al. 2007; Bilban et al. 2008). In the

literature, CO biological properties are prevented by the addition of anti-oxidants,

inhibition of complex III or the use of respiration-deficient ρº cells (Chin et al. 2007;

Zuckerbraun et al. 2007; Bilban et al. 2008; Vieira et al. 2008). In neuronal primary

cultures, CO exposure provides cytoprotective PC with an increased resistance

against apoptosis and ROS are crucial signalling molecules (Vieira et al. 2008).

On one hand, CO presents anti-apoptotic properties in several models. On the

other hand, mitochondria are central executers of the programmed cell death

process, via the MMP. In the literature, the most described role of CO in

mitochondria is the generation of ROS, which are signalling factors. Since no data

concerning the direct action of CO on MMP are available, the present work has

explored the direct effect of CO into non-synaptic mitochondria (MMP modulation)

and, consequently, its ability to prevent apoptosis in astrocytes. The involvement of

ROS as signalling molecules for CO-PC triggering were investigated, as well as the

biochemical mechanisms involved in the MMP control by CO.


49

Ch

apte

r II

2. MATERIAL AND METHODS

2.1. Materials

All the chemicals were of analytical grade and were obtained from Sigma

(Germany) unless stated otherwise. Plastic tissue culture dishes were from Nunc

(Denmark); fetal bovine serum (FBS), glutamine, penicillin-streptomicin solution and

Dulbecco’s Minimum Essential Medium were obtained from Gibco (UK); Wistar rats

were purchased from Instituto de Higiene e Medicina Tropical (Lisboa, Portugal).

2.2. Cell culture

Primary cultures of astrocytes were prepared from 2-day-old rat cortex, as

described in Sa Santos et al. 2005. Briefly, cerebral hemispheres were carefully

freed of the meninges, washed in ice-cold PBS and mechanically disrupted. Single-

cell suspensions were plated in T-flasks (3 hemispheres/175 cm2) in Dulbecco’s

Minimum Essential Medium (DMEM) supplemented with 10% (v/v) fetal bovine

serum heat inactivated, 100 U/mL penicillin-streptomycin solution and glucose (to

obtain a final concentration of 10 mM). Cells were maintained in a humidified

atmosphere of 7% CO2 at 37ºC. After 8 days the phase dark cells growing on the

astrocytic cell layer were separated by vigorous shaking and removed. The

remaining astrocytes were detached by mild trypsinization using trypsin/EDTA

(0.25% wt/vol) and subcultured in T-flasks for another 2 weeks. Growth medium was

changed twice a week.

2.3. Isolation of non-synaptic mitochondria from cortex

Mitochondria were isolated from 300-350 g male Wistar adult rat according to

(Kristian et al. 2000). Briefly, cortex was removed and washed in a ice-cold isolation

buffer containing 225 mM manitol, 75 mM sucrose, 1 mM EGTA and 5 mM HEPES,

pH 7.4. The tissue was minced with scissors and manually homogenized with Potter

Elvehjem in isolation buffer. The homogenate was centrifuged at 1300 g for 3 min,

and the resuspended pellet was re-centrifuged 1300 g for 3 min. Both supernatants

Chapter II

50

were pooled together and centrifuged at 21200 g for 10 min. The remaining pellet

was resuspended in 3.5 mL of Percoll solution 15% and layered into centrifuge tubes

containing a preformed two-step discontinuous density gradient consisting of 3.7 mL

of 24% Percoll on top of 1.7 mL 40% Percoll. The gradient was centrifuged at 31700

g for 9 min. The mitochondrial fraction, located between the layers 24% and 40%

Percoll, was removed, diluted 1:8 in isolation buffer and centrifuged at 16700 g for 10

min. The pellet was resuspended in 10 mL of isolation buffer containing 5 mg/mL

BSA (to remove lipids) and centrifuged at 6800 g for 10 min. The mitochondrial pellet

was resuspended in 100 μL of isolation buffer and the total amount of protein was

quantified using BCA assay (Pierce, Illinois). All the steps were carried out at 4ºC.

All isolated mitochondria analysis were performed on modified brain buffer

(Kristian et al. 2000) containing 125 mM KCl, 2 mM K2HPO4, 1 mM MgCl2, 15 μM

EGTA, 20 mM Tris, 5 mM glutamate and 5 mM malate, pH 7.3, unless stated

otherwise. All CO treatment in isolated mitochondria was performed with final

concentration of 10 μM, during 15 min at room temperature (RT), unless stated

otherwise.

2.4. Preparation of CO solutions

Fresh stock solutions of CO gas were prepared each day and carefully sealed.

PBS (Phosphate Buffered Saline) was saturated by bubbling 100% of CO gas during

30 min to produce 10-3 M stock solution. The concentration of CO in solution was

determined spectrophotometrically by measuring the conversion of deoxymyoglobin

to carbon monoxymyoglobin, as previously described (Motterlini et al. 2002). CO 100

% was purchased as compressed gas (Linde, Germany).

2.5. Apoptosis induction/prevention

Astrocytes were treated with CO (50 μM) for 3 hours, or with ethacrynic acid

(EA, 50 μM) or carmustine (BCNU, 100 μM) for 1h, followed by medium exchange.

Then, apoptosis was induced with diamide at concentrations ranging from 50 to 250

µM or with tert-butylhydroperoxide (t-BHP) at concentrations from 80 to 280 μM for


51

Ch

apte

r II

18h. In some cases, β-carotene (1 μM) was used to modulate CO effect, by applying

this anti-oxidant to cells 1h prior CO treatment.

2.6. Assessment of apoptosis-associated parameters

To detect apoptosis induced by diamide or t-BHP, cell samples were collected

by trypsinisation and cells were gated by the forward and side scatter. Two dyes

were used 3,3’-dihexyloxacarbocyanine iodide (DiOC6(3), 20 nM, Invitrogen, UK) to

quantify the mitochondrial transmembrane potential (∆Ψm) and propidium iodide

(PI, 1 μg/ml, Invitrogen, UK) to determine cell viability, based on plasma membrane

integrity. A flow cytometer (Partec, Germany) was used to analyse apoptosis-

associated parameters. This cytometer contains a blue solid state laser (488 nm)

with FL1 green fluorescence channel for DiOC6(3) at 530 nm and a FL3 red

fluorescence channel for PI detection at 650 nm. The acquisition and analysis of the

results were performed with FlowMax (Partec) software

2.7. Measurement of ROS generation

ROS generation was followed by the conversion of 2’,7’-dichlorofluorescein

diacetate (H2DCFDA, Invitrogen, UK) to fluorescent 2’, 7’-dichlorofluorescein (DCF).

Astrocytes were treated for 3 h with CO, supernatant was removed and cells were

incubated for 20 min with 10 μM of H2DCFDA prepared in PBS. Cells were washed

twice and fluorescence was measured (λexc: 485 nm, λem: 530 nm) using FL500 96

Well Spectrofluorimeter. β-carotene (1 μM) was added 1h prior CO treatment. In the

case of isolated mitochondria, 25 μg of mitochondrial protein was incubated with 5

μM of H2DCFDA and 10, 50 or 250 μM of CO, in modified brain buffer. Fluorescence

(λexc: 485 nm, λem: 530 nm) was measured using Biotek Synergy 2 Spectrofluorimeter

during 30 min at 37ºC. ROS generation was calculated as an increase over baseline

levels, determined for untreated cells (100%). In some cases, β-carotene (1 μM) was

added to isolated mitochondria 10 minutes prior CO treatment.

Chapter II

52

2.8. Quantification of mitochondrial swelling

25 μg of mitochondrial protein was diluted in modified brain buffer containing or

not 10 μM of CO. After 15 minutes of incubation at RT, 5 or 15 μM of Ca2+ were

added and the decrease in optical density at 540 nm was immediately measured for

30 min at 37ºC, using Biotek Synergy 2 Spectrofluorimeter. 100% of swelling is

calculated based on the optical density decrease after 30 min between non-treated

and 15 M Ca2+ treated mitochondria.

2.9. Cytochrome c release detection

25 μg of protein from isolated mitochondria was diluted in modified brain buffer

containing or not 10 μM of CO. After 15 minutes of incubation at RT, 5 or 15 μM of

Ca2+ were added and mitochondria were incubated for 30 min at 37ºC. Samples

were centrifuged for 10 minutes at 10000 g and mitochondrial pellet was analysed by

immunoblotting with -cytochrome c.

2.10. Mitochondrial depolarisation detection

For depolarisation measurements, isolated mitochondria (25 μg) containing 1 μM

of rhodamine 123 in modified brain buffer were pre-treated with: 10 μM of CO, 5 μM

of cyclosporine A (CsA), 1 μM of β-carotene or 10 μM of EA. To depolarization

assessment by Rhodamine 123 dequenching several MMP inducers were added: 5

or 7.5 μM of Ca2+, 300 μM of atractyloside or 250 μM of diamide. The fluorescent

measurements (λexc: 485 nm, λem: 535 nm, Biotek Synergy 2 Spectrofluorimeter)

were followed for 30 min at 37ºC and are expressed in percentage relative to the

positive control 5 μM of Ca2+ (100%) at the indicated time point.

2.11. Inner membrane permeabilisation assay

Citrate synthase activity assay is used to assess the inner membrane

permeability, as described in Belzacq-Casagrande et al. 2009. Upon inner

mitochondrial membrane permeabilisation acetyl-CoA is able to enter into

mitochondrial matrix, reacting with citrate synthase. 5, 5’-dithio-bis 2-nitrobenzoic


53

Ch

apte

r II

acid (DTNB) and deacetyled acetyl-CoA reaction gives 5-thio-2-nitrobenzoate (TNB)

which can be followed by absorbance at 412 nm.

Briefly, 25 μg of protein from isolated mitochondria was incubated with CO (10

μM), GSSG (1 μM) or EA (25 μM), in modified brain buffer containing 100 μM of

DTNB, 300 μM of acetylCoA and 1 mM of oxaloacetate. Inner membrane

permeabilisation was induced by atractyloside at 300 μM or Ca2+ at 15 μM.

Whenever was the case, β-carotene (1 μM) was added 10 minutes prior CO

treatment. The absorbance at 412 nm was recorded for 20 minutes, using Biotek

Synergy 2 Spectrofluorimeter.

2.12. ADP/ATP translocase activity assessment

ADP/ATP translocase activity was assessed accordingly with Belzacq-

Casagrande et al. 2009. The ADP/ATP exchange rate was evaluated on 75 μg of

protein from isolated mitochondria diluted in ATP buffer (20 mM HEPES, pH 7.2, 5

mM succinate, 300 mM sucrose, 10 mM KCl, 1 mM MgCl2, 1 mM Pi, 10 μM EGTA, 2

μM rotenone). Isolated mitochondria were treated with 10 μM of CO, 1 or 100 μM of

GSSG or 10 μM of EA. ATP efflux triggered by externally added ADP was monitored

by fluorescence (λexc: 360 nm, λem: 465 nm) following NADP+ reduction occurring in

a solution containing 2.5 mM glucose, 1 E.U. hexokinase (E.C. 2.7.1.1.), 0.5 E.U.

glucose-6-phosphate-dehydrogenase and 0.2 mM NADPH, as described (Belzacq-

Casagrande et al. 2009). Influence of adenylate kinase-dependent ATP synthesis

was evaluated after treatment of isolated mitochondria by 10 μM of adenylate kinase

specific inhibitor Ap5A (P1P5-diadenosine-5’-pentaphosphate). The values obtained,

using Biotek Synergy 2 Spectrofluorimeter, during 30 min at 37ºC are expressed in

percentage relative to control (100%) at the time point indicated.

2.13. Glutathione content quantification

After treatment with CO at 10 μM or 50 μM, in the presence or absence of β-

carotene at 1 μM, reduced and oxidized glutathione in isolated mitochondria were

determined spectrophotometrically (Molecular Devices, SpectraMax 340) using the

Chapter II

54

procedure previously described (Hirrlinger et al. 2005). The assay measured the rate

of formation of TNB from DTNB, in the presence of NADPH and glutathione

reductase, at 412 nm, during 5 minutes. Glutathione disulfide (GSSG) is measured

as described earlier after derivatization of GSH with 2-vinylpiridine (2VP) (Dringen et

al. 1996). The values are expressed as GSSG/GSH ratio.

2.14. Mitochondria isolation from primary culture of astrocytes

Primary cultures of astrocytes were washed with ice-cold PBS collected by

trypsinisation. The samples were centrifuged at 200 g for 10 min, and cells were

whashed in PBS by centrifugation at 200 g for 10 min. The supernatant was

discarded and the pellet (cells) incubated in 3.5 mL of hypotonic buffer (0.15 mM

MgCl2, 10 mM KCl, 10 mM Tris-HCl, pH 7.6) at 4ºC for 5 min. After the addition of an

equal volume of homogenisation buffer (0.15mM MgCl2, 10 mM KCl, 10 mM Tris-

HCl, 0.4 mM PMSF, 250 mM saccharose, pH 7.6) twice concentrated, samples were

manually homogenised with Douncer potter. Cell extracts were centrifuged at 900 g

for 10 min; followed by supernatant centrifugation at 10000 g for 10 min. The

mitochondrial pellet was resuspended in 100 μL of homogenisation buffer and the

total amount of protein was quantified using BCA assay (Pierce, Illinois). All the steps

were carried out at 4ºC.

2.15. Immunoprecipitation

100 μg of mitochondrial protein (isolated from astrocytes or from rat cortex) were

incubated in 100 μL of homogenisation buffer containing 0.5% of Triton X-100 in the

presence of 20 μL of α-GSH (ViroGen, USA; 1 mg/mL) or 20 μL of α-ANT

monoclonal antibody (Mitosciences, USA) for 90 min at 37ºC, followed by

immunoprecipitation with 15 μL of protein A/G PLUS-Agarose beads (Santa Cruz

Biotechnology, UK) during 30 min at 37ºC. After 10 min of 10000 g centrifugation,

supernatant was discarded and pellet was washed four times with PBS. Proteins

attached to the beads were solubilized by Laemmli buffer for further Western blot

analysis.


55

Ch

apte

r II

2.16. Immunoblotting

Several samples (from cell extracts, mitochondria or immunoprecipitated protein)

were separated under reducing electrophoresis on a 1 mm NuPAGE® Novex BIS-

Tris Gel (Invitrogen, UK) and electrically transferred to a nitrocellulose membrane

(Hybond™-C extra, Amersham Biosciences). ANT, caspase-3, GST or cytochrome c

protein was stained with α-ANT monoclonal antibody (Mitosciences, USA), α-

caspase-3 (Sigma), α-GST (GE Healthcare, UK) or α-cytochrome c (Abcam), all of

them at 1/1000 dilution and 2 h of RT incubation. Blots were developed using the

ECL (enhanced chemiluminescence) detection system after incubation with HRP-

labeled anti-mouse IgG antibody (Amersham Bioscience, UK), 1/5000, 1 h of RT

incubation. The area and intensity of bands were quantified by densitometry analysis

(GraphPad Prism 4), and are presented as percentage relative to the positive control

(100%). These experiments have been repeated three times, with similar results.

2.17. Statistical analyses

The data concerning intact cells were carried out at least in three independent

preparations (cell isolation). All data related to isolated mitochondria were done in

triplicate from at least three independent animals. Mitochondrial data are presented

as a representative result of three independent assays. For the western blot

technique, a representative image of three independent assays is shown. All values

are mean ± SD, n≥3. Error bars, corresponding to standard deviation, are

represented in the figures. Statistical comparisons were performed using ANOVA:

single factor with replication, with p<0.05, n≥3. p<0.05 means that samples are

significantly different at a confidence level of 95%.

Chapter II

56

3. RESULTS

3.1. Carbon monoxide prevents apoptosis in astrocytes

Primary cultures of cortical astrocytes were treated with CO saturated PBS 3h

prior to apoptosis induction. Astrocytic apoptosis was induced by oxidative stress

with the pro-oxidant tert-butylhydroperoxide (Figure 2.1. and 2.2.) and the thiol

cross-linker diamide (Figure 2.2.) treatment during 18h. Both reagents have been

shown to induce cell death, at least, by acting at the mitochondrial level (Constantini

et al. 2000). The dissipation of mitochondrial membrane potential, Δψm, (quantified

by DiOC6(3)) and plasma membrane permeabilization (detected by iodide propidium

fluorescence), a marker for loss of viability, were assessed by flow cytometry.

CO is not toxic up to 100 μM, however, at this concentration does not confer

cytoprotection (Figure 2.1. – A and B). In addition, carbon monoxide prevents

astrocytic cell death up to 48h after induction (Figure 2.1. – C). Thus, CO role is not

limited to delay apoptosis in a short time window.


57

Ch

apte

r II

Figure 2.1. Carbon monoxide at 100 μM does not confer protection against astrocytic apoptosis

but 50 μM of CO confers protection against apoptosis in delayed periods of time. Primary

cultures of astrocytes cultured in 24 well-plates were pre-treated with 100 μM of CO for 3h, following

apoptosis induction by 18h exposure to (A, B) the pro-oxidant, t-BHP (from 0 to 280 μM). The

apoptotic hallmarks were assessed by flow cytometry. In (A) the percentage of cells presenting high

mitochondrial potential, detected by DiOC6(3), is expressed. In (B) the percentage of cells containing

intact plasma membrane (viable cells) is presented, assessed with PI fluorochrome. All values are

mean ± SD, n = 4 (A, B). (C) Primary cultures of astrocytes cultured in 24 well-plates were pre-treated

with 50 μM of CO for 3h, following apoptosis induction by the pro-oxidant, t-BHP (160 μM) for 48 h.

The percentage of viable cells (assessed by PI) is presented at 0, 6, 12, 24 and 48 h after t-BHP

addition. All values are mean±SD, n=3.

In contrast, 50 μM of CO protected astrocytes against cell death caused by

diamide (Figure 2.2. – A and B) and t-BHP (Figure 2.2. – C and D). In addition,

carbon monoxide prevents caspase-3 activation induced by diamide (Figure 2.2 –

E). In conclusion, CO does protect astrocytes from cell death induced by oxidative

stress; furthermore CO presents an extended time window of action.

(A) (B)

Chapter II

58

Figure 2.2. Carbon monoxide confers protection against apoptosis. Primary cultures of

astrocytes cultured in 24 well-plates were pre-treated with 50 μM of CO for 3h, following apoptosis

induction by 18h exposure to (A, B) the thiol cross-linker, diamide (from 0 to 250 μM) and to (C, D) the

pro-oxidant, t-BHP (from 0 to 280 μM). The apoptotic hallmarks were assessed by flow cytometry. In (A,

C) the percentage of cells presenting high mitochondrial potential, detected by DiOC6(3), is expressed.

In (B, D) the percentage of cells containing intact plasma membrane (viable cells) is presented,

assessed with PI fluorochrome. All values are mean±SD, n=4 (A, B) with *p<0.05 compared with control

and CO-treated cells for each concentration of diamide and (C, D) # p<0.05 compared with control and

CO-treated cells for each concentration of t-BHP. (E) Immunodetection of caspase-3 activation by its

cleavage into 12kDa fraction. First line corresponds to astrocytes treated with diamide at 200 μM (18h);

second line astrocytes pre-treated with 50 μM of CO (3h) followed by diamide induction of apoptosis and

third line control astrocytes.

(E)

(A) (B)

(D) (C)


59

Ch

apte

r II

3.2. ROS generation is crucial for CO-induced cytoprotection

Astrocytes were treated with 50 M of carbon monoxide and after 3 h,

intracellular ROS generation (specifically H2O2) was measured by the conversion of

2’,7’-dichlorofluorescein diacetate (H2DCFDA) to fluorescent DCF. CO induces an

increase in intracellular ROS levels of about 20%, which is prevented by 1 h of pre-

treatment with the anti-oxidant -carotene at 1 M (Figure 2.3 – A). In order to verify

the role of ROS and, thus, preconditioning mode of action of CO, 1 M of -carotene

was added to primary culture of astrocytes previous to CO treatment. Indeed,

inhibition of ROS generation decreases the anti-apoptotic effect of CO for diamide

(Figure 2.3 – B) and for t-BHP (Figure 2.3 – C) inductions. Thus, in intact cells, ROS

are imperative signalling molecules for prevention of apoptosis by CO, indicating that

a preconditioning-like mechanism is involved.

Chapter II

60

Figure 2.3. Relevance of ROS on carbon monoxide protective role. (A) CO induces

intracellular ROS generation and it is prevented by the anti-oxidant β-carotene. Primary cultures of

astroglial cells were pre-treated for 1h with 1 μM of β-carotene, followed by 50 μM CO addition during

3h, ROS quantification was performed using 2’,7’-dichlorofluorescein diacetate (H2DCFDA). (B, C)

Astrocytes were subjected to 1 μM of β-carotene for 1h, followed by treatment with 50 μM of CO for 3h,

and apoptosis was induced with diamide (B) or t-BHP (C) for 18h. Mitochondrial potential and viability

were assessed by flow cytometry, using DiOC and PI respectively. All values are mean ± SD, n=3, (A)

*p<0.05 compared with control or with β-carotene and CO-treated cells. (B) with *p<0.05 compared with

control or with β-carotene and CO-treated cells for high ΔΨm and ** p <0.05 compared with control or

with β-carotene and CO-treated cells for viability. (C) *p<0.05 compared with control or with β-carotene

and CO-treated cells for high ΔΨm and **p<0.05 compared with control or with β-carotene and CO-

treated cells for viability.

(A)

(B) (C)


61

Ch

apte

r II

3.3. CO inhibits mitochondrial membrane permeabilisation (MMP) in isolated

non-synaptic mitochondria

Several different approaches were used to assess direct or indirectly

mitochondrial membrane permeabilisation: mitochondrial depolarisation, inner

membrane permeabilisation, cytochrome c release and mitochondrial swelling. Non-

synaptic mitochondria isolated from brain cortex (Kristian et al. 2000) were treated

with 10 μM of CO during 15 min prior to addition of diamide, atractyloside (a ligand of

ANT that prevents ADP/ATP translocation and induces its pore forming function) or

calcium to induce MMP. Loss of ΔΨm, or mitochondrial depolarisation, was

measured using the methodology based on dequenching of the fluorescent probe

Rhodamine 123 (Belzacq-Casagrande et al. 2009). Loss of ΔΨm induced by

atractyloside was prevented by prior addition of CO (Figure 2.4 – A). CO also

inhibits mitochondrial depolarisation induced by diamide and Ca2+ (Figure 2.4 – B).

To quantify this effect, Ca2+ at 5 μM was normalised to 100% of depolarisation

(Figure 2.4 – B). Moreover, cyclosporine A (CsA) also prevents ΔΨm loss, indicating

the involvement of the permeability transition pore (Figure 2.4 - B) (Crompton et al.

1999). Changes in the inner membrane permeability (the opening of a large channel

for molecules up to ~ 800 Da) were assessed via an enzymatic assay based on the

accessibility of citrate synthase, which is a soluble matrix enzyme (Belzacq-

Casagrande et al. 2009). The atractyloside induction of inner membrane

permeabilisation is partially prevented by CO (Figure 2.4 – C). Finally, CO also

inhibits mitochondrial swelling triggered by 5 or 15 μM of Ca2+ (Figure 2.4 – D); as

well as there is a partial prevention of cytochrome c release from mitochondria pre-

treated with CO when challenged with Ca2+ at 15 μM (Figure 2.4 – E). CO treatment

at 10 μM (alone) in isolated mitochondria had no effect on swelling, mitochondrial

depolarisation or pore formation through the inner membrane (Figure 2.4). In

conclusion, CO inhibits mitochondrial swelling, cytochrome c release, loss of ΔΨm

and inner membrane permeabilisation. Thus, CO prevents apoptosis, at least, via a

Chapter II

62

direct effect on mitochondria, i.e., by reducing mitochondrial membrane

permeabilisation (MMP).

Figure 2.4. Carbon monoxide effect on the mitochondrial membrane depolarization, inner

membrane permeabilisation, mitochondrial swelling and cytochrome c release. All 4 experimental

assays were performed using isolated non-synaptic mitochondria in modified brain buffer. (A)

Representative micrograph for Rhodamine 123 fluorescence change (λexc: 485 nm, λem: 535 nm),

measured for 30 min at 37ºC, in the absence or presence of 10 μM CO and 300 μM atractyloside or 5

μM Ca2+. (B) Quantitative expression of Rhodamine 123 fluorescent measurements at 15 min of

incubation. Isolated mitochondria were pre-treated with 10 μM CO or 1 μM CsA, then 0, 5 or 7.5 μM

Ca2+, 300 μM atractyloside or 250 μM diamide was added. The values are expressed in relative

percentage to 5 μM Ca2+ (100%). All values are mean±SD, n=3; *p<0.05 compared with control

mitochondria for each inducer. (C) An enzymatic assay based on citrate synthase activity was used to

follow inner membrane permeabilisation. Measurements were performed at 412 nm in the absence or

presence of 10 μM CO and 300 μM atractyloside, at 37ºC for 20 min. All values are mean±SD, n=3. (D)

Mitochondrial swelling was measured by absorbance at 540 nm, at 37ºC for 30 min the effect of 15 μM

Ca2+ was normalised to 100% of swelling. Mitochondria were treated in the presence or absence of Ca2+

at 5 or 15 μM and/or CO at 10 μM. All values are mean±SD, n=3; *p<0.05 compared with control and

CO-treated mitochondria. (E) Mitochondria, in the absence or presence of 10 μM of CO, were treated

with Ca2+ at 15 μM, at 37ºC for 30 min, followed by centrifugation to separate mitochondrial pellet for

immunodetection of cytochrome c release.

(A) (B)

(E)

(A) (B)

(C) (D)


63

Ch

apte

r II

3.4. ROS are important molecules for CO prevention of MMP

In isolated non-synaptic mitochondria, treatment with CO at 10, 50 or 250 μM

induced ROS generation in a dose-response manner; and pre-treatment with β-

carotene (1 μM) prevented ROS formation by CO (Figure 2.5 – A). Inhibition of

mitochondrial depolarisation by CO was lost when mitochondria were treated with the

anti-oxidant β-carotene prior to CO exposure (Figure 2.5 – B). Still, CO became

unable to prevent the atractyloside induced-opening of a large channel in the inner

membrane when β-carotene was added to mitochondria before CO treatment

(Figure 2.5 – C). These findings support the hypothesis that ROS generation by CO

is necessary for its MMP prevention in isolated mitochondria. Therefore, ROS are

also imperative CO-signalling molecules at mitochondrial level.

Chapter II

64

Figure 2.5. Influence of ROS on CO effect at mitochondrial level. In (A) mitochondria were treated

with 10, 50 or 250 μM of CO in the presence or absence of 1 μM of β-carotene, followed by ROS

quantification using 2’,7’-dichlorofluorescein diacetate (H2DCFDA, λexc: 485 nm, λem: 530 nm). The

values are expressed in percentage relative to control (100%). All values are mean±SD, n=4, *p<0.05

compared with control, **p<0.05 compared with control, #p<0.05 compared with control, ##p<0.05

compared with CO 10 µM and ***p<0.05 compared with CO 50 µM. (B) Mitochondria were pre-treated

with 1 μM of β-carotene and 10 μM of CO, then atractyloside at 300 μM or diamide at 250 μM was

added. The fluorescent measurements (λexc: 485 nm, λem: 535 nm) are expressed in relative percentage

to 5 μM of Ca2+ (100%) at 15 min of incubation. All values are mean±SD, n=3; *p<0.05 compared with

control and with β-carotene and CO-treated mitochondria. (C) Inner membrane permeabilisation was

assessed according to Belzacq-Casagrande et al. 2009. Measurements were performed at 412 nm in

the absence or presence of 10 μM of CO and 300 μM of atractyloside for during 20 min at 37ºC. All

values are mean±SD, n=3.

3.5. CO facilitates ADP/ATP translocation function of ANT

In order to clarify the mechanisms involved in CO inhibition of MMP, the influence

of this gas on adenine nucleotide translocase (ANT) activity was measured using an

(C)

(A) (B)


65

Ch

apte

r II

enzymatic based assay (Belzacq-Casagrande et al. 2009). ANT is a double function

protein located in the mitochondrial inner membrane. ANT’s physiological role

consists in exchanging ADP against ATP in a stoichiometric manner; in contrast, in

response to diverse stimuli, its activity can switch to a pore forming protein,

modulating MMP (Belzacq et al. 2003). Mitochondria treated with CO present an

increased ANT translocase activity (Figure 2.6), providing evidence that CO

prevents the opening of a non specific pore through the inner membrane by directly

acting on ANT and enforcing its physiological activity.

Figure 2.6. Carbon monoxide effect on ADP/ATP translocase activity of ANT. The results were

obtained using isolated non-synaptic mitochondria treated with 10 μM of CO and ADP/ATP

translocation was assessed according to Belzacq-Casagrande et al. 2009. ADP is added to

mitochondria and diffuses into the intermembrane space through VDAC. Once into the intermembrane

space, ADP can be transformed by adenylate kinase (AK) in AMP and ATP or exchange against ATP

by adenine nucleotide translocator (ANT). The values are expressed in relative percentage to control

(100%) at 15 min of incubation and are mean±SD, n=3, with *p<0.05 compared with control

mitochondria.

3.6. CO augments GSSG/GSH ratio in isolated mitochondria

Based on the fact that: (i) CO generates ROS in isolated mitochondria and (ii)

glutathione is one of the most efficient anti-oxidant systems in the cell, as well as at

mitochondrial level (Dringen 2000), total glutathione (GSH) and oxidized glutathione

(GSSG) were measured after CO treatment in isolated mitochondria. The total

amount of mitochondrial GSH is not altered in the presence of carbon monoxide; this

result correlates with the fact that glutathione synthesis occurs in the cytosol, while at

mitochondrial level, only GSH recycling takes place. In contrast, mitochondrial levels

of GSSG increase after CO exposure in a dose-response manner, which enhances

Chapter II

66

GSSG/GSH ratio (Figure 2.7). In addition, mitochondrial pre-treatment with β-

carotene prevents GSSG/GSH ratio augmentation due to the presence of CO

(Figure 2.7). These data suggest that GSSG is also a candidate factor for signalling

preconditioning and apoptosis prevention triggered by CO.

Figure 2.7. Carbon monoxide effect on mitochondrial GSSG/GSH ratio. After CO treatment (10 or

50 M) in the presence or absence of -carotene (1 M), oxidized and reduced glutathione

quantification was performed using a microtiter plate assay, as described in the Material and Methods

section. The values are mean±SD, n=3, with *p<0.05 compared with control, **p<0.05 compared with

mitochondria treated with 10 μM of CO, #p<0.05 compared with mitochondria without β-carotene

treatment and 10 μM CO treatment and ##p<0.05 compared with mitochondria without β-carotene

treatment and 50 μM CO treatment.

3.7. GSSG signalling and protein glutathionylation are involved in the

modulation of ANT activity and MMP by CO

In order to challenge the hypothesis of GSSG as signalling molecule in the CO

cytoprotective pathway, three distinct approaches were studied: (i) the effect of

GSSG on ADP/ATP translocation by ANT, (ii) modulation of inner membrane

permeability by small amounts of GSSG and (iii) the covalent modification of ANT by

glutathionylation of thiol groups. Small amounts of oxidized glutathione (1 μM)

increase the translocase activity of ANT (Figure 2.8. – A), which is similar to the CO

effect upon this enzyme. Furthermore, mitochondria treated with ethacrynic acid

(EA), which prevents glutathione recycling (Muyderman et al. 2004) and increases

GSSG levels, also facilitates ADP/ATP translocation by ANT (Figure 2.8. – A). Thus,

there might be a direct effect of GSSG on ANT. However, higher concentrations of


67

Ch

apte

r II

GSSG (100 μM) prevent ADT/ATP exchange activity (Figure 2.8. – A). Still, oxidized

glutathione (1 μM) and EA partially prevent inner membrane permeabilisation

challenged by atractyloside and Ca2+ (Figure 2.8. – B and C). Thus, small amounts

of GSSG seem to modulate ANT activity and prevent MMP.

GSSG/GSH ratio was calculated for different conditions in order to correlate the

effect of CO on glutathione levels and the addition of 1 μM GSSG. GSSG/GSH ratio

in non-treated mitochondria is 0.080 and in CO (10 μM)-treated mitochondria, it

increases to 0.135; while addition of 1 μM GSSG in mitochondrial preparation

corresponds to a GSSG/GSH ratio of 0.426, which is three-time higher than the ratio

induced by 10 μM CO. However, oxidized glutathione is added into the media, it

does not correspond to glutathione concentration in the mitochondrial matrix.

Therefore, and based on these similar values (same order of magnitude), one can

consider that addition of GSSG at 1 μM might mimic the augmentation of

GSSG/GSH ratio due to CO induction. These data are an additional support for the

hypothesis that GSSG are signaling molecules in the CO-induced prevention of

MMP.

In addition, an increase of GSSG levels allows the formation of protein mixed

disulfides, or protein glutathionylation. Reversible protein glutathionylation is a post-

translational process involved in the cellular response to redox changes, which can

protect cysteine residues against irreversible damage due to oxidative stress

(Gallogly et al. 2007). In addition, ANT presents critical thiol residues (Costantini et

al. 2000), which are important for the control of protein function and are candidate to

be glutathionylated. Purified mitochondria from control astrocytes or from CO-treated

astrocytes were incubated with α-GSH to immunoprecipitate glutathionylated

proteins, followed by immunodetection of ANT in Western blot assay (Figure 2.8. –

D). Moreover, purified mitochondria from non treated astrocytes were incubated with

small amounts of GSSG (1 μM), followed by immunoprecipitation with α-GSH and

detected with α-ANT by Western blot (Figure 2.8. –D). The presence of CO or

GSSG augments the levels of glutathionylated ANT (Figure 2. 8. – D).

Chapter II

68

In order to verify if diamide-targeted thiol residues of ANT are protected by

glutathionylation, a competition assay was performed. Isolated mitochondria were

treated with diamide and followed by CO exposure. In fact, diamide pre-treatment

decreases the ANT glutathionylation levels induced by CO (Figure 2.8. – E);

suggesting that diamide acts on the same cysteine residue(s) that is (are)

glutathionylated by CO. Taken all together, these data provide evidence that CO

increases GSSG levels, which covalently modifies ANT by glutathionylation,

facilitating ADP/ATP translocation and preventing inner membrane permeabilisation.

In contrast, and to further confirm the functional role of ANT glutathionylation by

CO, the same experimental conditions used to test CO modulation of MMP (Figure

2.4) were used to detect ANT glutathionylation. Isolated non-synaptic mitochondria

were first pre-treated with CO (10 or 50 μM) or GSSG (1 μM) at RT for 15 min,

followed by diamide addition (100 μM) at 37ºC for 30 min. Immunoprecipitation was

performed showing that ANT glutathionylation still occurs after diamide treatment

(Figure 2.8. - F).


69

Ch

apte

r II

Figure 2.8. Role of ANT glutathionylation in MMP modulation. (A) ADP/ATP translocation was

followed in isolated mitochondria in the presence of 1 μM or 100 μM of GSSG or 10 μM of EA. The

values are expressed in relative percentage to control (100%) at 15 min of incubation at 37ºC, and are

mean±SD, n=3, with *p<0.05 compared with control. (B, C). Isolated non-synaptic mitochondria were

treated with GSSG at 1 μM (B) or with EA at 25 μM (C) during 10 min followed by atractyloside (300

μM) or Ca2+ (5 μM) addition in order to induce inner membrane permeabilisation, which was assessed

according to (Belzacq-Casagrande et al. 2009). Measurements were performed at 412 nm for during

20 min at 37ºC. All values are mean±SD, n=3. (D) Primary cultures of astrocytes were treated with 0,

10, 50 μM of CO following mitochondria isolation; additionally 1 μM of GSSG was added to

mitochondria isolated from control astrocytes. Glutathionylated proteins ( -GSH) were

immunoprecipitated in mitochondria isolated from astrocytes and ANT was immunodetected by

Western blot from the immunoprecipitated proteins. The area and intensity of bands were quantified by

densitometry analysis (GraphPad Prism 4), and are presented as relative percentage to the positive

control (100%). This experiment has been repeated three times, with similar results. (E) Isolated non-

synaptic mitochondria were treated in the presence or absence of 100 μM diamide during 15 min,

followed by CO (10 μM) incubation for 15 min; then glutathionylated proteins ( -GSH) were

immunoprecipitated and ANT was immunodetected by Western blot from the immunoprecipitated

proteins. This experiment has been repeated three times, with similar results. (F) Isolated non-synaptic

mitochondria were pre-treated with 10 or 50 μM CO or with 1 μM GSSG for 15 min, followed by

diamide (100 μM) incubation at 37ºC for 30 min; then glutathionylated proteins (α-GSH) were

immunoprecipitated and ANT was immunodetected by Western blot from the immunoprecipitated

proteins. This experiment has been repeated three times, with similar results.

(A) (B)

(C) (D)

(E) (F)

Chapter II

70

3.8. Role of glutathione S-transferase (GST)

In the literature glutathione S-transferase (GST) is described to physically interact

with ANT and to be an endogenous repressor of apoptosis (Verrier et al. 2004). ANT

has been co-purified with GST from rat brain, co-immunoprecipitated from a colon

carcinoma cell line and the functional cooperation between both proteins has been

mimic by reconstitution into proteoliposomes (Verrier et al. 2004). On the other hand,

GST is a potential candidate enzyme to be involved in the ANT glutathionylation.

Thus, the effect of CO in the interaction between ANT and GST was assessed.

Purified mitochondria from control or from CO-treated astrocytes were

immunoprecipitated with α-ANT, followed by GST immunodetection in Western blot

assay (Figure 2.9). However, CO does not alter the physical interaction of ANT–

GST. Further future work about CO implication on GST activity induction must be

performed to disclose the role of GST in CO-induced ANT glutathionylation.

Figure 2.9. Effect of CO in ANT-GST interaction. Primary cultures of astrocytes were treated with 0,

10, 50 μM of CO following mitochondria isolation; ANT ( -ANT) was immunoprecipitated in

mitochondria isolated from astrocytes and GST was immunodetected by Western blot from the

immunoprecipitated proteins. This experiment has been repeated three times, with similar results.

3.9. Prevention of mitochondrial GSH recycling protects astrocytes from cell

death and inhibits MMP

To verify the role of mitochondrial GSSG as signalling molecules in the

modulation of apoptosis, inhibitor factors of GSH recycling were used: ethacrynic

acid (EA, specific for mitochondrial recycling) (Muyderman et al. 2004) or carmustine

(BCNU). Astrocytes were incubated with EA or BCNU prior to apoptosis induction


71

Ch

apte

r II

with t-BHP (Figure 2.10 – A). EA inhibits mitochondrial depolarisation and loss of

viability in astrocytes, while BCNU presents no effect (Figure 2.10. – A).

Furthermore, in isolated non-synaptic mitochondria, the presence of EA also

prevents dissipation of m induced by atractyloside (Figure 2.10. – B). It is

important to highlight that the EA concentration used here for intact cells (50 M) is

half of these used by Muyderman and colleagues (100 M) where they have shown

a total prevention of mitochondrial GSH recycling (Muyderman et al. 2004). Thus, the

protective effect of EA in the present work might be due to a partial inhibition of GSH

recycling, slightly increasing GSSG levels. In addition, isolated mitochondria from

EA-treated astrocytes revealed higher levels of ANT glutathionylation than from non

treated astrocytes (Figure 2.10. – C). Taken all together, these results indicate that

slight levels of GSSG in the mitochondria are important for prevention of MMP and

cell survival signalling, probably by targeting ANT.

Chapter II

72

Figure 2.10. Effect of mitochondrial GSSG in cell death, MMP and post-translational ANT

modifications. (A) The percentage of astrocytic survival when subjected to 50 μM of EA or 100 μM of

BCNU for 1h, followed by medium exchange and treatment with t-BHP (200 μM ) for 18h.

Mitochondrial potential and viability were assessed by flow cytometry, using DiOC and PI, respectively.

All values are mean±SD, n=4, with *p<0.05 compared with control cells treated with t-BHP for ΔΨm

high and **p<0.05 compared with control cells treated with t-BHP for viability. (B) Depolarisation assay.

Isolated mitochondria were pre-treated with 10 μM of EA for 10 min followed by 300 μM of

atractyloside or 5 μM of Ca2+ treatment at 37ºC. The fluorescent measurements (λexc: 485 nm, λem: 535

nm) are expressed in relative percentage to 5 μM of Ca2+ (100%) at 15 min of incubation. All values

are mean±SD, n=4, with *p<0.05 compared with atractyloside-treated mitochondria. (C) Primary

cultures of astrocytes were treated with 0 or 50 μM of EA following mitochondria isolation;

glutathionylated proteins ( -GSH) were immunoprecipitated in mitochondria isolated from astrocytes

and ANT was immunodetected by Western blot from the immunoprecipitated proteins. This experiment

has been repeated three times, with similar results.

4. DISCUSSION

The present study has demonstrated that CO confers protection against oxidative

stress-induced apoptosis in primary culture of astrocytes (Figure 2.2). For the first

time, it has been shown that there is a direct anti-apoptotic effect of CO upon

mitochondria, by preventing mitochondrial membrane permeabilisation (MMP), which

is a key event in the intrinsic apoptotic pathway. The data reported here revealed that

CO inhibits mitochondrial swelling, cytochrome c release, dissipation of ΔΨm, as

(A) (B)

(C)


73

Ch

apte

r II

well as the opening of a non specific pore through the inner membrane in isolated

non-synaptic mitochondria (Figure 2.4).

Previously, we have shown that CO prevented neuronal apoptosis by inducing a

preconditioning-like effect, model in which reactive oxygen species appeared to be

signalling molecules (Vieira et al. 2008). Herein, it has been demonstrated that (i) CO

induces ROS production in astrocytes and (ii) inhibition of ROS generation by an

anti-oxidant addition (β-carotene) reverses the anti-apoptotic effect of CO. In intact

cells, ROS are necessary for CO to delay apoptosis (Figure 2.3). Also, at subcellular

level (isolated mitochondria) ROS appear to be critical for CO to reduce MMP

(Figure 2.5). Thus, ROS generation is crucial for CO signalling.

ANT is a key protein involved in the control of the permeability transition pore,

leading to the release of pro-apoptotic factors into the cytosol (Vieira et al. 2000).

ANT interacts with either the pro-apoptotic protein Bax or the anti-apoptotic protein

Bcl-2, which both influence ANT in opposite ways. Bax facilitates the opening of a

pore through the inner membrane, while Bcl-2 increases translocation activity of ANT

(Marzo et al. 1998; Brenner et al. 2000; Belzacq et al. 2003). CO increases the

ADP/ATP translocation by ANT (Figure 2.6), which is comparable to Bcl-2-ANT

model of MMP regulation. CO appears to change ANT conformation, which

stimulates translocation activity and inhibits channel function. This conformation

might be the c-conformation first described by Brandolin et al. (Brandolin et al. 1993).

This is in agreement with the literature, as it has been demonstrated that HO-1

expression increases the activity of ADP/ATP transporter in renal mitochondria in

experimental diabetes (Di Noia et al. 2006). Therefore, CO modulates MMP by, at

least, directly acting on ANT.

CO augments the mitochondrial GSSG/GSH ratio in a dose-response manner

(Figure 2.7). This GSSG/GSH ratio increase is prevented by β-carotene addition.

Additionally, glutathione redox changes can lead to the regulated formation of mixed

disulfides between protein thiol and glutathione disulfide (protein glutathionylation).

The progressive glutathionylation of key proteins is proposed as a molecular switch

by which cells respond in an immediate and reversible fashion to oxidative stress

Chapter II

74

(Gallogly and Mieyal 2007). Protein glutathionylation can be considered as a

physiological signalling function, in the same way as the phosphorylation process.

On the other hand, critical thiol residues of ANT can be oxidized and/or derivatized in

order to modulate permeability transition (Costantini et al. 2000; McStay et al. 2002).

Herein it was shown that CO and GSSG induce ANT glutathionylation (Figure 2.8. –

D). Moreover, in a functional approach, it has been demonstrated that small

concentrations of GSSG (1 μM) or EA (inhibitor of mitochondrial GSH recycling)

stimulates ADP/ATP translocation activity of ANT (Figure 2.8. – A) and prevents

inner membrane permeabilisation (Figure 2.8. – B and C) as does CO. Therefore,

one can speculate that glutathionylation of ANT protects critical thiol groups against

oxidation and/or cross linking between residues, avoiding conformation changes,

such as the formation of ANT dimers, which are responsible for a non specific pore

formation (Costantini et al. 2000). In fact, diamide-pre-treatment decreases ANT

glutathionylation levels induced by CO in isolated mitochondria (Figure 2.8. – E),

which suggests that the diamide-targeted cysteine residue(s) might be the same that

is (are) glutathionylated by CO. In contrast, higher concentrations of GSSG (100

μM) inhibit ADP/ATP translocation by ANT (Figure 2.8. – A), which is in agreement

with Vesce and colleagues who have shown that acute GSH depletion decreases

ATP transport through the inner membrane (Vesce et al. 2005).

Furthermore, one can state that 1 μM addition of GSSG mimics the effect of CO

(10 μM) treatment in isolated mitochondria. In mitochondria, GSSG/GSH ratio

increases from 0.080 to 0.135 upon CO exposure; while addition of 1 μM GSSG

corresponds to a GSSG/GSH ratio of 0.426. Although there is a three-time increase,

these values have the same order of magnitude. Moreover, GSSG addition is done

into the medium, and GSSG/GSH ratio is calculated in the mitochondrial matrix. In

conclusion, addition of small amount of GSSG mimics endogenous GSSG

generation triggered by CO, acting as signalling factors.

A slight increase in mitochondrial GSSG levels is also demonstrated to be

important for cell signalling in functional approaches. Partial inhibition of GSH


75

Ch

apte

r II

recycling at mitochondrial level, by EA addition, protects astrocytes from cell death

induced by oxidative stress (Figure 2.10. – A) and protects mitochondria from MMP

induced by atractyloside (Figure 2.10. – B). However, when a general inhibitor of

GSH recycling (BCNU) is used, no protection is found (Figure 2.10. – B).

Furthermore, EA increases ANT glutathionylation levels (Figure 2.10. – C). Taken

together, these data suggest that little amounts of mitochondrial GSSG can act as

signalling molecules, in much the same way as ROS do.

Several examples of protein glutathionylation or de-glutathionylation involved in

apoptosis control can be found in the literature: (i) glutathionylation prevents

caspase-3 activation (Huang et al. 2008); (ii) glutathionylation of complex II

decreases after myocardial ischemia and reperfusion, limiting the electron transfer

activity of this complex (Chen et al. 2007) or (iii) reversible glutathionylation of

complex I increases mitochondrial superoxide formation (Taylor et al. 2003). In

accordance with our data, Piantadosi and colleagues (Piantadosi et al. 2006) have

found that rats exposed to small amounts of CO presented higher levels of protein

mixed disulfides in liver mitochondria. Further data are necessary to clarify the

existence of other target proteins to be glutathionylated in the CO modulation of

apoptosis. Some possible targets are Bcl-2 family proteins or the different isoforms of

ANT, since ANT1 and ANT3 have been identified as a pro-apoptotic isoform,

whereas ANT2 is anti-apoptotic (Le Bras et al. 2006).

GST is considered as an endogenous repressor of apoptosis, because its

interaction with ANT is lost during the apoptotic process and, on the other hand,

cancer cell lines present higher levels of GST interacting with ANT (Verrier et al.

2004). Ablation of GST expression increases apoptosis induced by oxidative stress

with 4-hydroxynonenal (Vaillancourt et al. 2008). Moreover, GST is also a promising

candidate enzyme to be involved in the ANT glutathionylation process. By

immunoprecipitation assays it was confirmed that ANT physically interacts with GST;

however, CO does not alter this interaction (Figure 2.10). Whether CO induces an

augmentation of GST activity remains to be determined.

Chapter II

76

In this work, CO protects astrocytes from cell death by inhibiting MMP, and the

process can be divided in at least three main steps: (i) ROS as signalling molecules,

(ii) mitochondrial GSSG as transducing factors and (iii) ANT as effector protein. CO

appears to trigger a preconditioning-like event, by activating the cellular endogenous

protective mechanisms. It is important to highlight that the mechanisms described in

the present study are related to early preconditioning response since the time frame

window analysed is short. ROS generation, mitochondrial GSSG increase and ANT

glutathionylation were assessed between minutes to 3 hours after CO treatment.

However, late preconditioning is not excluded because when apoptosis is induced 24

hours after CO treatment, this molecule is still able to confer cell protection

(unpublished data). In addition, it is expected that late preconditioning involves the

expression of cellular anti-oxidant enzymes. Indeed, hypoxia-induced PC increases

expression of superoxide dismutase and/or glutathione peroxidase (Stroev et al.

2005; Choi et al. 2007), and continuous CO exposure in rats increases SOD2

expression (Piantadosi et al. 2006). Thus, in future work, the cellular anti-oxidant

machinery behaviour in response to CO will be explored. Still, it can be speculated

that oxidized glutathione increases locally in mitochondria in a first time window of

cellular response to CO, and in a second step (late preconditioning) cellular reduced

glutathione levels might increase to prevent oxidative injury.

In summary, CO presents anti-apoptotic properties in astrocytes by directly

preventing mitochondrial membrane permeabilisation, with GSSG as transducing

factor, and acting on ANT function via critical thiol residue glutathionylation. CO is a

potential anti-apoptotic factor against cerebral hypoxia-ischemia and reperfusion;

furthermore, it can be used as a tool to disclose the cellular pathways involved in the

preconditioning phenomenon able to confer cytoprotection.

5. ACKNOWLEDGMENTS

This work was supported by the Portuguese Fundação para a Ciência e

Tecnologia project PTDC/SAU-NEU/64327/2006, and HLAV’s

SFRH/BPD/27125/2006 and CSFQ’s SFRH/BD/43387/2008 fellowships. The authors


77

Ch

apte

r II

express their gratitude to João Seixas from Alfama, Portugal, for measurements of

CO in solution; to Dr Lígia Martins from Instituto de Tecnologia Química e Biológia,

Portugal, for providing Biotek Synergy 2 Spectrofluorimeter and to Sofia Almeida

from IBET, Portugal, for technical support.

6. REFERENCES

Bannenberg, G. L. and H. L. Vieira (2009). "Therapeutic applications of the gaseous mediators carbon monoxide and hydrogen sulfide." Expert Opin Ther Pat 19(5): 663-82.

Belzacq-Casagrande, A. S., C. Martel, et al. (2009). "Pharmacological screening and enzymatic assays for apoptosis." Front Biosci 14: 3550-62.

Belzacq, A. S., H. L. Vieira, et al. (2003). "Bcl-2 and Bax modulate adenine nucleotide translocase activity." Cancer Res 63(2): 541-6.

Bernardi, P., V. Petronilli, et al. (2001). "A mitochondrial perspective on cell death." Trends Biochem Sci 26(2): 112-7.

Bilban, M., A. Haschemi, et al. (2008). "Heme oxygenase and carbon monoxide initiate homeostatic signaling." J Mol Med 86(3): 267-79.

Boehning, D., C. Moon, et al. (2003). "Carbon monoxide neurotransmission activated by CK2 phosphorylation of heme oxygenase-2." Neuron 40(1): 129-37.

Brandolin, G., A. Le Saux, et al. (1993). "Chemical, immunological, enzymatic, and genetic approaches to studying the arrangement of the peptide chain of the ADP/ATP carrier in the mitochondrial membrane." J Bioenerg Biomembr 25(5): 459-72.

Brenner, C., H. Cadiou, et al. (2000). "Bcl-2 and Bax regulate the channel activity of the mitochondrial adenine nucleotide translocator." Oncogene 19(3): 329-36.


Chen, Y. R., C. L. Chen, et al. (2007). "Mitochondrial complex II in the post-ischemic heart: oxidative injury and the role of protein S-glutathionylation." J Biol Chem 282(45): 32640-54.

Chin, B. Y., G. Jiang, et al. (2007). "Hypoxia-inducible factor 1alpha stabilization by carbon monoxide results in cytoprotective preconditioning." Proc Natl Acad Sci U S A 104(12): 5109-14.

Chapter II

78

Choi, Y. S., K. O. Cho, et al. (2007). "Ischemic preconditioning in the rat hippocampus increases antioxidant activities but does not affect the level of hydroxyl radicals during subsequent severe ischemia." Exp Mol Med 39(4): 556-63.

Costantini, P., A. S. Belzacq, et al. (2000). "Oxidation of a critical thiol residue of the adenine nucleotide translocator enforces Bcl-2-independent permeability transition pore opening and apoptosis." Oncogene 19(2): 307-14.

Costantini, P., B. V. Chernyak, et al. (1996). "Modulation of the mitochondrial permeability transition pore by pyridine nucleotides and dithiol oxidation at two separate sites." J Biol Chem 271(12): 6746-51.

Crompton, M., S. Virji, et al. (1999). "The mitochondrial permeability transition pore." Biochem Soc Symp 66: 167-79.

D'Amico, G., F. Lam, et al. (2006). "Inhibition of cellular respiration by endogenously produced carbon monoxide." J Cell Sci 119(Pt 11): 2291-8.

Di Noia, M. A., S. Van Driesche, et al. (2006). "Heme oxygenase-1 enhances renal mitochondrial transport carriers and cytochrome C oxidase activity in experimental diabetes." J Biol Chem 281(23): 15687-93.


Dore, S. (2002). "Decreased activity of the antioxidant heme oxygenase enzyme: implications in ischemia and in Alzheimer's disease." Free Radic Biol Med 32(12): 1276-82.

Dringen, R. (2000). "Metabolism and functions of glutathione in brain." Prog Neurobiol 62(6): 649-71.

Dringen, R. and B. Hamprecht (1996). "Glutathione content as an indicator for the presence of metabolic pathways of amino acids in astroglial cultures." J Neurochem 67(4): 1375-82.

Gallogly, M. M. and J. J. Mieyal (2007). "Mechanisms of reversible protein glutathionylation in redox signaling and oxidative stress." Curr Opin Pharmacol 7(4): 381-91.


Hirrlinger, J. and R. Dringen (2005). "Multidrug resistance protein 1-mediated export of glutathione and glutathione disulfide from brain astrocytes." Methods Enzymol 400: 395-409.

Huang, Z., J. T. Pinto, et al. (2008). "Inhibition of caspase-3 activity and activation by protein glutathionylation." Biochem Pharmacol 75(11): 2234-44.


79

Ch

apte

r II


Kristian, T., J. Gertsch, et al. (2000). "Characteristics of the calcium-triggered mitochondrial permeability transition in nonsynaptic brain mitochondria: effect of cyclosporin A and ubiquinone O." J Neurochem 74(5): 1999-2009.

Kroemer, G., L. Galluzzi, et al. (2007). "Mitochondrial membrane permeabilization in cell death." Physiol Rev 87(1): 99-163.

Le Bras, M., A. Borgne-Sanchez, et al. (2006). "Chemosensitization by knockdown of adenine nucleotide translocase-2." Cancer Res 66(18): 9143-52.

Marzo, I., C. Brenner, et al. (1998). "Bax and adenine nucleotide translocator cooperate in the mitochondrial control of apoptosis." Science 281(5385): 2027-31.

McStay, G. P., S. J. Clarke, et al. (2002). "Role of critical thiol groups on the matrix surface of the adenine nucleotide translocase in the mechanism of the mitochondrial permeability transition pore." Biochem J 367(Pt 2): 541-8.


Muyderman, H., M. Nilsson, et al. (2004). "Highly selective and prolonged depletion of mitochondrial glutathione in astrocytes markedly increases sensitivity to peroxynitrite." J Neurosci 24(37): 8019-28.

Piantadosi, C. A., M. S. Carraway, et al. (2006). "Carbon monoxide, oxidative stress, and mitochondrial permeability pore transition." Free Radic Biol Med 40(8): 1332-9.


Sa Santos, S., L. L. Fonseca, et al. (2005). "Culturing primary brain astrocytes under a fully controlled environment in a novel bioreactor." J Neurosci Res 79(1-2): 26-32.

Sandouka, A., E. Balogun, et al. (2005). "Carbon monoxide-releasing molecules (CO-RMs) modulate respiration in isolated mitochondria." Cell Mol Biol (Noisy-le-grand) 51(4): 425-32.

Schipper, H. M. (2004). "Heme oxygenase expression in human central nervous system disorders." Free Radic Biol Med 37(12): 1995-2011.

Stroev, S. A., T. S. Gluschenko, et al. (2005). "The effect of preconditioning on the Cu, Zn superoxide dismutase expression and enzyme activity in rat brain at the early period after severe hypobaric hypoxia." Neurosci Res 53(1): 39-47.

Taylor, E. R., F. Hurrell, et al. (2003). "Reversible glutathionylation of complex I increases mitochondrial superoxide formation." J Biol Chem 278(22): 19603-10.

Chapter II

80

Vaillancourt, F., H. Fahmi, et al. (2008). "4-Hydroxynonenal induces apoptosis in human osteoarthritic chondrocytes: the protective role of glutathione-S-transferase." Arthritis Res Ther 10(5): R107.

Verrier, F., A. Deniaud, et al. (2004). "Dynamic evolution of the adenine nucleotide translocase interactome during chemotherapy-induced apoptosis." Oncogene 23(49): 8049-64.

Vesce, S., M. B. Jekabsons, et al. (2005). "Acute glutathione depletion restricts mitochondrial ATP export in cerebellar granule neurons." J Biol Chem 280(46): 38720-8.

Vieira, H. L., D. Haouzi, et al. (2000). "Permeabilization of the mitochondrial inner membrane during apoptosis: impact of the adenine nucleotide translocator." Cell Death Differ 7(12): 1146-54.


Wiegand, F., W. Liao, et al. (1999). "Respiratory chain inhibition induces tolerance to focal cerebral ischemia." J Cereb Blood Flow Metab 19(11): 1229-37.

Zoratti, M. and I. Szabo (1995). "The mitochondrial permeability transition." Biochim Biophys Acta 1241(2): 139-76.

Zuckerbraun, B. S., B. Y. Chin, et al. (2007). "Carbon monoxide signals via inhibition of cytochrome c oxidase and generation of mitochondrial reactive oxygen species." Faseb J 21(4): 1099-106.

III CARBON MONOXIDE DIRECT EFFECT ON NON-SYNAPTIC MITOCHONDRIA:

mitochondrial metabolism reinforcement


Carbon monoxide modulates apoptosis by reinforcing oxidative metabolism in astrocytes: role of Bcl-2

Ana S. Almeida, Cláudia S.F. Queiroga, Marcos F.Q. Sousa, Paula M. Alves and

Helena L.A. Vieira (2012) Journal of Biological Chemistry, 287(14): 10761-10770.

“Mitochondria occupy a central position in the biology of cells and are crucial to life, as we know it”.

J. William O. Ballard, 2004

MITOCHONDRIAL METABOLISM REINFORCEMENT

83

Ch

apte

r III

ABSTRACT

Modulation of cerebral cell metabolism for improving the outcome of hypoxia-

ischemia and reperfusion is a strategy yet to be explored. Because carbon monoxide

(CO) is known to prevent cerebral cell death, herein the role of CO in the modulation

of astrocytic metabolism, in particular, at the level of mitochondria was investigated.

Low concentrations of CO partially inhibited oxidative stress-induced apoptosis in

astrocytes, by preventing caspase-3 activation, mitochondrial potential depolarization

and plasmatic membrane permeability. CO exposure enhanced intracellular ATP

generation, which was accompanied by an increase in specific oxygen consumption,

a decrease in lactate production and a reduction in glucose utilization, indicating an

improvement of oxidative phosphorylation. Accordingly, CO increased cytochrome c

oxidase (COX) enzymatic specific activity and stimulated mitochondrial biogenesis.

In astrocytes, COX interacts with Bcl-2, which was verified by immunoprecipitation;

this interaction is superior after 24 h of CO treatment. Furthermore, CO enhanced

Bcl-2 expression in astrocytes. By silencing Bcl-2 expression with siRNA

transfection, CO effects in astrocytes were prevented, namely: (i) inhibition of

apoptosis, (ii) increase on ATP generation, (iii) stimulation of COX activity and (iv)

mitochondrial biogenesis. Thus, Bcl-2 expression is crucial for CO modulation of

oxidative metabolism and for conferring cytoprotection. In conclusion, CO protects

astrocytes against oxidative stress-induced apoptosis by improving cellular

metabolism, in particular mitochondrial oxidative phosphorylation.

Chapter III

84

CONTENTS

1. Introduction ........................................................................................................... 85

2. Material and methods ........................................................................................... 86

2.1. Materials ....................................................................................................... 86

2.2. Cell culture in monolayer .............................................................................. 87

2.3. Cell culture in Bioreactor ............................................................................... 87

2.4. Measurement of oxygen specific consumption (qO2) ................................... 88

2.5. Lactate/glucose ratio ..................................................................................... 88

2.6. siRNA transfection ........................................................................................ 88

2.7. Isolation of non-synaptic mitochondria from cortex ....................................... 89

2.8. Preparation of CO solutions .......................................................................... 90

2.9. Assessment of apoptosis-associated parameters by flow cytometry ............ 90

2.10. Immunoprecipitation ................................................................................... 90

2.11. Immunoblotting ........................................................................................... 91

2.12. Mitochondrion isolation from primary culture of astrocytes ......................... 91

2.13. ATP quantification ....................................................................................... 91

2.14. Polymerase chain reaction ......................................................................... 92

2.15. Cytochrome c oxidase activity .................................................................... 92

2.16. Statistical analyses ..................................................................................... 93

3. Results ................................................................................................................. 93

3.1. Carbon monoxide prevents apoptosis and increases intracellular ATP ........ 93

3.2. Carbon monoxide improves oxidative metabolism ....................................... 95

3.3. Role of Bcl-2 in oxidative phosphorylation .................................................... 99

4. Discussion .......................................................................................................... 104

5. Acknowledgments .............................................................................................. 108

6. References ......................................................................................................... 108

Cláudia S.F. Queiroga had carried out all the experimental part on the astrocytic survival, as well as involved on the decisions on how to execute the experiments, on the discussion and

interpretation of the results.


85

Ch

apte

r III

1. INTRODUCTION

Proposing carbon monoxide (CO) as a therapeutic molecule is counter-intuitive at

first glance, since for many decades, it has been primarily seen as a toxic gas and

“silent-killer” due to its high affinity for hemoglobin. However, CO has been identified

as an endogenous product of heme degradation by heme-oxygenase (HO) activity.

Furthermore, CO is largely recognized as a homeostatic molecule, modulating

inflammation, apoptosis and proliferation (Soares et al. 2009; Motterlini et al. 2010).

Three main areas of potential therapeutic applications have been extensively

studied: cardiovascular diseases, inflammatory disorders and organ transplantation

(Bannenberg et al. 2009; Motterlini and Otterbein 2010).

The cytoprotective and, in particular, anti-apoptotic role of CO is widely described

in the airways and cardiovascular system (Ryter et al. 2006). The anti-apoptotic

protein Bcl-2 also seems to be involved in CO-induced cytoprotection. CO-stimulated

Bcl-2 expression has conferred protection in a lung model of ischemia-reperfusion

(Zhang et al. 2003), while overexpression of HO-1 was neuroprotective in a model of

permanent middle cerebral artery occlusion in transgenic mice by increasing Bcl-2

levels in neurons (Panahian et al. 1999). In the central nervous system (CNS), low

concentrations of CO suppressed neuroinflammation in a model of multiple sclerosis

(experimental autoimmune encephalomyelitis) (Chora et al. 2007) and induced

vasodilatation in a model of epileptic seizures in newborn piglets (Zimmermann et al.

2007). In primary cultures of neurons and astrocytes, CO induced preconditioning by

de novo protein synthesis and by post-translational protein modification, respectively,

preventing apoptosis (Vieira et al. 2008; Queiroga et al. 2010). In an adult model of

cerebral ischemia, brain lesion was less marked in CO-pre-treated animals (Zeynalov

et al. 2009; Wang et al. 2011).

However, the association between CO-induced metabolic changes and its

cytoprotective role remains unclear. In hepatocytes, adenosine triphosphate (ATP)

production is stimulated by increasing enzymatic activity of heme-oxygenase or by

exogenously administration of CO (Tsui et al. 2005; Tsui et al. 2007), which

Chapter III

86

enhances resistance against hepatic apoptosis. On the other hand, CO has been

shown to target and inhibit cytochrome c oxidase, generating ROS, which are

important signalling molecules for CO’s mode of action (Taille et al. 2005; D'Amico et

al. 2006; Zuckerbraun et al. 2007; Bilban et al. 2008). In cardiomyocytes, CO triggers

mitochondrial biogenesis in a ROS-dependent mode (Suliman et al. 2007) and

prevents murine doxorubicin cardiomyopathy (Suliman et al. 2007). Still, ROS (in

particular hydrogen peroxide and anion superoxide) are crucial intracellular signalling

molecules for CO to prevent apoptosis in astrocytic and neuronal primary cultures

(Vieira et al. 2008; Queiroga et al. 2010).

Hypoxia-ischemia and reperfusion, due to stroke in adults and to perinatal

complications in newborns, are the main cause of brain damage. Cerebral damage is

a result of oxygen and tissue energy depletion, leading to acidosis, inflammation,

glutamate excitotoxicity, and generation of ROS (Vannucci et al. 2004; Dirnagl et al.

2009). Stimulation of angiogenesis is the single strategy based on improving

metabolism for treating cerebral hypoxia-ischemia and reperfusion (Vannucci and

Hagberg 2004). Thus, novel strategies targeting cellular metabolic performance

represent a window of opportunity for addressing and improving brain ischemia

outcome. Furthermore, astrocytes are the most metabolic active cells in the CNS,

and are involved in brain structural support, repair after trauma and maintenance of

normal neuronal transmission and metabolism. Herein the role of CO in astrocytic

metabolism to confer cytoprotection and prevent damage in a model of hypoxia-

ischemia and reperfusion (HIR) is explored. The hypothesis lies on an enhancement

of oxidative metabolism by CO, via modulation of cytochrome c oxidase activity and

mitochondrial biogenesis; both events are dependent on Bcl-2 expression.


2.1. Materials

All of the chemicals were of analytical grade and were obtained from Sigma-

Aldrich (Munich, Germany) unless stated otherwise. Plastic tissue culture dishes


87

Ch

apte

r III

were from Nunc (Roskilde, Denmark); fetal bovine serum (FBS),

penicillin/streptomycin (P/S) solution, and Dulbecco’s minimum essential medium

were obtained from Gibco (Paisley, UK); and Wistar rats were purchased from

Instituto de Higiene e Medicina Tropical (Lisboa, Portugal).

2.2. Cell culture in monolayer

Primary cultures of astrocytes were prepared from 2-day-old rat cortex, as

described (McCarthy et al. 1980; Sa Santos et al. 2005). Briefly, cerebral

hemispheres were carefully freed of the meninges, washed in ice cold phosphate-

buffered saline (PBS), and mechanically disrupted. Single-cell suspensions were

plated in T-flasks (three hemispheres/175 cm2) in Dulbecco’s minimum essential

medium (DMEM) supplemented with 10% (v/v) FBS (heat-inactivated), 100 units/ml

penicillin/streptomycin solution, and glucose (to obtain a final concentration of 10

mM). Cells were maintained in a humidified atmosphere of 7% CO2 at 37 °C. After 8

days, the phase dark cells growing on the astrocytic cell layer were separated by

vigorous shaking and removed. The remaining astrocytes were detached by mild

trypsinization using trypsin/EDTA (0.25%, w/v) and subcultured in Tflasks for another

2 weeks. Growth medium was changed twice a week.

2.3. Cell culture in Bioreactor

After 3 weeks of astrocytes isolation in t-flasks, cells were harvested by mild

trypsinisation and immobilized in Cytodex 3 microcarriers (3 g/L, GE Healthcare)

using a cell inoculum of approximately 0.35 x 106 cell / mL. Immobilized cells were

initially cultivated in spinner-flask at 100 rpm, in an incubator at 37ºC with 7% CO2 in

air. 50 % of the culture volume was exchanged twice a week and cells were allowed

to grow until reach confluence, prior to the experiments in bioreactor, for assessing

glucose consumption, lactate production and specific oxygen consumption. To carry

out the assays in bioreactor, fully controlled cell culture environment was guarantee

by the use of commercially available bioreactors (Biostat Q-Plus, Sartorius-Stedim,

Germany) with 250 mL working volume and equipped with 3-blade impeller. Partial

Chapter III

88

pressure of oxygen (pO2), pH and temperature were monitored using adequate

probes (both from Mettler-Toledo, Urdorf, Switzerland). pO2 was maintained

constant at 30% of air saturation via surface aeration with a mixture of N2. pH and

temperature were controlled at 7.2 and 37ºC by CO2 injection and water recirculation

in the vessel jacket, respectively. Stirring was set to 100 rpm. For bioreactor control

and data acquisition MFCS/Win vs 2.1 software was used (Sartorius AG).

2.4. Measurement of oxygen specific consumption (qO2)

Oxygen uptake rate (OUR) was continuously assessed during the culture by

using equation (1):

OUR = kLa x (C*-C)

where kLa is the mass transfer coefficient previously calculated for the used

culture conditions (data not shown) as described in (Atkinson et al. 1987); C* is

saturated oxygen concentration and C is oxygen concentration in solution.

Then the qO2 value was determined using equation (2):

qO2 = OUR/Xv

OUR and Xv (viable cell concentration) values used for qO2 correspond to the

same specific time point of the culture.

2.5. Lactate/glucose ratio

Total glucose and lactate concentrations in the culture supernatant (from

bioreactor system) were determined with automated enzymatic assays (YSI 7100

Multiparameter Bioanalytical System; Dayton, OH, USA). The rate between lactate

production and glucose consumption was obtained by linear regression of the

metabolites concentrations.

2.6. siRNA transfection

Bcl-2 expression was silenced by bcl-2 coding siRNA transfection according to

manufacturer’s instructions (Invitrogen, UK). Astrocytes at 40% of confluence were

transfected using Lipofectamine™ RNAiMAX and Opti-MEM® medium (Invitrogen,


89

Ch

apte

r III

UK); for 2, 3.9 or 79 cm2 of astrocytic culture area 6, 12 or 237 pmol of siRNA were

used, respectively. At room temperature (RT) siRNA and culture medium were gently

mixed with lipofectamine for forming liposomes, and then astrocytes were transfected

in the absence of antibiotics. 24, 48 and 72 h after transfection Bcl-2 expression was

assessed by western blot assay, silencing was more efficient between 24 and 48 h,

then Bcl-2 silenced astrocytes were used in this time frame.

2.7. Isolation of non-synaptic mitochondria from cortex

Enriched fractions of non-synaptic mitochondria were isolated from 300–350 g

male Wistar adult rats according to Sims 1990; Kristian et al. 2000. Briefly, the cortex

was removed and washed in a ice-cold isolation buffer containing 225 mM manitol,

75 mM sucrose, 1 mM EGTA, and 5 mM HEPES, pH 7.4. The tissue was minced

with scissors and manually homogenized with Potter-Elvehjem in isolation buffer.

The homogenate was centrifuged at 1300 g for 3 min, and the resuspended pellet

was recentrifuged a 1300 g for 3 min. Both supernatants were pooled together and

centrifuged at 21,200 g for 10 min. The remaining pellet was resuspended in 3.5 ml

of 15% Percoll solution and layered into centrifuge tubes containing a preformed two-

step discontinuous density gradient consisting of 3.7 ml of 24% Percoll on top of 1.7

ml of 40% Percoll. The gradient was centrifuged at 31,700 g for 9 min. The

mitochondrial fraction, located between the layers of 24 and 40% Percoll, was

removed, diluted 1:8 in isolation buffer and centrifuged at 16,700 g for 10 min. The

pellet was resuspended in 10 ml of isolation buffer containing 5 mg/ml bovine serum

albumin (to remove lipids) and centrifuged at 6,800 g for 10 min. The mitochondrial

pellet was resuspended in 100 μl of isolation buffer, and the total amount of protein

was quantified using a BCA assay (Pierce). All of the steps were carried out at 4°C.

All isolated mitochondria analyses were performed on modified brain buffer (Kristian

et al. 2000) containing 125 mM KCl, 2 mM K2HPO4,1 mM MgCl2, 15 M EGTA, 20 mM

Tris, 5 mM glutamate, and 5 mM malate, pH 7.3, unless stated otherwise. All CO

treatment in isolated mitochondria was performed with a final concentration of 10 μM

for 5, 30 and 60 min at 37ºC.

Chapter III

90



PBS was saturated by bubbling 100% of CO gas for 30 min to produce 10-3 M stock

solution. The concentration of CO in solution was determined spectrophotometrically

by measuring the conversion of deoxymyoglobin to carbon monoxymyoglobin, as

described previously (Motterlini, 2002). 100% CO was purchased as compressed

gas (Linde, Germany).

2.9. Assessment of apoptosis-associated parameters by flow cytometry

To detect apoptosis induced by tert-butylhydroperoxide (t-BHP), cell samples

were collected by trypsinization, and cells were gated by the forward and side

scatter. Two dyes were used: 3,3’-dihexyloxacarbocyanine iodide (DiOC6(3); 20 nM)

(Invitrogen, UK) to quantify the mitochondrial transmembrane potential (ΔΨm) and

propidium iodide (PI; 1 μg/ml) (Invitrogen, UK) to determine cell viability, based on

plasma membrane integrity. A flow cytometer (Partec, Germany) was used to

analyze apoptosis-associated parameters. This cytometer contains a blue solid state

laser (488 nm) with FL1 green fluorescence channel for DiOC6 (3) at 530 nm and a

FL3 red fluorescence channel for PI detection at 650 nm. The acquisition and

analysis of the results were performed with FlowMax® (Partec, Germany) software.

2.10. Immunoprecipitation

100 μg of mitochondrial protein (isolated from astrocytes or from rat cortex) were

incubated in 100 μL of homogenisation buffer containing 0.5% of Triton X-100 in the

presence of 30 μL of α-COX (Santa Cruz Biotechnology, Germany; 200μg/mL) for

90 min at 37ºC, followed by immunoprecipitation with 15 μL of protein A/G PLUS-

Agarose beads (Santa Cruz Biotechnology, UK) during 30 min at 37ºC. After 10 min

of 10000 g centrifugation, supernatant was discarded and pellet was washed four

times with PBS. Proteins attached to the beads were solubilised by Laemmli buffer

for further Western blot analysis.


91

Ch

apte

r III


Several samples (from cell extracts, mitochondria or immunoprecipitated protein)

were separated under reducing electrophoresis on a 1 mm NuPAGE® Novex BIS-

Tris Gel (Invitrogen, UK) and electrically transferred to a nitrocellulose membrane

(Hybond™-C extra, Amersham Biosciences). Caspase-3 or Bcl-2 protein was stained

with α-active caspase-3 (Sigma C8487, France) or α-Bcl-2 (Santa Cruz

Biotechnology, Germany), at 1/1000 dilution for 2 h at RT. Blots were developed

using the ECL (enhanced chemiluminescence) detection system after incubation with

HRP-labeled anti-mouse IgG antibody (Amersham Bioscience, UK), 1/5000, 1 h of

RT incubation. These experiments have been repeated three times, with similar

results.

2.12. Mitochondrion isolation from primary culture of astrocytes

Primary cultures of astrocytes were pre-treated 3 h and 24 h with CO (final

concentration of 50 μM). Cells were washed with ice-cold PBS and collected by

trypsinization. The samples were centrifuged at 200 g for 10 min, and cells were

washed in PBS by centrifugation at 200 g for 10 min. The supernatant was

discarded, and the pellet (cells) was incubated in 3.5 ml of hypotonic buffer (0.15 mM

MgCl2, 10 mM KCl, 10 mM Tris-HCl, pH 7.6) at 4°C for 5 min. After the addition of an

equal volume of homogenization buffer (0.15 mM MgCl2 ,10 mM KCl, 10 mM Tris-

HCl, 0.4 mM phenylmethylsulfonyl fluoride, 250 mM saccharose, pH 7.6) twice

concentrated, samples were manually homogenized with a Dounce Potter

homogenizer. Cell extracts were centrifuged at 900 g for 10 min, followed by

supernatant centrifugation at 10,000 g for 10 min. The mitochondrial pellet was

resuspended in 100 μl of homogenization buffer, and the total amount of protein was

quantified using BCA assay (Pierce, Illinois). All of the steps were carried out at 4°C.

2.13. ATP quantification

Intracellular ATP of primary astrocytes pre-treated with CO (final concentration

of 50 μM) was quantified using “ATPlite 1 step – Luminescence ATP Detection Assay

Chapter III

92

System” (PerkinElmer, Gruningem, Netherlands), according to manufacturer’s

instructions.

2.14. Polymerase chain reaction

Genomic DNA was extracted from astrocytes using “High Pure PCR Template

Preparation Kit” (Roche Diagnostics, Mannheim, Germany). Polymerase chain

reaction (PCR) was performed using specific forward and reverse primes designed

for mitochondrial cytochrome b gene: 5’-CATCAGTCA CCCACATCTGC-3’ and 5’-

GGTTAGCGGGTGTATAATTG-3’, and for GAPDH gene: 5’-CCTTCATTG

ACCTCAACTACAT-3’ and 5’-CCAAAGTTGTCATGGATGACC-3’, respectively. “Fast

Start DNA Master Plus SYBR Green I” (Roche Diagnostics, Mannheim, Germany)

was used with the experimental run protocol: denaturation program was 95°C for 10

min, followed by 45 cycles of 95°C for 15’’, 60°C for 6’’ and 72°C for 20’’. For

evaluation of Bcl-2 expression, mRNA was extracted from astrocytes using “High

Pure RNA isolation kit” (Roche Diagnostics, Mannheim, Germany) and cDNA

synthesis was performed using “Transcriptor High Fidelity cDNA synthesis kit”

(Roche Diagnostics, Mannheim, Germany). Polymerase chain reaction (PCR) was

performed using specific forward and reverse primers designed for Bcl-2 gene:5’-

GGTGGAGGAACTCTTCAGGG-3’and 5’-GAGACAGCCAGGAGAAATCA-3’, and for

cyclophilin A gene: 5’-ATGGCAAATGCTGGACCAAA-3’ and 5’-

GCCTTCTTTCACCTTCCCAAA-3', respectively. “Fast Start DNA Master Plus SYBR

Green I” (Roche Diagnostics, Mannheim, Germany) was used with the experimental

run protocol: denaturation program was 95 °C for 10’, followed by 45 cycles of 95 °C

for 15’’, 66 °C for 10’’ and 72 °C for 15’’.

2.15. Cytochrome c oxidase activity

To assess CO early effect on cytochrome c oxidase (COX) activity (5’, 30’ and

1h), 100 μg of non-synaptic mitochondria from cortex were treated with 10 μM of CO

followed by COX activity measurements. For late effect, mitochondria from pre-

treated astrocytes were isolated, and then 100 μg of mitochondria were used to


93

Ch

apte

r III

quantify COX enzymatic activity. In both cases, COX activity was quantified using

“Cytochrome c Oxidase Assay Kit” (Sigma-Aldrich, Saint Louis, Missouri, USA),

according to manufacturer’s instructions.

2.16. Statistical analyses

The data concerning astrocytic culture were carried out at least in three

independent preparations (cell isolation). All data related to isolated non-synaptic

mitochondria were done in triplicate from at least three independent animals. For the

western blot technique, a representative image of three independent assays is

shown. All values are mean ± SD, n≥3. Error bars, corresponding to standard

deviation, are represented in the figures. Statistical comparisons were performed

using ANOVA: single factor with replication, with p<0.05, n≥3. p<0.05 means that

samples are significantly different at a confidence level of 95%.

3. RESULTS

3.1. Carbon monoxide prevents apoptosis and increases intracellular ATP

3h prior to apoptosis induction, primary culture of astrocytes was pre-treated

with CO-saturated solutions at a final concentration of 50 μM. Then, astrocytes were

challenged with the pro-oxidant tert-butylhydroperoxide (80 to 240 μM) during 20 h to

trigger apoptosis by oxidative stress. The assessed hallmarks of apoptosis were

dissipation of mitochondrial membrane potential (ΔΨm), caspase-3 activation and

plasma membrane permeabilization, a late event on the apoptotic process, (also

called secondary necrosis). Dissipation of ΔΨm (quantified by DiOC6(3)) and plasma

membrane permeabilization (detected by iodide propidium fluorescence) were

measured by flow cytometry. The presence of CO partially prevents dissipation of

ΔΨm and permeabilization of plasma membrane (Figure 3.1. – A). Caspase-3

activation was assessed by Western blot assay, in CO-treated astrocytic culture

there is less activated caspase-3 (Figure 3.1. – B). Thus, in accordance to our

previous work (Queiroga et al. 2010), CO prevents astrocytic apoptosis.

Chapter III

94

Figure 3.1. Carbon monoxide confers protection against apoptosis. Primary cultures of astrocytes

cultured in 24 well-plates were pre-treated with 50 μM of CO for 3 h, following apoptosis induction by

20 h exposure to the pro-oxidant, t-BHP (from 0 to 240 μM). The apoptotic hallmarks were assessed

by flow cytometry. In (A) the percentage of cells presenting high mitochondrial potential (detected by

DiOC6(3)) and containing intact plasma membrane (viable cells - assessed with PI) is presented. All

values are mean±SD, n=4 with *p<0.05 compared with control and CO-treated cells for each

concentration of t-BHP. (B) Representative picture of immunodetection of caspase-3 activation by its

cleavage into 17 kDa fraction. First line corresponds to astrocytes treated with t-BHP at 160 μM (20 h);

second line astrocytes pre-treated with 50 μM of CO (3 h) followed by t-BHP induction of apoptosis.

It is worthy of note that upon opening the sealed vial of CO saturated solution,

CO quickly diffuses out from the cell culture system; after 30 min more than 50% is

already lost in the atmosphere (João Seixas, personal communication). Additionally,

up to 48 h after CO treatment astrocytes still present increased resistance against

cell death (Queiroga et al. 2010). Hence, CO action might occur by activating

endogenous mechanisms and by altering gene expression. In addition, CO’s effect

on astrocytes appears not to be limited to shifting cell death signalling pathways, but

this gaseoustransmitter also changes cell metabolism. Intracellular ATP

concentration is higher at 3 h and 24 h after CO treatment than in non-treated

astrocytes (Figure 3.2). Thus, cellular metabolism alterations seem to be involved in

prevention of astrocytic apoptosis by CO via an increase of cellular energetic supply.

(A) (B)


95

Ch

apte

r III

Figure 3.2. CO increases ATP generation in astrocytes. Astrocytes were treated with 50 μM of CO

for 3 and 24 h, followed by ATP assessment. ATP concentration is represented per μg of protein from

cellular extract. All values are mean±SD, n=3 with *p<0.05 compared with control and CO-treated cells

for 3 and 24 h.

3.2. Carbon monoxide improves oxidative metabolism

Glycolysis comprises a series of biochemical reactions by which glucose is

converted into pyruvate, which may be further, converted into metabolic products

such as lactate or enter into tricarboxylic acid (TCA) cycle. In order to disclose the

metabolic pathway involved in the increase of ATP induced by CO and to follow

whether the cellular metabolic shift is further glycolytic or oxidative, the levels of

lactate production and glucose consumption were monitored. Specific rates of

glucose consumption or lactate production (μmol h-1 per cell) were calculated

throughout 36 h after CO treatment (50 μM) in astrocytic cultures performed in

bioreactors, and data are summarized in Table 3.1. Upon CO addition, the lactate

production/glucose consumption ratio decreased, meaning that higher amounts of

pyruvate entered and were metabolized by TCA cycle (Maranga et al. 2006).

Additionally, the specific rate of oxygen consumption (qO2) was calculated in the

presence of CO in astrocytes cultured for 36h in the bioreactor system. CO treatment

increases oxygen consumption by astrocytes in about 20% (Table 3.1). Given that

CO decreases the lactate production/glucose consumption ratio and increases

Chapter III

96

specific oxygen consumption, the CO-induced enhance in ATP production appears to

be derived from oxidative phosphorylation improvement.

Table 3.1. Metabolic hallmarks

Time range 0 to 36h of astrocytic culture

Glucose consumption (pmol h-1/cell) ±s.d.

Lactate production (pmol h-1/cell) ±s.d.

Lac/Glc qO2

(μmol/106cell h) ±s.d.

No treatment 0.2612±0.0022 0.4460±0.0003 1.708 0.142±0.006

CO treatment (50 µM) 0.2923±0.0337 0.3831±0.0426 1.311 0.170±0.0130

Furthermore, to verify the importance of glycolytic metabolism in astrocytes after

CO treatment, glycolysis was limited by two different strategies: (i) using glucose-free

medium in the presence of deoxyglucose (which competes with the remaining

glucose from the serum) or (ii) same strategy as previous, with the addition of

pyruvate to reinforce TCA cycle and oxidative phosphorylation. After 2 h of astrocytic

culture under glycolysis limiting conditions, CO was added to the culture medium. In

both strategies, CO treatment still increases the levels of ATP in astrocytes after 3

and 24 h (Figure 3.3. – A). Thus, CO-induced ATP enhance is not due to an

improvement on glycolytic metabolism. Additionally, glycolysis limitation does not

prevent CO-triggered cytoprotection in astrocytes because there is inhibition of

astrocytic apoptosis by CO when glucose is not available (Figure 3.3. – B and C).


97

Ch

apte

r III

Figure 3.3. Effect of CO on ATP production and protection against cell death under glycolysis

limiting conditions. Astrocytes were cultured in glucose-free media complemented with 2 mM of

deoxyglucose (for inhibiting small amounts of glucose presented in FBS) (A and B) and glucose-free

media with deoxyglucose and 2 mM of pyruvate (for directly feeding the TCA cycle) (A and C) for 2 h.

(A) Then astrocytes were treated with 50 μM of CO for 3 and 24 h, followed by ATP assessment. ATP

concentration is represented per μg of protein from cellular extract. All values are mean±SD, n=3 with

*p<0.05 compared with control and CO-treated cells for 3 and 24 h, under normal conditions and

glycolysis-limiting conditions. After culturing astrocytes in glucose-free media complemented with 2 mM

of deoxyglucose (B) and glucose-free media with deoxyglucose and 2 mM of pyruvate (C) for 2 h, 50

μM of CO was added for 3 h, followed by cell death induction with t- BHP (0 to 240 μM). The apoptotic

hallmarks were assessed by flow cytometry as in Figure 1. All values are mean±SD, n=3 with *p<0.05

compared with control and CO-treated cells for each concentration of t-BHP.

The effect of CO on cytochrome c oxidase (COX) activity was measured to

assess the influence of this gaseoustransmitter on oxidative phosphorylation. Two

distinct approaches were followed. First, non-synaptic mitochondria isolated from rat

(A)

(B) (C)

Chapter III

98

cerebral cortex were treated with CO at 10 μM, and COX activity was measured at 5,

30 and 60 min after treatment. Although being a less physiological approach, this

strategy allows the assessment of the mitochondrial early response to CO (in

particular at the level of COX activity). Immediately after CO addition (5 min), COX

activity slightly decreases (Figure 3.4. – A), which is in accordance with the literature

(Alonso et al. 2003; D'Amico et al. 2006; Bilban et al. 2008). Although being a small

effect, it is statistically significant. After 30 and 60 min of CO treatment this inhibition

is reverted (Figure 3.4. – A).

In a second strategy, astrocytes were treated with 50 μM of CO for 3h or 24h,

followed by mitochondria isolation for COX activity assessment. In this more

physiological approach, CO increased specific COX activity at 3 and 24 h (Figure

3.4. – B). Therefore, COX activity presents a two-step response to low

concentrations of CO: in the first minutes CO slightly inhibits its enzymatic activity in

isolated mitochondria. While, after 30 or 60 min of CO treatment in the case of

isolated mitochondria, and 3 or 24h when gas treatment is done in the whole intact

astrocytes, low concentrations of CO appear to improve COX activity. This two-step

response is in accordance to our previous work in isolated liver mitochondria,

showing that CO transiently inhibited COX activity up to 10 min after treatment,

followed by an enzymatic activity improvement at 30 min (Queiroga et al. 2011).

In addition to COX activity assays, the influence of CO treatment on cellular

mitochondrial population was also assessed. Quantitative real-time PCR was used to

quantify mitochondrial coding gene for cytochrome b (Cyt b), which estimates

mitochondrial biogenesis. Indeed, 3h of CO treatment induced a 50% increased on

mitochondrial Cyt b DNA in astrocytes, meaning that CO stimulates mitochondrial

DNA replication and, consequently, this organelle biogenesis. At 24h there is a slight

decrease on the amount of Cyt b DNA (Figure 3.4. – C). These data are in

accordance with Suliman and colleagues who have shown that in cardiomyocytes

CO induces mitochondrial biogenesis in a ROS-dependent manner (Suliman et al.

2007; Suliman et al. 2007). Taken all together, CO improves oxidative cell


99

Ch

apte

r III

metabolism and increases ATP production by two distinct mechanisms: accelerating

specific enzymatic activity of COX and increasing cellular mitochondrial population.

Figure 3.4. Effect of CO on cytochrome c oxidase activity and mitochondria biogenesis. (A) Non-

synaptic mitochondria were isolated from rat cortex, 100 μg were treated with 10 μM of CO and COX

enzymatic activity was assessed at 5, 30 and 60 min. All values are mean±SD, n=3 with *p<0.05

compared with control and CO-treated cells for 5, 30 and 60 min. (B) Astrocytes were treated with 50

μM of CO for 3 and 24 h, followed by mitochondria isolation (100 μg) and COX activity measurements.

All values are mean±SD, n=3 with *p<0.05 compared with control and CO-treated cells for 3 and 24 h.

(C) Astrocytes were treated with 50 μM of CO for 3 and 24 h, followed by DNA extraction for measuring

mitochondrial cytochrome b gene to assess mitochondrial DNA amount, which is represented by fold

increase when compared to control without CO treatment. All values are mean±SD, n=4 with *p<0.05

compared with control and CO-treated cells for 3 and 24 h.

3.3. Role of Bcl-2 in oxidative phosphorylation

CO modulates the expression of the anti-apoptotic protein Bcl-2, preventing cell

death in a lung model (Panahian et al. 1999; Zhang et al. 2003). On the other hand,

Chen and colleagues have demonstrated that Bcl-2 promotes survival of cancer cells

(A) (B)

(C)

Chapter III

100

by elevating mitochondrial respiration through the direct interaction with COX and

improvement of its activity (Chen et al. 2007; Chen et al. 2010). Thus, we

hypothesized that CO could also increase Bcl-2 expression in astrocytes and that

Bcl-2 could be involved on CO-triggered improvement of COX activity. CO

modulation of Bcl-2 expression was evaluated by reverse transcriptase quantitative

PCR. Astrocytes were treated with 50 μM of CO for 3 and 24 h, then total mRNA was

purified, the corresponding cDNA was synthesized by reverse transcriptase activity

and quantified by real time PCR. CO induced an increased on Bcl-2 mRNA (Figure

3.5. – A). In order to assess the physical interaction of COX and Bcl-2 upon CO

action, immunoprecipitation assays were conducted. Purified mitochondria from

control astrocytes or from CO-treated astrocytes for 3 and 24h were incubated with

α-COX to immunoprecipitate the attached proteins to cytochrome c oxidase, followed

by immunodetection of Bcl-2 by Western blot assay. The presence of CO augments

the interaction between Bcl-2 and COX after 24 h of CO treatment (Figure 3.5. – B);

however after 3 h the increase in protein-protein interaction is not significant.

Therefore, the augmentation of Bcl-2-COX interaction might occur due to an increase

on Bcl-2 expression. Taken together, Bcl-2 is involved in CO-stimulated cytochrome

c oxidase activity.


101

Ch

apte

r III

Figure 3.5. Role of CO in expression of Bcl-2 and Bcl-2-COX interaction. (A) mRNA of Bcl-2 was

quantified at 3 and 24h of CO treatment at 50 μM. Values are represented in comparison to control

astrocytes without CO treatment. All values are mean±SD, n=3 with *p<0.05 compared with control

and CO-treated cells for 3 and 24 h. (B) COX was immunoprecipitated in mitochondria isolated from

astrocytes treated with CO at 50 μM for 3 and 24 h, and Bcl-2 was immunodetected by Western blot

from the immunoprecipitated proteins. The area and intensity of bands were quantified by densitometry

analysis (GraphPad Prism 4), and are presented as relative percentage to the positive control (100%).

All values are mean±SD, n=3 with *p<0.05 compared with control and CO-treated cells for 24 h.

To validate this hypothesis, Bcl-2 expression was transiently silenced by cell

transfection with small interference RNA (siRNA) coding for Bcl-2. Silencing was

confirmed by protein expression analysis via Western blot assay 24 and 48 h after

transfection (data not shown). Silencing of Bcl-2 expression prevented the

cytoprotective effect of CO, which was demonstrated by flow cytometry analysis,

upon oxidative stress induction. CO-pre-treatment was not able to inhibit plasma

membrane permeabilization (detected by iodide propidium fluorescence) or

dissipation of mitochondrial membrane potential, ΔΨm, (quantified by DiOC6(3))

(A)

(B)

Chapter III

102

(Figure 3.6. – A). Furthermore, whenever Bcl-2 expression is silenced CO did not

prevent caspase-3 activation induced by pro-oxidant addition (Figure 3.6. – B). The

role of Bcl-2 is not limited to modulation of astrocytic apoptosis after oxidative stress

challenge. Bcl-2 expression is also relevant for: (i) CO-induced ATP production, (ii)

CO-increased COX activity and (iii) CO-stimulated mitochondrial biogenesis. Indeed,

Bcl-2 silencing prevented ATP generation enhancement by CO treatment (Figure

3.6. – C). Still, Bcl-2 expression is also crucial for CO to increase COX activity

following gas exposition, since by silencing Bcl-2 no increase on this enzymatic

activity was observed (Figure 3.6. – D). Finally, by silencing Bcl-2, CO did not

stimulate any more mitochondria biogenesis after gas exposition (Figure 3.6. – E). In

conclusion, Bcl-2 expression and interaction with COX are crucial for CO

improvement of oxidative phosphorylation.


103

Ch

apte

r III

Figure 3.6. Role of Bcl-2 in CO-induced astrocytic metabolism modulation. Bcl-2 expression was

silenced by siRNA transfection and CO treatment was performed between 24 and 48 h after transfection

to warranty gene silencing. Control and Bcl-2-silenced astrocytes were treated with CO for 3 h and

challenged to death with 160 μM of t-BHP for 20 h. (A) Cell death was assessed by flow cytometry

(detected by DiOC6(3) and PI). All values are mean±SD, n=4 with *p<0.05 compared with control and

CO-treated cells and #p<0.05 compared with Bcl-2 silenced astrocytes and CO-treated Bcl-2 silenced

astrocytes. (B) Representative picture of immunodetection of caspase-3 activation by its cleavage into

12 kDa fraction. Astrocytes were challenged to death with 160 μM of t-BHP (line 1); pre-treated with CO

(line 2), Bcl-2 was silenced and astrocytes treated with t-BHP (line 3) and Bcl-2-silenced astrocytes

(A)

(B)

(C) (D)

(E)

Chapter III

104

pretreated with CO followed by t-BHP cell death induction (line 4). (C) Bcl-2 silenced and control

astrocytes were treated with 50 μM of CO for 3 h, followed by ATP assessment. ATP concentration is

represented per μg of protein from cellular extract. All values are mean±SD, n=3 with *p<0.05

compared with non treated and CO-treated cells for 3 h and #p<0.05 compared with Bcl-2 silenced

astrocytes and control astrocytes, both treated with CO. (D) 100 μg of mitochondria were isolated from

control astrocytes and Bcl-2 silenced astrocytes, both treated or not with 50 μM of CO for 24 h for COX

activity measurement. All values are mean±SD, n=5 with *p<0.05 compared with control and CO-

treated cells. (E) Bcl-2 silenced and control astrocytes were treated with 50 μM of CO for 3 and 24 h,

followed by DNA extraction for measuring mitochondrial cytochrome b gene to assess mitochondrial

DNA amount, which is represented by fold increase when compared to control without Bcl-2

4. DISCUSSION

Previously, we have demonstrated that CO confers protection against astrocytic

cell death by directly acting on mitochondria and preventing mitochondrial membrane

permeabilization, which is a key event in the intrinsic apoptotic pathway (Queiroga et

al. 2010). The present work focuses on the role of CO in astrocytic metabolism

modulation, in particular at mitochondrial level.

Pretreatment of astrocytes with low doses of CO for 3h (Figure 3.1.) or up to 48h

(Queiroga et al. 2010) improved cellular response against stress and prevented

apoptosis by preconditioning induction; as previously published by us and others in

several tissues (Stein et al. 2005; Taille et al. 2005; Chin et al. 2007; Vieira et al.

2008; Queiroga et al. 2010).

In order to disclose the role of CO in astrocytic metabolism, the levels of ATP, the

major cellular energy carrier, was assessed. Astrocytic ATP generation increases

following CO treatment (Figure 3.2.). The two major pathways for ATP generation

are glycolysis and oxidative phosphorylation. During 36h after CO addition in

astrocytic culture, the ratio between lactate production and glucose consumption has

decreased, while the oxygen consumption levels have increased, indicating that CO

stimulated oxidative metabolism in contrast to glycolysis (Table 3.1.). In addition, CO

was able to increase ATP production and to prevent astrocytic cell death whenever

glycolysis was inhibited (Figure 3.3.), suggesting that glycolysis is not affected by

this gaseoustransmitter.


105

Ch

apte

r III

While in cancer cells, glycolysis promotes cytoprotection; in cerebral hypoxia-

ischemia, glycolysis does not appear to present similar function. In response to

ischemia, glycolysis (which is necessary to maintain ATP levels under low levels of

oxygen) leads to lactate accumulation and neural cell death by acidosis (Vannucci

and Hagberg 2004). The improvement of oxidative metabolism by restoring high-

energy stores (in particular ATP) without any glycolysis stimulation appears to confer

neuroprotection in a cerebral preconditioning model (Vannucci et al. 1998). In

myocardium ischemia, CO reduced glycolytic metabolism response (Ahlstrom et al.

2009). Furthermore, in macrophages, CO activated hypoxia-inducing factor-1 (HIF-1)

without increasing the rate of glycolysis (Chin et al. 2007). Additionally, Fukuda and

colleagues have shown that activation of HIF-1 was involved in the regulation of

cytochrome c oxidase subunits to optimize the efficiency of respiration in cancer cells

(Fukuda et al. 2007). Taken all together, from the present data and from the

literature, CO stimulates oxidative phosphorylation for preventing apoptosis in

astrocytes.

In agreement with the previous data, CO also accelerates COX activity (Figure

3.4. – A and B). In a short time response (5 min) CO slightly decreased COX activity

in isolated mitochondria, which is in accordance with several published works (Taille

et al. 2005; D'Amico et al. 2006; Zuckerbraun et al. 2007; Bilban et al. 2008). While,

after 30 min in isolated mitochondria and 3 and 24h in the case of CO-treatment of

intact astrocytes, specific COX activity has increased (Figure 3.4. – A and B). Also

in hepatic mitochondria, COX presented a two-phase response to low doses of CO:

at 5 min there was a decrease on its enzymatic activity, while after 30 min the effect

was inverted (Queiroga et al. 2011). One can speculate that CO role on COX activity

might depend on two factors: (i) time response and (ii) gaseoustransmitter

concentration, giving rise to distinct enzymatic responses. Accordingly, CO also

accelerated ATP/ADP translocase activity of ANT in non-synaptic mitochondria

(Queiroga et al. 2010). Furthermore, Di Noia and colleagues have demonstrated that

HO-1 overexpression enhanced renal oxidative phosphorylation by increasing

mitochondrial transport carriers, in particular ANT, and cytochrome c oxidase activity

Chapter III

106

in experimental diabetes (Di Noia et al. 2006). In conclusion, previously published

and the present data indicate that CO improves mitochondrial respiratory chain.

Although there is a clear effect of CO on COX activity, the molecular target of CO

remains uncertain.

CO stimulation of oxidative metabolism is not limited to COX activity

augmentation, but this gas also induced mitochondrial biogenesis in astrocytes

(Figure 3.4. – C). Accordingly, Suliman and colleagues have demonstrated CO

stimulated mitochondrial biogenesis in cardiomyocytes (Suliman et al. 2007). Herein,

mitochondria rapidly respond to CO, since mitochondrial DNA replication was

detected after 3h of CO treatment. In conclusion, CO stimulates oxidative

phosphorylation by a double effect: (i) accelerates COX enzymatic activity and (ii)

increases cellular mitochondrial population.

CO treatment enhanced the expression of the anti apoptotic protein Bcl-2 (Figure

3.5. – A). Furthermore, in cancer cells, Bcl-2 physically interacts with COX,

increasing its enzymatic activity and oxidative phosphorylation, which induces a pro-

oxidant state and cytoprotection (Chen and Pervaiz 2007; Chen and Pervaiz 2010).

In astrocytes, Bcl-2 also interacts with COX, and its interaction clearly increased at

24 h after CO treatment (Figure 3.5. – B), which is in accordance with higher levels

of Bcl-2 expression. In contrast, at 3h of CO-treatment no increase on COX-Bcl-2

interaction was observed.

In order to validate the role of Bcl-2 in CO modulation of oxidative metabolism,

astrocytes were transfected with siRNA (coding for Bcl-2) for transiently silencing this

gene expression. It is not surprising that by silencing the anti-apoptotic protein Bcl-2

the extension of cell death was higher and CO did not protect astrocytes against

apoptosis (Figure 3.6. – A and B). The CO-induced metabolism modulation, such

as: increase on ATP generation, enhancement of COX activity and stimulation of

mitochondrial DNA replication (mitochondrial biogenesis) was at least partially

prevented whenever Bcl-2 was silenced (Figure 3.6. – C-E). In conclusion, Bcl-2

expression is crucial for CO-triggered metabolism regulation and cytoprotection in

astrocytes. It is worthy of note that Bcl-2 is important for CO mode of action not only


107

Ch

apte

r III

when it is overexpressed, but also when it is expressed under constitutive levels for

two main reasons. First, modulation of cell metabolism by CO occurs at 3 h after gas

exposition, indicating an early cell response that does not involve protein expression.

Second, Bcl-2 silencing prevented CO metabolic effects on astrocytes at 3 h.

Therefore, there are two types of cell response to CO: (i) early preconditioning

(3h) probably involving ROS signaling, post-translational protein modifications or

intracellular protein localization and (ii) late preconditioning (24h) including changes

in gene expression.

A preferential cellular target for CO is the mitochondrion. CO generates

mitochondrial ROS for cell signaling (Taille et al. 2005; D'Amico et al. 2006;

Zuckerbraun et al. 2007; Bilban et al. 2008; Vieira et al. 2008; Queiroga et al. 2010)

prevents mitochondrial membrane permeabilization (Queiroga et al. 2010; Queiroga

et al. 2011) and stimulates mitochondrial biogenesis (Suliman et al. 2007; Suliman et

al. 2007). Herein, CO acts at mitochondrial level: (i) improves oxidative metabolism,

(ii) enhances mitochondrial population, (iii) modulates COX activity, (iv) increases

cellular oxygen consumption and (v) stimulates ATP production. However, the

molecular target of CO is still unclear. The most accepted hypothesis is that CO can

direct target COX inducing ROS generation at the level of complex III. CO-induced

ROS can become toxic or signalling depending on its concentration. However,

Iacono and colleagues have proposed that CO can protect cell against oxidative

stress by uncoupling mitochondria and decreasing ROS generation (Lo Iacono et al.

2011). Thus, one can speculate that CO would present “feed-back control system”,

by generating ROS to signal preconditioning; but also capable of limiting ROS

generation by uncoupling mitochondria and avoiding further oxidative stress. Future

investigation will be necessary to disclose this subject.

In conclusion, modulation of astrocytic metabolism appears as a novel and

promising strategy to protect the brain against HIR. Moreover, astrocytes are the

most metabolic active cells in the CNS; and the importance of astrocytic metabolism

modulation is not limited to its cell autonomous functioning, but is also crucial for the

maintenance of normal neuronal transmission and metabolism.

Chapter III

108

5. ACKNOWLEDGMENTS

This work was supported by the Portuguese Fundação para a Ciência e

Tecnologia project PTDC/SAUNEU/098747/2008, and HLAV’s

SFRH/BPD/27125/2006 and CSFQ’s SFRH/BD/43387/2008 fellowships. The authors

express their gratitude to João Seixas from Alfama, Portugal, for measurements of

CO in solution.

6. REFERENCES

Ahlstrom, K., B. Biber, et al. (2009). "Metabolic responses in ischemic myocardium after inhalation of carbon monoxide." Acta Anaesthesiol Scand 53(8): 1036-42.

Alonso, J. R., F. Cardellach, et al. (2003). "Carbon monoxide specifically inhibits cytochrome c oxidase of human mitochondrial respiratory chain." Pharmacol Toxicol 93(3): 142-6.

Atkinson, B. and F. Mavituna (1987). Biochemical Engineering Fundamentals. New York, McGraw-Hill, Inc.

Bannenberg, G. L. and H. L. Vieira (2009). "Therapeutic applications of the gaseous mediators carbon monoxide and hydrogen sulfide." Expert Opin Ther Pat 19(5): 663-82.

Bilban, M., A. Haschemi, et al. (2008). "Heme oxygenase and carbon monoxide initiate homeostatic signaling." J Mol Med 86(3): 267-79.

Chen, Z. X. and S. Pervaiz (2007). "Bcl-2 induces pro-oxidant state by engaging mitochondrial respiration in tumor cells." Cell Death Differ 14(9): 1617-27.

Chen, Z. X. and S. Pervaiz (2010). "Involvement of cytochrome c oxidase subunits Va and Vb in the regulation of cancer cell metabolism by Bcl-2." Cell Death Differ 17(3): 408-20.

Chin, B. Y., G. Jiang, et al. (2007). "Hypoxia-inducible factor 1alpha stabilization by carbon monoxide results in cytoprotective preconditioning." Proc Natl Acad Sci U S A 104(12): 5109-14.


D'Amico, G., F. Lam, et al. (2006). "Inhibition of cellular respiration by endogenously produced carbon monoxide." J Cell Sci 119(Pt 11): 2291-8.

Di Noia, M. A., S. Van Driesche, et al. (2006). "Heme oxygenase-1 enhances renal mitochondrial transport carriers and cytochrome C oxidase activity in experimental diabetes." J Biol Chem 281(23): 15687-93.


109

Ch

apte

r III

Dirnagl, U., K. Becker, et al. (2009). "Preconditioning and tolerance against cerebral ischaemia: from experimental strategies to clinical use." Lancet Neurol 8(4): 398-412.

Fukuda, R., H. Zhang, et al. (2007). "HIF-1 regulates cytochrome oxidase subunits to optimize efficiency of respiration in hypoxic cells." Cell 129(1): 111-22.

Kristian, T., J. Gertsch, et al. (2000). "Characteristics of the calcium-triggered mitochondrial permeability transition in nonsynaptic brain mitochondria: effect of cyclosporin A and ubiquinone O." J Neurochem 74(5): 1999-2009.

Lo Iacono, L., J. Boczkowski, et al. (2011). "A carbon monoxide-releasing molecule (CORM-3) uncouples mitochondrial respiration and modulates the production of reactive oxygen species." Free Radic Biol Med 50(11): 1556-64.

Maranga, L. and C. F. Goochee (2006). "Metabolism of PER.C6 cells cultivated under fed-batch conditions at low glucose and glutamine levels." Biotechnol Bioeng 94(1): 139-50.

McCarthy, K. D. and J. de Vellis (1980). "Preparation of separate astroglial and oligodendroglial cell cultures from rat cerebral tissue." J Cell Biol 85(3): 890-902.


Panahian, N., M. Yoshiura, et al. (1999). "Overexpression of heme oxygenase-1 is neuroprotective in a model of permanent middle cerebral artery occlusion in transgenic mice." J Neurochem 72(3): 1187-203.

Queiroga, C. S., A. S. Almeida, et al. (2011). "Carbon monoxide prevents hepatic mitochondrial membrane permeabilization." BMC Cell Biol 12: 10.

Queiroga, C. S., A. S. Almeida, et al. (2010). "Glutathionylation of adenine nucleotide translocase induced by carbon monoxide prevents mitochondrial membrane permeabilization and apoptosis." J Biol Chem 285(22): 17077-88.


Sa Santos, S., L. L. Fonseca, et al. (2005). "Culturing primary brain astrocytes under a fully controlled environment in a novel bioreactor." J Neurosci Res 79(1-2): 26-32.

Sims, N. R. (1990). "Rapid isolation of metabolically active mitochondria from rat brain and subregions using Percoll density gradient centrifugation." J Neurochem 55(2): 698-707.

Soares, M. P. and F. H. Bach (2009). "Heme oxygenase-1: from biology to therapeutic potential." Trends Mol Med 15(2): 50-8.

Chapter III

110

Stein, A. B., Y. Guo, et al. (2005). "Administration of a CO-releasing molecule induces late preconditioning against myocardial infarction." Journal of Molecular and Cellular Cardiology 38(1): 127-134.

Suliman, H. B., M. S. Carraway, et al. (2007). "The CO/HO system reverses inhibition of mitochondrial biogenesis and prevents murine doxorubicin cardiomyopathy." J Clin Invest 117(12): 3730-41.

Suliman, H. B., M. S. Carraway, et al. (2007). "A new activating role for CO in cardiac mitochondrial biogenesis." J Cell Sci 120(Pt 2): 299-308.

Taille, C., J. El-Benna, et al. (2005). "Mitochondrial respiratory chain and NAD(P)H oxidase are targets for the antiproliferative effect of carbon monoxide in human airway smooth muscle." J Biol Chem 280(27): 25350-60.

Tsui, T. Y., A. Obed, et al. (2007). "Carbon monoxide inhalation rescues mice from fulminant hepatitis through improving hepatic energy metabolism." Shock 27(2): 165-71.

Tsui, T. Y., Y. T. Siu, et al. (2005). "Heme oxygenase-1-derived carbon monoxide stimulates adenosine triphosphate generation in human hepatocyte." Biochem Biophys Res Commun 336(3): 898-902.

Vannucci, R. C., J. Towfighi, et al. (1998). "Hypoxic preconditioning and hypoxic-ischemic brain damage in the immature rat: pathologic and metabolic correlates." J Neurochem 71(3): 1215-20.

Vannucci, S. and H. Hagberg (2004). "Hypoxia-ischemia in the immature brain." J Exp Biol.: 3149-3154.




Zhang, X., P. Shan, et al. (2003). "Carbon monoxide modulates Fas/Fas ligand, caspases, and Bcl-2 family proteins via the p38alpha mitogen-activated protein kinase pathway during ischemia-reperfusion lung injury." J Biol Chem 278(24): 22061-70.



111

Ch

apte

r III


Chapter III

112

IV NON-CELL AUTONOMOUS ROLE

OF CARBON MONOXIDE: Purinergic signalling

This chapter is based on data to be published as:

Non-cell autonomous effect of CO-treated astrocytes on neuronal survival:

purinergic signalling

Cláudia S.F. Queiroga, Raquel M.A. Alves, Paula M. Alves and Helena L.A. Vieira,

unpublished data

“Glia already knows how to save neurons, whereas neuroscientists have no clue”

Allaman, 2011

CARBON MONOXIDE NON-CELL AUTONOMOUS ROLE

115

Ch

apte

r IV

ABSTRACT

Low doses of carbon monoxide (CO) play a beneficial role in several cerebral

models, including in vivo model of perinatal cerebral ischemia, in vitro primary culture

of neurons and astrocytes. CO generates small amounts of reactive oxygen species

(ROS), which act as signalling intermediaries and induce a preconditioning state.

Herein, a model of primary co-culture of neurons and astrocytes (2D) is used to

explore the role of CO in cell-to-cell communication. Neuronal survival was assessed

after being co-cultured with astrocytes pre-treated or not with CO. Neuronal cell

death was triggered by tert-butylhydroperoxide (t-BHP), an oxidant molecule, for

mimicking oxidative stress, which occurs in response to ischemia. CO-pre-treated

astrocytes reduced neuronal cell death in the co-culture systems. In this system

there is no physical contact between neurons and astrocytes, thus CO seems to

induce the release of some neuroprotective factor(s) from astrocytes. The classical

energetic molecule ATP and its degradation product, adenosine, are the candidate

neuroprotective molecules. ATP content in the co-culture media after CO treatment

was assessed by luciferase assay, showing alterations on their levels. In

monoculture of neurons, the addition of adenosine or ATP (αβmeATP resistant to

degradation) into the media increased neuronal survival. Furthermore, several

chemical inhibitors of purinergic receptors (both P1 and P2), namely SCH58261

(SCH), Suramin or 8-cyclopentyl-1,3-dipropylxanthine (DPCPX) partially reverted

CO-induced neuroprotection via astrocytes in co-cultures. Moreover, the non-cell

autonomous effect of CO-treated astrocytes on neuronal survival decreased after

astrocytic treatment with a blocker of connexin 43 or of equilibrative adenosine

transporters, for preventing ATP or adenosine release from astrocytes, respectively.

Likewise, prevention of ectonucleotidase activity limited CO-induced neuroprotection

via astrocytes. Furthermore, the possible involvement of TrK B receptors as a

neuronal downstream event was verified. Overall, one can conclude that CO

neuroprotective role is not limited to a cell autonomous effect but this gasotransmitter

also modulates purinergic signalling for increasing astrocytic protection against

neuronal cell death.

Chapter IV

116

CONTENTS

1. Introduction ......................................................................................................... 117

2. Material and methods ......................................................................................... 119

2.1. Materials ..................................................................................................... 119

2.2. Primary cultures of brain cells ..................................................................... 120

2.3. Preparation of CO solutions ........................................................................ 121

2.4. Cell death induction/prevention and detection ............................................ 121

2.5. ATP quantification ....................................................................................... 122

2.6. Statistical analysis of data .......................................................................... 123

3. Results ............................................................................................................... 123

3.1. Non-cell autonomous neuroprotection of carbon monoxide via astrocytes . 123

3.2. Carbon monoxide influences ATP extracellular content ............................. 125

3.3. Adenosine and αβmeATP protect neuronal mono-cultures against oxidative

stress ................................................................................................................. 126

3.4. Effect of neuronal purinergic receptors on CO protection ........................... 127

3.5. Effect of adenosine, ATP release and ATP metabolization on CO protection

........................................................................................................................... 128

3.6. TrK B receptors are involved in the CO protection ..................................... 130

4. Discussion .......................................................................................................... 130


6. References ......................................................................................................... 133


117

Ch

apte

r IV

1. INTRODUCTION

Astrocytes have an active role in brain homeostasis and the intercellular

communication between neurons and astrocytes is tightly regulated to assure the

correct maintenance of this important organ. Any dysfunction can affect several

cellular populations, as well as the flow of survival messages (Allaman et al. 2011).

Preconditioning (PC) is an endogenous therapeutic strategy already in clinical

trials (Dirnagl et al. 2009). It can be defined as an insult below damage threshold that

induces a tolerance state in the organism, antagonizing the deleterious mechanisms

early in time (Kirino 2002; Alkan et al. 2008). PC can be of early or late response

lasting for minutes, hours or days, respectively. The cell has to be subjected to an

initial stimulus. The cell senses the stimulus, transduces and effects it, shifting the

overall cellular effect from death to survival. The complexity of PC pathways is

growing as well as the importance of the knowledge of this neuroprotective response

for the design of new therapies.

Carbon monoxide (CO) is an endogeneous product, resultant from heme

degradation by heme-oxygenase (HO) activity. There are two main isoforms of HO

described: HO-1 (inducible) and HO-2 (constitutive). Oxidative stress activates HO-2

and increases HO-1 expression (Ryter et al. 2006). HO is classified as an antioxidant

enzyme, and its important role in cell survival has been studied in many systems

such as brain (Dore 2002), heart (Piantadosi et al. 2008), intestine (Nakao et al.

2008), liver (Babu et al. 2007) and lung (Morse et al. 2009). Besides CO, heme

degradation produces biliverdin and free iron. As biliverdin is a strong antioxidant

molecule, the cerebral cytoprotection conferred by HO has been claimed to be due to

biliverdin and, more recently, to CO. Indeed, a number of beneficial properties of CO

are described in the literature, namely anti-inflammation, anti-apoptosis and anti-

proliferation (Motterlini et al. 2010). In the central nervous system (CNS) low

amounts of CO prevents inflammation (Chora et al. 2007), vasoconstriction

(Zimmermann et al. 2007) and cerebral damage after ischemia (Zeynalov et al. 2009;

Queiroga et al. 2012). Moreover, in primary cultures of neurons, CO prevented

Chapter IV

118

apoptosis in a preconditioning manner (Vieira et al. 2008). Likewise in astrocytes, CO

limited apoptosis by preventing mitochondrial membrane permeabilization (MMP)

and the subsequent release of pro-apoptotic factors into the cytosol (Queiroga et al.

2010). Also, in astrocytes, CO increases ATP intracellular levels, improving cellular

metabolism. This improvement is due to strengthening mitochondrial oxidative

phosphorylation, inducing mitochondrial biogenesis and increasing cytochrome c

oxidase (COX) activity via COX-Bcl-2 interaction (Almeida et al. 2012).

ATP is a classical energy molecule but, recently, its role as signalling molecule

has gained importance. This molecule is crucial for the astrocyte-astrocyte,

astrocyte-microglia and astrocyte-neuron communication (Shinozaki et al. 2005;

Abbracchio et al. 2006; Fields et al. 2006; Sebastiao et al. 2009) and is released by

neurons and glia in response to neurotransmitter stimulation, hypoxia, inflammation

or mechanical stress (Fields and Burnstock 2006). Likewise ATP is associated to

Ca2+ waves in astrocytes and is involved in the modulation of several intracellular

pathways since it regulates two important second messengers, cAMP and

cytoplasmic Ca2+. Extracellular ATP acts as a chemoattractant (Fields and Burnstock

2006), can induce the expression of genes important for cell survival (McKee et al.

2006), can protect astrocytes from oxidative stress (Fields and Burnstock 2006;

Schock et al. 2007) and prevents neuronal excitotoxicity (Schock et al. 2007). In

addition, the ischemic preconditioning protective mechanism involves the increasing

availability of energy substrates (Kavianipour et al. 2003).

Adenosine, which is an ATP degradation product, is also an important signalling

and neuroprotective factor in the brain. There are two sources of extracellular

adenosine: ATP metabolization by ectonucleotidases and the release from the

cytosol through equilibrative nucleoside transporters (ENTs) (Fields and Burnstock

2006; Sebastiao and Ribeiro 2009). Mild hypoxia increases adenosine release

(McKee et al. 2006) in a defensive way, since adenosine is a well described

neuroprotector (Sebastiao and Ribeiro 2009).

There are two main classes of purinergic receptors: P1, which respond to

adenosine and are associated with G-proteins and P2, which are activated by ATP.


119

Ch

apte

r IV

The P1 family has 4 different receptors: A1, A2A, A2B and A3. A1 and A2A receptors are

considered as high-affinity receptors to adenosine, whereas A2B and A3 are receptors

of low-affinity (Fields and Burnstock 2006). The P2 receptors can be ion channels

(P2X or ionotropic) or G-protein coupled (P2Y or metabotropic). ATP has a dual role,

by acting directly on P2 receptors or it can be metabolized to adenosine acting on P1

receptors. These two purinergic molecules often have antagonistic actions as a way

to refine the regulation mechanism (Fields and Burnstock 2006).

Still, there is controversy regarding the receptor’s action on the cellular outcome.

A2A receptor activation is described as a protective process in mice models of

Parkinson Disease (Yu et al. 2008) and in HIR in kidneys (Day et al. 2003); while, in

cerebral ischemia (Pedata et al. 2005; Melani et al. 2009), rat cerebellar granule

neurons (Fatokun et al. 2007; Fatokun et al. 2008) and hippocampus of male rats

(Stone et al. 2007) its activation is noxious.

Based on the premise that: (i) ATP acts as intercellular signalling molecule

(Schock et al. 2007), (ii) adenosine has several beneficial effects (Sebastiao and

Ribeiro 2009), (iii) PC mechanisms are related to purinergic receptors (Sebastiao

and Ribeiro 2009), (iv) intracellular ATP levels increase in response to CO (Almeida

et al. 2012), the purinergic role on the non-cell autonomous effect of CO was

pursued.


2.1. Materials

All the chemicals were of analytical grade and were obtained from Sigma

(Germany) unless stated otherwise. Plastic tissue culture dishes were from Nunc

(Denmark); cell culture inserts were from BD Falcon (USA), fetal bovine serum

(FBS), glutamine, penicillin-streptomicin solution and Dulbecco’s Minimum Essential

Medium were obtained from Gibco (UK).

Chapter IV

120

2.2. Primary cultures of brain cells

Astroglial cells: primary cultures of astrocytes were prepared from 2-day-old rat

brains. Cerebral hemispheres were carefully freed of the meninges, washed in ice-

cold PBS and mechanically disrupted. Single-cell suspensions were plated in T-

flasks (3 hemispheres/175 cm2) in Dulbecco’s Minimum Essential Medium (DMEM)

supplemented with 10 mM of glucose, 1% (v/v) penicillin-streptomycin solution and

10% (v/v) fetal bovine serum heat inactivated. Cells were maintained in a humidified

atmosphere of 7% CO2 at 37ºC. After 8 days the phase dark cells growing on the

astrocytic cell layer were separated by vigorous shaking and removed as described

(McCarthy et al. 1980). The remaining astrocytes were detached by mild

trypsinization using trypsin/EDTA (0.25% wt/vol) and subcultured in T-flasks for

another 3 weeks. Growth medium was changed twice a week. Wistar rats were

purchased from Faculdade de Ciências Médicas (Lisboa, Portugal).

Neuronal cells: cerebellar granule cells were isolated from cerebellum of 7-day-

old mice. The brain tissue was trypsinized followed by trituration in a DNase solution

containing a trypsin inhibitor from soybeans. Cells were suspended (1.25 x 106

cells/mL) and cultured in Basal Medium Eagle’s (BME) containing 12 mM of glucose,

7.3 µM p-aminobenzoic acid, 4 µg/L insulin, 2 mM glutamine, 1% (v/v) penicillin-

streptomycin solution and 10% (v/v) fetal bovine serum heat inactivated. Cells were

maintained in a humidified atmosphere of 7% CO2 at 37ºC. Cytosine arabinoside (20

µM) was added after 24-48h to prevent glial cell proliferation. Neurons are mature

between 7 to 11 days in vitro. Wistar rats were purchased from Faculdade de

Ciências Médicas (Lisboa, Portugal).

Co-cultures of neurons and astrocytes: the co-cultures were initiated by

transferring cell culture inserts (0.4 µm) containing astroglial cells into a well with

neuronal primary cultures. Small molecules are allowed to pass through the

membrane but the astrocyte-to-neuron contact is prevented.


121

Ch

apte

r IV



PBS was saturated by bubbling 100% of CO gas for 30 min to produce 10-3 M stock

solution. The concentration of CO in solution was determined spectrophotometrically

by measuring the conversion of deoxymyoglobin to carbon monoxymyoglobin, as

described previously (Motterlini et al. 2002). 100% CO was purchased as

compressed gas (Linde, Germany).

2.4. Cell death induction/prevention and detection

Neuronal cells: 5 μM of adenosine or 10 μM of α,β-methyleneadenosine 5’-

triphosphate lithium salt (αβmeATP) were added to neuronal mono-cultures 10

minutes or 15 minutes, respectively, before tert-butylhydroperoxide (t-BHP) cell

death induction, at 10 and 20 μM. Apoptosis-related parameters were analysed 18h

after.

Co-cultures: Astrocytes were pre-treated with 50 µM of CO 3 hours prior the co-

cultures establishment, when neuronal apoptosis was induced with t-BHP at 20 to 80

µM for 18h. Then apoptosis-associated parameters were analysed.

Cell death-associated hallmarkers: In both in vitro models cell death-associated

parameters were analysed by fluorescence microscopic using Hoechst 33342 (Hoe,

2 mM) for chromatin condensation assessment and propidium iodide (PI, 1 μg/ml,

Invitrogen, UK) to determine cell viability, based on the plasma membrane integrity.

The results are expressed in percentage relative to the control (100%). Several

compounds were used to modulate CO effect and are listed in Table 4.1. The cells

were observed on a Leica DMRB microscope (Leica, Wetzlar, Germany) using a filter

cube giving an UV excitation range with a bandpass of 340–380 nm of wavelength.

For each coverslip, 8–10 fields containing around 200 cells were counted (a total of

at least 1500 counted cells).

Chapter IV

122

Table 4.1. List of compounds used to modulate CO effect. Time-line of events representated in Fig.

4.1.

Compound Action Final

concentration

Target cell

population

Time of addition

(before co-cultures

establishment)

Suramin P2X antagonist 30 μM Neurons 15 min

SCH58261 (SCH) A2A antagonist 1 μM Neurons 15 min

8-cyclopentyl-1,3-

dipropylxanthine

(DPCPX)

A1 antagonist 25 μM Neurons 20 min

18α-glycyrrhetinic acid

(AGA)

Connexin 43

inhibitor 15 μM Astrocytes 5 min

S-(ρ-nitrobenzyl)-6-

thioinosine (NBTI)

Adenosine

Equilibrative

Nucleotide

Transporter inhibitor

5 μM Neurons 30 min

ARL67156 (ARL) Ectonucleotidase

inhibitor 50 μM Neurons 30 min

K252a TrK B receptor

antagonist 200 nM Neurons 30 min

2.5. ATP quantification

Adenosine triphosphate quantity was assessed with a PerkinElmer kit. It is a

luminescent assay, based on a luciferase reaction. ATP reacts with luciferin, emitting

luminescent light that can be detected and it is proportional to the ATP content

present in the co-culture supernatant. The results are expressed in percentage

relative to the control (100%).


123

Ch

apte

r IV

2.6. Statistical analysis of data

All experiments were carried out at least in triplicate; values are mean ± SD, n≥3.

Error bars, corresponding to standard deviation. Statistical comparisons were

performed using ANOVA: single factor with replication, with p<0.05, n≥3. p<0.05

means that samples are significantly different at a confidence level of 95%.

The experimental time-line is representated in Figure 4.1.

Figure 4.1. Representation of experimental time-line. The chemical compounds were added at the

respective timing and concentration, whenever was the case and not in the same experiment.

3. RESULTS

3.1. Non-cell autonomous neuroprotection of carbon monoxide via astrocytes

In order to better mimic the in vivo environment, the used in vitro model is based

on a co-culture system with primary cultures of neurons and astrocytes, which are

together in the same environment, sharing metabolites but without any physical

Chapter IV

124

interaction. 50 μM of CO was added to primary cultures of astrocytes. After 3h the

co-culture was established and neurons were challenged to death by oxidative

stress, (tert-butylhydroperoxide addition). After 18h, neuronal survival levels were

higher in the case of CO-treated astrocytes, in particular at highest concentrations of

t-BHP (Figure 4.2. – A and B). Thus, CO improves astrocytic neuroprotective

function in order to prevent neuronal apoptosis in a higher extent.

One can speculate whether CO stimulates astrocytes for releasing pro-survival

molecules or for uptaking toxic factors, improving neuronal environment and,

therefore, improving neuronal survival. In order to clarify this question, neurons were

deadly-challenged in the presence of astrocytic conditioned media (50% of the total

neuronal media) with or without CO pre-treatment. The physical presence of

astroglial cells is important for neuronal population (Figure 4.2. – C and D). Also,

astrocytes are not excelling neuronal environment per se, since neuronal survival

was decreased in the presence of conditioned media. This is in accordance with the

fact that astrocytes are more metabolically active than neurons, consuming nutrients

and excreting toxic metabolic products in a higher rate. In the case of CO-treated

astrocytes, the presence of this gaseoustransmitter re-established the percentage of

cell survival (Figure 4.2. – C and D). Taken together, CO has an important and

modulator effect on astrocytes, which can be beneficial for neurons, improving the

release of neuroprotective factors.


125

Ch

apte

r IV

Figure 4.2. Effect of astroglial pre-treatment with carbon monoxide in neuronal apoptosis.

Primary cultures of astrocytes were pre-treated during 3h with 50 μM of CO, followed by co-culture

establishment and 18h of t-BHP (20 to 60 μM) treatment. Neuronal apoptotic hallmarks were analysed

by fluorescent microscopy (A), (C) chromatin condensation, measured by Hoechst 33342 and (B), (D)

loss of viability, measured by propidium iodide. In (A) and (B) it was shown that CO pre-treated

astrocytes increase in a higher extent neuronal survival. According to results shown in (C) and (D), CO

is exerting its effect by stimulating astrocytes to release a pro-survival factor. Quantification was based

on counting the viable nucleus, nucleus presenting chromatin condensation and unviable cells

(presenting red stained nucleus), and for each cover slip, at least 600 cells were counted. All values are

mean±SD, n=5, *p<0.05, compared with control and **p<0.05, compared with co-culture.

3.2. Carbon monoxide influences ATP extracellular content

Several factors have been described as implicated in astrocytes-neurons

communication (Theodosis et al. 2008; Allaman et al. 2011). Previously, we have

demonstrated that CO reinforces oxidative phoshphorylation and ATP intracellular

(C) (D)

(A) (B)

Chapter IV

126

content in primary cultures of astrocytes (Almeida et al. 2012). In addition, as (i)

extracellular ATP is a signalling molecule, (ii) adenosine is a neuroprotector molecule

and (iii) purinergic receptors have been described as implicated in protective

mechanisms, these are strong candidate molecules for being involved in the cross-

talk between astrocytes and neurons triggered by CO and crucial for neuronal

survival. In accordance, carbon monoxide pre-treatment influences ATP extracellular

content in the co-cultures (Figure 4.3.). At 1h, the presence of CO decreased ATP

amount in the co-culture supernatant, whereas at 4h, the ATP amount in the

extracellular environment is higher for CO-treated astrocytes. This can be easily

explained if CO is stimulating ATP metabolization, in particular in the first period

when co-culture is established. At 4h, the equilibrium is already re-established and

ATP can be accumulating.

Figure 4.3. CO influences ATP and adenosine content in co-culture supernatant. Astrocytes were

treated with 50 μM of CO for 3h, followed by co-culture establishment. Co-culture supernatant was

collected at 1, 4 and 24 h after co-culture establishment and ATP quantified as described in Material

and methods section. All values are mean±SD, n=3 with *p<0.05 compared with control.

3.3. Adenosine and αβmeATP protect neuronal mono-cultures against

oxidative stress

In accordance with previous reports, the addition of 5 μM of adenosine and 10

μM of αβmeATP (ATP analogous, resistant to degradation) increases neuronal

survival in monocultures of neurons (Figure 4.4.).


127

Ch

apte

r IV

Figure 4.4. Adenosine and αβmeATP protect neurons against cell death. Neuronal cultures were

treated with 5 μM of adenosine and 10 μMαβmeATP for 10 and 15 minutes, respectively. Then cell

death was induced by t-BHP. Neuronal apoptotic hallmarks were analysed by fluorescent microscopy,

such as chromatin condensation (A) and cell viability (B). Quantification was based on counting the

viable nucleus, nucleus presenting chromatin condensation and unviable cells (presenting red stained

nucleus), and for each cover slip, at least 600 cells were counted. All values are mean±SD, n=5, with

*p<0.05, compared with control.

One may consider that adenosine experiment is more physiological since

αβmeATP does not exist in the brain.

3.4. Effect of neuronal purinergic receptors on CO protection

There are two main classes of purinergic receptors: P1, which respond to

adenosine and are associated with G-proteins and P2, which are activated by ATP.

From the P1 family, the high-affinity receptors (A2A and A1) were the elected ones.

While for the P2 family, the experimental evidences are contradictory; therefore we

did not choose a particular receptor but a subfamily of receptors, P2X.

It was pursued the effect of A2A and P2X antagonists in CO protective

mechanism (Figure 4.5.). Both in the presence of SCH58261 (SCH, A2A antagonist)

and suramin (P2X antagonist), the protection conferred by the CO pre-treatment was

reverted. This result confirms that these receptors are implicated in the neuronal

protection that CO pre-treatment in astrocytes confers.

(A) (B)

Chapter IV

128

25 μM of 8-cyclopentyl-1,3-dipropylxanthine (DPCPX), an A1 antagonist, was

also tested (Figure 4.5.). However, DPCPX had no effect on CO-induced protection,

excluding the involvement of A1 receptor in this pathway.

Figure 4.5. Effect of SCH58261 (SCH), suramin and8-cyclopentyl-1,3-dipropylxanthine (DPCPX)in

the CO mechanism. Primary cultures of astrocytes were pre-treated during 3h with 50 μM of CO. 1 μM

of SCH, 30 μM of suramin or 25 μM of DPCPX were added to primary cultures of neurons 15 , 15 or 20

min, respectively before co-culture establishment, followed by 18h of t-BHP (20 to 60 μM) treatment.

Neuronal apoptotic hallmarks were analysed by fluorescent microscopy, such as chromatin

condensation (A) and cell viability (B). Quantification was based on counting the viable nucleus, nucleus

presenting chromatin condensation and unviable cells (presenting red stained nucleus), and for each

cover slip, at least 600 cells were counted. All values are mean±SD, n=5, with *p<0.05, compared with

control and **p<0.05, compared with co-culture, #p<0.05, compared with co-culture with astrocytes pre-

treated with CO.

3.5. Effect of adenosine, ATP release and ATP metabolization on CO protection

CO is inducing a PC state in astrocytes that signal neurons towards an increase

on survival. ATP, adenosine, P2X and A2A receptors are involved in this beneficial

communication. Nevertheless, it is not clear whether ATP, adenosine or both are

released by astrocytes. ATP can be released through connexin 43 and adenosine is

released by equilibrative nucleotide transporter (ENT). Thus, whenever connexin 43

and ENT were inhibited by 15 μM of 18α-glycyrrhetinic acid (AGA) and by 5 μM of S-

(ρ-nitrobenzyl)-6-thioinosine (NBTI), respectively (Figure 4.6.), ATP and adenosine

release inhibition reverted CO protection. Furthermore, NBTI caused a great extent

(A) (B)


129

Ch

apte

r IV

of reversion, presenting a much lower survival percentage than the co-cultures ones.

Thus these data indicate that ENT inhibition might affect other neuronal events.

Furthermore, adenosine can also be generated by ATP metabolization through

ectonucleotidase action. Therefore, ectonucleotidase inhibition with 50 μM, not only

reverted CO protection, but also achieved values similar to the co-culture conditions

(Figure 4.6.). Thus, ATP metabolization has a great importance for CO-astroglial

protection in neurons, which is in accordance with previous results (Figure 4.3.)

Altogether, one can speculate that the main CO-induced pathway is by

stimulation of ATP release from astrocytes that is then metabolized to adenosine,

which will activate its neuronal receptors A2A, in order to initiate downstream events

towards cell protection.

Figure 4.6. Effect of 18α-glycyrrhetinic acid (AGA), S-(ρ-nitrobenzyl)-6-thioinosine (NBTI) and

ARL67156 (ARL) in the CO mechanism. Primary cultures of astrocytes were pre-treated during 3h

with 50 μM of CO. 15 μM of AGA was added to primary cultures of astrocytes, 5 min before CO

treatment. 5 μM of NBTI or 50 μM of ARL were added to primary cultures of neurons 30 min before co-

culture establishment. In both experiments, it was followed by 18h of t-BHP (20 and 40 μM) treatment.

Neuronal apoptotic hallmarks were analysed by fluorescent microscopy, such as chromatin

condensation (A) and cell viability (B). Quantification was based on counting the viable nucleus,

nucleus presenting chromatin condensation and unviable cells (presenting red stained nucleus), and for

each cover slip, at least 600 cells were counted. All values are mean±SD, n=5, with *p<0.05, compared

with control and **p<0.05, compared with co-culture, #p<0.05, compared with co-culture with astrocytes

pre-treated with CO.

(A) (B)

Chapter IV

130

3.6. TrK B receptors are involved in the CO protection

Brain-derived neurotrophic factor (BDNF) has been implicated in activity-

dependent synaptic plasticity (Caldeira et al. 2007) and has been associated to

protective outcomes through TrK B receptors activation (Gomes et al. 2012). In fact,

the inhibition of TrK B receptors (Figure 4.7) reverts CO protection, confirming its

involvement as a neuronal downstream event in this protective pathway.

Figure 4.7. Effect of K252a in the CO mechanism. Primary cultures of astrocytes were pre-treated

during 3h with 50 μM of CO. 200 nM of K252awas added to primary cultures of neurons 30 min before

co-culture establishment, followed by 18h of t-BHP (20 to 60 μM) treatment. Neuronal apoptotic

hallmarks were analysed by fluorescent microscopy, such as chromatin condensation (A) and cell

viability (B). Quantification was based on counting the viable nucleus, nucleus presenting chromatin

condensation and unviable cells (presenting red stained nucleus), and for each cover slip, at least 600

cells were counted. All values are mean±SD, n=5, with *p<0.05, compared with control and **p<0.05,

compared with co-culture, #p<0.05, compared with co-culture with astrocytes pre-treated with CO.

4. DISCUSSION

The communication between neurons and astrocytes has been extensively

studied (Shinozaki et al. 2005). Astrocytes are well-known for the important function

as nutritional, structural and signalling support for neurons. They are responsible for

providing nutrients like glutamine (glutamine-glutamate cycle) and lactate (astrocyte-

(A) (B)


131

Ch

apte

r IV

neuron lactate shuttle), but also for scavenging the waste and toxic compounds

resultant from neuronal metabolism (Belanger et al. 2011). In many pathological

contexts astrocytes are able to rescue neurons from death (Bezzi et al. 2001;

Allaman et al. 2011). However, to our knowledge, for the first time it was shown that

carbon monoxide has a non-cell autonomous effect, acting on astrocytes, modulating

their metabolism so that a released astrocytic factor(s) or action can prevent

neuronal death. Moreover, the main molecular events were disclosed: (i) ATP is

released from astrocytes (Figure 4.3.), (ii) ATP is metabolized into adenosine

(Figure 4.6.), (iii) A2A and TrK B receptors are activated (Figures 4.5. and 4.7.).

Metabolized adenosine is the principal molecule acting in neurons, following CO-

induced non-cell autonomous effect. This is in agreement with reports of up-

regulation of ectonucleotidases after brain ischemia, in order to provide cerebral

protection (Shinozaki et al. 2005). A2A receptor activation is also in accordance with

the literature as Boeck et al (Boeck et al. 2005) described that adenosine resultant

from ectonucleotidase activity has preference for A2A receptors whilst adenosine

released prefers A1 receptors. In order to further confirm A2A role, genetic

approaches must be done, such as silencing this receptor and verifying neuronal

protection induced by astrocytic CO treatment.

Because A2A receptors can provoke translocation and activation of TrK B

receptors in a BDNF-independent manner (Assaife-Lopes et al. 2010); more

experiments need to be performed to demonstrate if TrK B receptors activation in the

present model is dependent or independent of BDNF actions. One possible approach

can be the assessment of the pool of pro-BDNF/BDNF in neurons, which are

cultivated in co-cultures.

One may also consider that the described pathway is not the unique that occurs.

P2X receptor was not excluded from being involved in the CO-induced paracrine

protection (Figure 4.3.). In addition, NBTI indicates a more extensive action than

expected (Figure 4.5.), however, one cannot conclude that equilibrative adenosine

transporters are not releasing more adenosine in response to CO-preconditioning.

Possibly, CO triggers all the referred events: ATP and adenosine release, ATP

Chapter IV

132

metabolization or not and A2A and P2X activation. Moreover, others ways of ATP and

adenosine release can be explored. Nevertheless, the combination of our results are

clear in indicating a major set of contributors for the positive outcome.

Adenosine binding to A2A receptor activates HO-1, which, via CO generation,

alters A2A receptor expression. Also, a positive feedback loop among adenosine, HO-

1, CO and A2A receptor has been describe in the chronological resolution in the

inflammatory response in macrophages (Haschemi et al. 2007). Based on these

authors, one may hypothesize that the extense amount of evidences on adenosine

neuroprotective role can also be due to the HO-1/CO contribution. In the present

study, it can be proposed a very interesting and novel effect. CO induces

preconditioning in astrocytes to exert a non-cell autonomous effect on neurons,

through A2A receptor activation, which will augment CO content in the target neurons.

Therefore, it can be hypothesized that neuronal protection can also occur due to CO

action. As the oxidative stress has been already induced when neuronal A2A

receptors are activated, CO is not inducing neuronal preconditioning as already

described by our laboratory (Vieira et al. 2008). Thus, it can be a focus of scientific

interest to study which mechanisms CO triggers.

Others astrocyte-neuron communication interplayers may be considered for

future studies. Ruscher and colleagues describe one paracrine mean of ischemic

tolerance induction. Hypoxia-inducible factor-1 (HIF-1) is a transcriptor factor

activated under hypoxia, with multiple gene targets. In astrocytes, HIF-1 activation

increases erythropoietin (EPO) production, which is released, activating its neuronal

receptor. Subsequently, JAK-2, PI3K are activated, regulating gene transcription and

phosphorylation. Finally, hypoxia-induced apoptosis in neurons is inhibited (Ruscher

et al. 2002). Estrogen also presents a non-cell autonomous role, by stimulating the

astrocytic release of neuronal growth factors (Sortino et al. 2005).

In conclusion, in this work, purinergic signalling and the main players were

disclosed in the paracrine protective communication induced by CO. Furthermore, it

is demonstrated the capability of CO modulating astrocytic non-cell autonomous


133

Ch

apte

r IV

effect, taking advantage of the complex, but efficient cell-to-cell communication,

which opens a new field of CO studies and possible applications.

5. ACKNOWLEDGMENTS

This work was supported by the Portuguese Fundaçãopara a Ciência e

Tecnologia project PTDC/SAUNEU/098747/2008, and HLAV’s

SFRH/BPD/27125/2006 and CSFQ’s SFRH/BD/43387/2008 fellowships.

6. REFERENCES

Abbracchio, M. P. and S. Ceruti (2006). "Roles of P2 receptors in glial cells: focus on astrocytes."Purinergic Signal 2(4): 595-604.

Alkan, T., B. Goren, et al. (2008). "Effects of hypoxic preconditioning in antioxidant enzyme activities in hypoxic-ischemic brain damage in immature rats."Turk Neurosurg 18(2): 165-71.

Allaman, I., M. Belanger, et al. (2011). "Astrocyte-neuron metabolic relationships: for better and for worse."Trends Neurosci 34(2): 76-87.

Almeida, S. S., C. S. Queiroga, et al. (2012). "Carbon monoxide modulates apoptosis by reinforcing oxidative metabolism in astrocytes: role of BCL-2."J Biol Chem 287(14): 10761-10770.

Assaife-Lopes, N., V. C. Sousa, et al. (2010). "Activation of adenosine A2A receptors induces TrkB translocation and increases BDNF-mediated phospho-TrkB localization in lipid rafts: implications for neuromodulation."J Neurosci 30(25): 8468-80.

Babu, A. N., S. S. Damle, et al. (2007). "Hemoglobin-based oxygen carrier induces hepatic heme oxygenase 1 expression in Kupffer cells."Surgery 142(2): 289-94.

Belanger, M., I. Allaman, et al. (2011). "Brain energy metabolism: focus on astrocyte-neuron metabolic cooperation."Cell Metab 14(6): 724-38.

Bezzi, P. and A. Volterra (2001). "A neuron-glia signalling network in the active brain."Curr Opin Neurobiol 11(3): 387-94.

Chapter IV

134

Boeck, C. R., E. H. Kroth, et al. (2005). "Adenosine receptors co-operate with NMDA preconditioning to protect cerebellar granule cells against glutamate neurotoxicity."Neuropharmacology 49(1): 17-24.

Caldeira, M. V., C. V. Melo, et al. (2007). "Brain-derived neurotrophic factor regulates the expression and synaptic delivery of alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid receptor subunits in hippocampal neurons."J Biol Chem 282(17): 12619-28.

Carvalho, A. L., M. V. Caldeira, et al. (2008). "Role of the brain-derived neurotrophic factor at glutamatergic synapses."Br J Pharmacol 153 Suppl 1: S310-24.

Chora, A. A., P. Fontoura, et al. (2007). "Heme oxygenase-1 and carbon monoxide suppress autoimmune neuroinflammation."J Clin Invest 117(2): 438-47.

Day, Y. J., L. Huang, et al. (2003). "Renal protection from ischemia mediated by A2A adenosine receptors on bone marrow-derived cells."J Clin Invest 112(6): 883-91.

Dirnagl, U., K. Becker, et al. (2009). "Preconditioning and tolerance against cerebral ischaemia: from experimental strategies to clinical use."Lancet Neurol 8(4): 398-412.

Dore, S. (2002). "Decreased activity of the antioxidant heme oxygenase enzyme: implications in ischemia and in Alzheimer's disease."Free Radic Biol Med 32(12): 1276-82.

Fatokun, A. A., T. W. Stone, et al. (2007). "Cell death in rat cerebellar granule neurons induced by hydrogen peroxide in vitro: mechanisms and protection by adenosine receptor ligands."Brain Res 1132(1): 193-202.

Fatokun, A. A., T. W. Stone, et al. (2008). "Adenosine receptor ligands protect against a combination of apoptotic and necrotic cell death in cerebellar granule neurons."Exp Brain Res 186(1): 151-60.

Fields, R. D. and G. Burnstock (2006). "Purinergic signalling in neuron-glia interactions."Nat Rev Neurosci 7(6): 423-36.

Gomes, J. R., J. T. Costa, et al. (2012). "Excitotoxicity downregulates TrkB.FL signaling and upregulates the neuroprotective truncated TrkB receptors in cultured hippocampal and striatal neurons."J Neurosci 32(13): 4610-22.

Haschemi, A., O. Wagner, et al. (2007). "Cross-regulation of carbon monoxide and the adenosine A2a receptor in macrophages."J Immunol 178(9): 5921-9.


135

Ch

apte

r IV

Inoue, K., S. Koizumi, et al. (2007). "The role of nucleotides in the neuron--glia communication responsible for the brain functions."J Neurochem 102(5): 1447-58.

Kavianipour, M., G. Ronquist, et al. (2003). "Ischaemic preconditioning alters the energy metabolism and protects the ischaemic myocardium in a stepwise fashion."Acta Physiol Scand 178(2): 129-37.

Kirino, T. (2002). "Ischemic tolerance."J Cereb Blood Flow Metab 22(11): 1283-96.

McCarthy, K. D. and J. de Vellis (1980). "Preparation of separate astroglial and oligodendroglial cell cultures from rat cerebral tissue."J Cell Biol 85(3): 890-902.

McKee, S. C., C. S. Thompson, et al. (2006). "Regulation of expression of early growth response transcription factors in rat primary cortical neurons by extracellular ATP."Brain Res 1088(1): 1-11.

Melani, A., S. Cipriani, et al. (2009). "Selective adenosine A2a receptor antagonism reduces JNK activation in oligodendrocytes after cerebral ischaemia."Brain 132(Pt 6): 1480-95.

Morse, D., L. Lin, et al. (2009). "Heme oxygenase-1, a critical arbitrator of cell death pathways in lung injury and disease."Free Radic Biol Med 47(1): 1-12.

Motterlini, R., J. E. Clark, et al. (2002). "Carbon monoxide-releasing molecules: characterization of biochemical and vascular activities."Circ Res 90(2): E17-24.

Motterlini, R. and L. E. Otterbein (2010). "The therapeutic potential of carbon monoxide."Nat Rev Drug Discov 9(9): 728-43.

Nakao, A., D. J. Kaczorowski, et al. (2008). "Application of heme oxygenase-1, carbon monoxide and biliverdin for the prevention of intestinal ischemia/reperfusion injury."J Clin Biochem Nutr 42(2): 78-88.

Pascual, O., K. B. Casper, et al. (2005). "Astrocytic purinergic signaling coordinates synaptic networks."Science 310(5745): 113-6.

Pedata, F., M. Gianfriddo, et al. (2005). "The protective effect of adenosine A2A receptor antagonism in cerebral ischemia."Neurol Res 27(2): 169-74.

Piantadosi, C. A., M. S. Carraway, et al. (2008). "Heme oxygenase-1 regulates cardiac mitochondrial biogenesis via Nrf2-mediated transcriptional control of nuclear respiratory factor-1."Circ Res 103(11): 1232-40.

Chapter IV

136

Queiroga, C. S., A. S. Almeida, et al. (2010). "Glutathionylation of adenine nucleotide translocase induced by carbon monoxide prevents mitochondrial membrane permeabilization and apoptosis."J Biol Chem 285(22): 17077-88.

Queiroga, C. S. F., S. Tomasi, et al. (2012). "Preconditioning triggered by carbon monoxide (CO) provides neuronal protection following perinatal hypoxia-ischemia."PLoS One.

Ryter, S. W., J. Alam, et al. (2006). "Heme oxygenase-1/carbon monoxide: from basic science to therapeutic applications."Physiol Rev 86(2): 583-650.

Schock, S. C., D. Leblanc, et al. (2008). "ATP release by way of connexin 36 hemichannels mediates ischemic tolerance in vitro."Biochem Biophys Res Commun 368(1): 138-44.

Schock, S. C., N. Munyao, et al. (2007). "Cortical spreading depression releases ATP into the extracellular space and purinergic receptor activation contributes to the induction of ischemic tolerance."Brain Res 1168: 129-38.

Sebastiao, A. M. and J. A. Ribeiro (2009). "Triggering neurotrophic factor actions through adenosine A2A receptor activation: implications for neuroprotection."Br J Pharmacol 158(1): 15-22.

Shinozaki, Y., S. Koizumi, et al. (2005). "Cytoprotection against oxidative stress-induced damage of astrocytes by extracellular ATP via P2Y1 receptors."Glia 49(2): 288-300.

Stone, T. W. and W. M. Behan (2007). "Interleukin-1beta but not tumor necrosis factor-alpha potentiates neuronal damage by quinolinic acid: protection by an adenosine A2A receptor antagonist."J Neurosci Res 85(5): 1077-85.

Tanaka, H., H. Yokota, et al. (2004). "Ischemic preconditioning: neuronal survival in the face of caspase-3 activation."J Neurosci 24(11): 2750-9.

Thauerer, B., S. Zur Nedden, et al. (2012). "Purine nucleosides: endogenous neuroprotectants in hypoxic brain."J Neurochem 121(3): 329-42.

Theodosis, D. T., D. A. Poulain, et al. (2008). "Activity-dependent structural and functional plasticity of astrocyte-neuron interactions."Physiol Rev 88(3): 983-1008.

Vieira, H. L. A., C. S. F. Queiroga, et al. (2008). "Preconditioning induced by Carbon Monoxide provides neuronal protection against Apoptosis."J Neurochem.


137

Ch

apte

r IV

Yu, L. L. and Z. Cheng (2008). "Application of electron spin resonance (ESR) spectrometry in nutraceutical and food research."Mol Nutr Food Res 52(1): 62-78.

Zeynalov, E., Z. A. Shah, et al. (2009). "Heme oxygenase 1 is associated with ischemic preconditioning-induced protection against brain ischemia."Neurobiol Dis 35(2): 264-9.

Zimmermann, A., C. W. Leffler, et al. (2007). "Cerebroprotective effects of the CO-releasing molecule CORM-A1 against seizure-induced neonatal vascular injury."Am J Physiol Heart Circ Physiol 293(4): H2501-7.

Chapter IV

138

V CARBON MONOXIDE EFFECT ON PERINATAL HYPOXIA-ISCHEMIA:

apoptosis prevention


Preconditioning triggered by carbon monoxide (CO) provides neuronal protection following perinatal hypoxia-ischemia

Cláudia S.F. Queiroga, Simone Tomasi, Marius Widerøe, Paula M. Alves,

Alessandro Vercelli and Helena L.A. Vieira (2012) PLoS One, 7(8):e42632.

“The dose makes the poison.” Paracelsus 11th century

NEURONAL PROTECTION FOLLOWING PERINATAL HYPOXIA-ISCHEMIA

141

Ch

apte

r V

ABSTRACT

Perinatal hypoxia-ischemia is a major cause of acute mortality in newborns and

cognitive and motor impairments in children. Cerebral hypoxia-ischemia leads to

excitotoxicity and necrotic and apoptotic cell death, in which mitochondria play a

major role. Increased resistance against major damage can be achieved by

preconditioning triggered by subtle insults. CO, a toxic molecule that is also

generated endogenously, may have a role in preconditioning as low doses can

protect against inflammation and apoptosis. In this study, the role of CO-induced

preconditioning on neurons was addressed in vitro and in vivo.

The effect of 1h of CO treatment on neuronal death (plasmatic membrane

permeabilization and chromatin condensation) and bcl-2 expression was studied in

cerebellar granule cells undergoing to glutamate-induced apoptosis. CO’s role was

studied in vivo in the Rice-Vannucci model of neonatal hypoxia-ischemia (common

carotid artery ligature + 75 min at 8% oxygen). Apoptotic cells, assessed by Nissl

staining were counted with a stereological approach and cleaved caspase 3-positive

profiles in the hippocampus were assessed. Apoptotic hallmarks were analyzed in

hippocampal extracts by Western Blot.

CO inhibited excitotoxicity-induced cell death and increased Bcl-2 mRNA in

primary cultures of neurons. In vivo, CO prevented hypoxia-ischemia induced

apoptosis in the hippocampus, limited cytochrome c released from mitochondria and

reduced activation of caspase-3. Still, Bcl-2 protein levels were higher in

hippocampus of CO pre-treated rat pups.

Our results show that CO preconditioning elicits a molecular cascade that limits

neuronal apoptosis. This could represent an innovative therapeutic strategy for high-

risk cerebral hypoxia-ischemia patients, in particular neonates.

Chapter V

142

CONTENTS

1. Introduction ......................................................................................................... 143

2. Material and methods ......................................................................................... 145

In vitro experiments ................................................................................................ 145

2.1. Materials ..................................................................................................... 145

2.2. Cell culture .................................................................................................. 145

2.3. Preparation of CO solutions ........................................................................ 146

2.4. Cell treatments, induction of apoptosis and assessment of apoptosis-

associated parameters ...................................................................................... 146

2.5. Real Time Quantitative PCR ....................................................................... 147

In vivo experiments................................................................................................. 147

2.6. Animals ....................................................................................................... 147

2.7. Experimental plan ....................................................................................... 148

2.8. Carbon monoxide exposure ........................................................................ 149

2.9. Model of neonatal cerebral hypoxia-ischemia ............................................. 150

2.10. Tissue processing ..................................................................................... 150

2.11. Histological and immunohistochemical assessment ................................. 151

2.12. Stereological counts in the hippocampus ................................................. 151

2.13. Immunoblotting ......................................................................................... 153

2.14. Statistical analysis of data ........................................................................ 154

3. Results ............................................................................................................... 154

3.1. Carbon monoxide prevents excitotoxicity-induced cell death and induces Bcl-

2 expression in primary cultures of neurons ...................................................... 154

3.2. Effect of preconditioning on HI-induced apoptosis in vivo .......................... 156

3.3. Effect of carbon monoxide on expression of apoptotic markers ................. 158

4. Discussion .......................................................................................................... 160


6. References ......................................................................................................... 164


143

Ch

apte

r V

1. INTRODUCTION

Perinatal hypoxia-ischemia (HI) remains a major cause of acute mortality in

newborns and of cognitive and motor impairments in children (Vannucci et al. 1999).

Cerebral damage results from oxygen and tissue energy depletion that lead to:

acidosis, inflammation, glutamate excitotoxicity, cell death and generation of reactive

oxygen species (ROS) during reperfusion (Vannucci SJ 2004). Cerebral hypoxia-

ischemia induces distinct types of cell death: within the ischemic core, rapid cell

death occurs mainly as necrosis; while in the penumbra, the region around the

ischemic core, delayed apoptotic cell death takes place hours and days after the

insult, contributing to secondary damage (Taylor et al. 2008). In the developing brain,

apoptosis also plays a homeostatic role, and many pro-apoptotic factors are normally

up-regulated during early stages of maturation (Tsujimoto et al. 2006; Galluzzi et al.

2007). Therefore, apoptosis is closely related to the injury response after hypoxia-

ischemia in the immature brain of newborn infants (Carloni et al. 2008). Mitochondria

play a major role in death of mammalian cells (Kroemer 2003). During the apoptotic

process and upon mitochondrial membrane permeability, several biochemical

molecules confined to the inter-membrane space are released to the cytosol thus

activating proteases and nucleases. For example, cytochrome c released from

mitochondria interacts with apoptotic protease activating factor 1 (Apaf-1) and

caspase-9 to form the apoptosome that activates caspase-3 leading to cell death

(Galluzzi et al. 2009).

Currently, hypothermia is the only treatment used clinically for minimizing

cerebral damage after perinatal hypoxia-ischemia, but it has limited efficiency and its

use has also limitations (Gunn et al. 2008). Other therapies, which either can be

used alone or in combination with hypothermia, are therefore needed.

Preconditioning (PC) induction consists of an insult that does not cause damage, but

triggers a protective state (tolerance) that increase cellular resistance against a

subsequent and more severe challenge (Kirino 2002; Alkan et al. 2008). PC can

induce an early response (minutes or hours) or a late response within days including

Chapter V

144

de novo protein synthesis (Gidday 2006). Furthermore, clinical studies with patients

suffering from transient ischemic attacks (TIA) (Dirnagl et al. 2009) and animal

models (Gidday 2006; Stenzel-Poore et al. 2007; Dirnagl et al. 2008) have suggested

that cerebral tolerance induced by a PC state is an efficient strategy to protect brain

tissue against HI. Thus, preconditioning processes are promising alternatives for

therapy in patients at high risk of suffering HI. Indeed, perinatal HI may eventually be

predicted based on known risk factors associated with previous ischemic episodes

including intrauterine fetal distress and hypoxic-ischemic insults during birth (Heazell

et al. 2008); Bonifacio et al. 2011). Also, PC-based therapies could be useful for

neonates going through major heart surgery with associated risks of global cerebral

ischemia (Kirino 2002; Dirnagl et al. 2009; Bonifacio et al. 2011).

Carbon monoxide (CO) is commonly known to be toxic. This is due to its high

affinity for haem-proteins, which can compromise oxygen delivery to tissues

(carboxy-haemglobin) or can decrease oxidative phosphorylation at the cellular level

by binding to cytochrome c oxidase (Ahlstrom et al. 2009). CO is an endogenous

molecule generated by haem-oxygenase (HO) activity along with the production of

free iron and biliverdin (Motterlini et al. 2010). Low doses of exogenous CO are

cytoprotective against inflammation and apoptosis, in particular following

cardiovascular incidents, organ rejection and autoimmune disease in several models

(Motterlini and Otterbein 2010). Also, in rat retinal ganglion cells, inhalation of 250

ppm of CO protected against ischemia-reperfusion injury (Biermann et al. 2012). In

the central nervous system (CNS), low amounts of CO limit neuroinflammation in a

model of multiple sclerosis (Chora et al. 2007) and induced vasodilation, presenting

cytoprotective effects in the cerebral circulation in a model of epileptic seizures in

newborn piglets (Zimmermann et al. 2007). CO treatment also decreased infarct

volume and brain damage in adult models of transient and permanent focal cerebral

ischemia when the animals were exposed to CO immediately after middle cerebral

artery occlusion (Zeynalov et al. 2009; Wang et al. 2011). Nevertheless, the cellular

mechanisms involved in CO-induced neuroprotection are still not fully understood. In

primary cultures of cerebellar neurons, CO triggers preconditioning and prevents


145

Ch

apte

r V

apoptosis by ROS signaling and modulation of soluble guanylyl cyclase, nitric oxide

synthase and mitochondrial ATP dependent potassium channel (Vieira et al. 2008).

Likewise, in primary cultures of astrocytes, CO inhibits apoptosis by directly targeting

mitochondria and preventing their membrane permeabilization, which is also

dependent on ROS and protein glutathionylation signaling (Queiroga et al. 2010).

Since preconditioning emerges as a promising strategy to limit brain damage

following perinatal ischemia, we have examined the ability of CO to induce

preconditioning and to limit apoptosis in the hippocampus in the present study. Pre-

treatment of rat pups with CO prevented hippocampal cell death via: an increase on

Bcl-2 expression, a decrease on cytochrome c translocation from mitochondria into

cytosol and an inhibition of caspase-3 activation. To our knowledge, this is the first

study to use CO preconditioning to prevent hypoxia-ischemia-induced neuronal

death in the developing brain.


In vitro experiments

2.1. Materials

All chemicals were purchased from Sigma-Aldrich (Munich, Germany) unless

stated otherwise. Plastic tissue culture dishes were from Nunc (Roskilde, Denmark);

fetal bovine serum (FBS), glutamine, penicillin-streptomycin solution and Dulbecco’s

Minimum Essential Medium (DMEM) were obtained from Gibco (Paisley, UK). CO

100 % was purchased as compressed gas (Linde, Germany).

2.2. Cell culture

Primary cerebellar granule cells were prepared as described by Schousboe

(Schousboe et al. 1989) from Wistar rats purchased from Instituto de Higiene e

Medicina Tropical (Lisboa, Portugal). Briefly, cells were isolated from postnatal day

7(P7) rat cerebella, after mild trypsinization followed by trituration in a DNase solution

containing soybean trypsin inhibitor. Cells were suspended (1x106 cells/mL) and

Chapter V

146

cultured in BME basal medium containing 12 mM glucose, 7.3 µM p-Aminobenzoic

acid, 4µg/L insulin, 2mM glutamine, 1% (vol/vol) penicillin-streptomycin solution and

10% (v/v) FBS. Cells were cultured in 24- and 96-well poly-D-lysine coated plates

and maintained in humidified atmosphere of 7% CO2 at 37ºC. To prevent glia

proliferation, cytosine arabinoside (20 µM) was added 48h after seeding. The

experiments were performed on 1-week-old cerebellar granule neuronal culture. All

experiments were performed at least in triplicate.


Fresh stock solutions of CO gas were prepared each day and sealed.

Phosphate-buffered saline (PBS) was saturated by bubbling 100% of CO gas for 30

minutes to produce a 10-3 M stock solution. The concentration of CO in solution was

determined spectrophotometrically by measuring the conversion of deoxymyoglobin

to carbon monoxymyoglobin, as described by Motterlini (Motterlini 2002). CO 100 %

was purchased as compressed gas (Linde, Germany).

2.4. Cell treatments, induction of apoptosis and assessment of apoptosis-

associated parameters

Neuronal cells were cultured on poly-D-lysine-coated coverslips. Neuronal

apoptosis was induced immediately after the end of CO exposure with 10 to 30 µM

of glutamate over a 24h period to mimic excitotoxicity, which is a consequence of

cerebral ischemia (Vieira et al. 2008). For inhibition of cell death, cerebellar granule

cells were treated with 10 µM CO for 1h prior to glutamate addition. Neurons were

stained with Hoechst 33342 (2 µM, Sigma) and Propidium Iodide (PI, 1 µM,

Molecular Probes, USA) followed by quantitative assessment of chromatin

condensation and cell viability, respectively. Cells were observed on a Leica DMRB

microscope using a filter with a bandpass of 340-380 nm (UV).


147

Ch

apte

r V

2.5. Real Time Quantitative PCR

After 6 or 24 h of CO treatment, RNA was extracted from primary cerebellar

neurons (3x106 cells) using the High Pure RNA isolation Kit (Roche, Germany);

cDNA was synthesized from RNA (Transcriptor High Fidelity cDNA Synthesis Kit,

Roche, Germany). For real time quantitative PCR (RT-qPCR), forward and reverse

primer sequences specific for bcl-2 gene consisted of 5’-

GGTGGAGGAACTCTTCAGGG-3’ and 5’-GAGACAGCCAGGAGAAATCA-3’,

respectively. An internal control used expression of cyclophilin A, a constitutive

protein, using forward and reverse sequences: 5’-ATGGCAAATGCTGGACCAAA-3’

and 5’-GCCTTCTTTCACCTTCCCAAA-3’. The RT-qPCRwas performed according to

manufacturer indications (LightCycler® FastStart DNA MasterPLUS SYBR Green I,

Roche Diagnostics, Germany). Amplification used the following protocol:

denaturation at 95ºC for 10 min; amplification for 35 cycles, at 95ºC for 15 s, 60ºC for

6 s, 72ºC for 15 s with a single fluorescent measurement; 95ºC for 15 s, 60ºC for 15

s, 95ºC for 15 s with a continuous fluorescence measurement for the melting curve;

finally the cooling step was at 40ºC. The results were expressed in percentage

relative to the control sample.

In vivo experiments

2.6. Animals

P4-P7 (postnatal day 4 to postnatal day 7) Wistar rat pups (n=74, Janvier,

France) weighing on average 27 ( 1,4) and 35 g ( 1,5) respectively were used.

Great differences in pup’s weight are due to the fast rate of growing at this period of

life. All animal experimental procedures were carried out in strict accordance with the

recommendations in the European Community Council Directive 86/609/EEC

(November 24, 1986), authorization 17/2010-B (June 30, 2010) by Italian Ministry of

Health and University of Turin institutional guidelines on animal welfare (law 116/92

on Care and Protection of Living Animals Undergoing Experimental or Other

Scientific Procedures). The protocol was approved by Turin University Bioethical

Chapter V

148

Committee. Animals were given ad libitum access to food and water and kept on

12h:12h light/dark cycle. Pup’s sex was not taken into account, and male and female

pups were used randomly for introducing variability in the study and for being more

representative of relevant patient populations. All efforts were made to minimize

suffering and limit the number of animals used.

2.7. Experimental plan

To test whether CO prevented HI-induced neurodegeneration, and to ensure

that CO administration did not cause toxicity, animals were randomly assigned to

four experimental groups: CONTROL group (sham surgery without hypoxia

exposure, n=22), CO+SHAM group (CO exposure prior to sham surgery without

hypoxia exposure, n=16), HI group (hypoxia-ischemia, n=17) and CO+HI group (CO

exposure prior to HI, n=19) (Figure 1). Animals were randomly assigned to each

group through computer-generated (Excel) randomization schedules before

performing any experimental procedure. During the HI procedure and CO exposure

pups in all experiment groups were kept separate from their mother the equal period

of time. Rats were sacrificed 6 and 24 hours after HI onset. All subsequent

procedures were done by researchers who were blinded to the experimental groups.


149

Ch

apte

r V

Figure 5.1. Experimental groups and time-points schematic representation. Control group, n=22,

untreated animals that did not suffer any treatment; Carbon Monoxide (CO) group, n=16, subjected to

3 exposures of 250 ppm, for 1h at P4, P5 and P6; Hypoxia-Ischemia (HI) group, n=17, animals that

underwent surgery and hypoxia (8% of O2 in nitrogen) exposure for 75 minutes; CO+HI group, n=19,

CO treatment plus hypoxia-ischemia. Animals were euthanized at 6 and 24h post-HI. Brains were

collected and analyzed for lesion volume and cell death markers, as described in the methods section.

Histo, for brains analyzed by histological methods; WB, for brains collected and processed for western

blot analysis.

2.8. Carbon monoxide exposure

Because this is the first study of CO as neuroprotective agent against perinatal

ischemia, using rat pups, CO exposure conditions (dose and time) were extrapolated

from several previous in vivo studies for liver (Zuckerbraun et al. 2003, Otterbein et

al. 2005), heart (Nakao et al. 2010) or adult brain (Wang et al. 2011). To induce a

preconditioning state and test the neuroprotective role of CO, rat pups were exposed

to 250 ppm of CO for 1h at P4, P5 and P6. During the exposure, gas concentration

was carefully monitored by a CO analyser (Interscan, Chatsworth, US) and

temperature was kept at 37ºC. At the end of each CO administration, rat pups were

returned to their cages. During CO exposure time, control pups (without CO

exposure) were also kept separated from their mothers.

Chapter V

150

2.9. Model of neonatal cerebral hypoxia-ischemia

HI was induced in P7 rat pups, according to the Rice-Vannucci modification of

the Levine procedure (Levine 1960; Rice et al. 1981). The P7 rat was chosen

because its level of cortical development closely resembles that of 32-34 week

gestation: cortical layering is complete and low grade myelination is visible (Vannucci

et al. 1997). Rat pups were anesthetized with isoflurane (Isoflurane Vet, Alcyon Italia,

Marene, Italy, 4% during induction, then maintained with 2.5%), presented in a

mixture of 30:70 O2/N2O, delivered with a face-mask throughout the surgery.

Through a mid-neck incision, the left common carotid artery (CCA) was exposed and

double-ligated by means of 4/0 silk suture in order to permanently interrupt the blood

flow. Care was taken to avoid damage to the adjacent vagal nerve. The procedure

was completed in 10-15 min. After 2h of recovery and feeding, pups were exposed to

humidified 8% O2 - 92% N2 gas mixture (Wideroe et al. 2009) for 75 minutes in a

home-made acrylic hypoxic chamber (2,7 L). During all surgical procedures and

exposure in the hypoxic chamber the body temperature of the pups was maintained

with a heating carpet. Pups were allowed to recover for 5 min in room air, before

returned to their mothers. Sham-operated animals underwent the same surgical

procedure without CCA ligation and put into a similar chamber for the same period of

time as the HI group to mimic the time away from the mother. The temperature was

monitored and maintained at 37ºC throughout all procedure. Cell death assessment

was performed at 6h and 24h after HI, based on our in vitro experimental data (data

not shown, (Vieira et al. 2008; Queiroga et al. 2010; Almeida et al. 2012).

2.10. Tissue processing

For histological and immunohistochemical staining, animals were deeply

anesthetized by intraperitoneal injection of chloral hydrate 24h after surgery and

transcardially perfused with PBS, followed by buffered paraformaldehyde (4% in 0.1

M phosphate buffer, PB, pH 7.4). Brains were post-fixed overnight in the same

solution, infiltrated with 30% sucrose in 0.1 M PB for cryoprotection, frozen, and

stored at -20°C. 50 μm-thick serial coronal sections were cut on the cryostat; every


151

Ch

apte

r V

sixth slice was mounted on gelatine-coated slides for histological and immunological

staining. For Western blot assays, animals were decapitated and brains dissected in

ice-cold water, kept in ice for 15 min, and then frozen at -80°C.

2.11. Histological and immunohistochemical assessment

To define ischemic boundaries and precisely identify apoptotic profiles, serial

sections were stained with cresyl violet. For immunohistochemistry, sections were

immersed in 10% normal donkey serum (NDS), then incubated overnight in

antibodies against cleaved-caspase 3 (1:200, Cell Signaling Technology, Beverly,

MA, US), S100β (1:1000, Sigma-Aldrich) or Ki67 (1:400, Novocastra, Newcastle, UK)

at 4°C; primary antibodies were prepared in 10% NDS solution. For Ki67

immunohistochemistry, sections were initially heated in 0.01 M citrate buffer (pH 6.0)

for 3 minutes at 800 W using a microwave oven prior to primary antibody incubation

to enhance antigen retrieval. Cy2- or Cy3-conjugated anti-rabbit or anti-mouse

secondary antibodies (1:200 and 1:400 respectively, 2h at room temperature,

Jackson ImmunoResearch Laboratories, West Grove, USA) were used to visualize

binding of primary antibodies; cell nuclei were counterstained with 4, 6-diaminodino-

2-phenylindole (DAPI, Sigma Aldrich). Control tissue was processed as above with

non-immune serum instead of the primary antibody. Immunoreacted sections were

examined with Nikon 90i epifluorescence microscope by an investigator blinded to

the experimental groups. Co-localization was determined on a Leica TCS SP5

confocal laser scanning microscope equipped with argon 488 nm, helium–neon 543

nm, and helium–neon 633 mn lasers. Double-immunolabeled cells were analyzed

using three-dimensional (3D) reconstructed images with the x–z and y–z orthogonal

projections, using Leica LAS AF software.

2.12. Stereological counts in the hippocampus

Cresyl violet-stained sections were analysed with 100x oil-immersion objective

for stereological estimation of apoptotic profiles. Sections were visualized using a

Nikon Eclipse E600 microscope equipped with a motorized stage controller; they

Chapter V

152

were photographed with a CCD camera (OptronicsMicrofire, Goleta, CA, USA).

Analyses were done by an investigator blinded to the experimental groups. We used

the optical fractionator method for stereological estimates of apoptotic profiles in the

hippocampal formation; the volume of hippocampus was calculated according to the

Cavalieri’s principle (Neurolucida 8.0 User’s Guide—Document Version NL0108-

08.10 Microbrightfield Inc., 1988-2008.); all analyses were done with use of

StereoInvestigator (Microbrightfield, Williston, VT, USA). Series were 300 μm

spaced; the first section of each series was dorsal to the hippocampal white matter;

subsequent collected sections were equidistant from this reference slice; each series

consisted of 9 sections. The procedure yields a series of systematically random

sections (a requirement for the optical fractionator methods, Lyster et al 2005; West

et al 1991). Stereological sampling was done according to Fitting (Fitting et al.,

2008). An initial pilot study optimized the sampling scheme. The coefficient of

sampling error (CE) was calculated to determine the appropriate number and size of

disector counting frames. When considering 9 sections per case, a sampling grid of

400x400 μm, with a 25x25 μm counting frame and a disector height of 20 μm

(disector volume=12500 μm3) was chosen; the CE varied between 0.01 and 0.1. To

avoid oversampling, two guard zones were set on top and bottom of sections, each

corresponding to 10% of the focally measured section thickness. Only neurons

bearing morphological changes strongly suggestive for apoptosis, namely cell

shrinkage, chromatin condensation and visible apoptotic bodies were considered

dying cells and counted. The estimated total (T) number of objects (i.e., apoptotic

profiles) was finally calculated in accordance to the following formula, as previously

described by Fitting (Fitting et al. 2008)

T = ΣQ x t/h x1/asfx1/ssf

where ΣQis the number of objects counted in the disectors, t is the section

thickness, h is the height of disector probe, asf is the ratio between counting frames

area and grid step along x and y axis, and ssf is the section sampling fraction and

was set to 1/6. To avoid bias due to asymmetrical cutting, the total estimated number


153

Ch

apte

r V

of objects was then normalized to the sampled volume and density values are

presented (Figure 5.2.)

Figure 5.2. Stereological measurement of apoptosis. To estimate apoptosis, the hippocampal

formation was traced in serial sections under 4x magnification, then counts were performed at 100x

magnification (A); to allow unbiased sampling, counting sites were randomly selected by software at

the end of tracing procedures (B). Each subsequent section was then superimposed and aligned to

this reference slice to allow unbiased 3D-reconstruction of the hippocampus (C-D).


The apoptotic hall-markers: (i) Bcl-2 expression, (ii) release of cytochrome c

from mitochondria and (iii) caspase activation, were assessed in hippocampus

isolated 6 and 24 h after the insult. Rat pups were first anesthetized then

decapitated, the hippocampus dissected and stored at -80ºC until analysis; tissue

was triturated manually with Potter Elvehjem in lysis buffer (15 mMTris-HCl, pH 7.6,

320 mM sucrose, 1 mM DTT, 1 mM MgCl2, 0.5% protease inhibitor, 3 mM EDTA-K,

30 μg/mL CsA). For total cell extract analysis, samples were sonicated before protein

quantification. For mitochondrial analysis, this protein extract was centrifuged at 800

g for 10 min at 4ºC, supernatant was re-centrifuged at 9200 g for 15 min at 4ºC and

the pellet containing mitochondria was stored for Western blot analysis. Protein

content (from total extract or mitochondrial fraction) was determined by BCA assay

(Pierce, Illinois). Proteins (20-40 μg) were separated on SDS-PAGE (12%

polyacrylamide), transferred to PVDF membranes, blocked in 5% skim milk in Tris-

buffered saline (containing 0.1% of Tween 20) for 1h at room temperature (RT).

Equal amounts of protein were confirmed by the internal control assessment of ß-

Chapter V

154

actin. The following primary antibodies were used overnight at 4°C: anti-Bcl-2 (Santa

Cruz Biotechnology, 1:5000); anti-cytochrome c (Abcam, 1:2000); anti-active

caspase-3 (Cell Signaling, 1:5000); anti ß-actin (Santa Cruz Biotechnology, 1:5000);

all were diluted in blocking solution. Blots were developed using the ECL

chemiluminescence kit (Amersham Bioscience, UK) after incubation with HRP-

labeled anti-mouse or anti-rabbit IgG (GE Healthcare, UK, 1:5000) for 1 hour at RT.

The area and intensity of bands were quantified by densitometry analysis (GraphPad

Prism 4), and were normalized to the positive control (100%). Each experiment was

repeated three times, and gave similar results.

2.14. Statistical analysis of data

All experiments were carried out at least in triplicate; values are mean ± SD,

n≥3. Error bars, corresponding to standard deviation. Statistical comparisons were

performed using ANOVA: single factor with replication, with P<0.05, n≥3. P<0.05

means that samples are significantly different at a confidence level of 95%. All

statistical comparisons were conducted using the SPSS package (version 18, SPSS

Inc., Chicago, IL, USA). For in vivo study, data distribution and equality of variances

were initially assessed by the Shapiro-Wilk (Shapiro et al. 1965) and the Levene

median (Levene 1960) tests, respectively. One-way ANOVA was applied to

determine overall significant differences in the number of apoptotic cells among

groups. Post-hoc analyses were conducted when p<0.05 by Fisher’s protected least

significant difference (PLSD) test.

3. RESULTS

3.1. Carbon monoxide prevents excitotoxicity-induced cell death and induces

Bcl-2 expression in primary cultures of neurons

Primary cultures of neurons were pre-treated for 1h with CO-saturated solution

at 10 µM. After 24h, chromatin condensation and loss of membrane integrity were

assessed by fluorescence microscopy (representative photos in Figure 5.3. – A),


155

Ch

apte

r V

and CO partially prevented neuronal cell death (Figure 5.3. – B). Additionally, Bcl-2

mRNA was measured by RT-Q-PCR, with increased expression levels at 6 h and 24

h (Figure 5.3. – C) after CO treatment; which is in accordance with CO-related

neuroprotection, since Bcl-2 is an anti-apoptotic protein.

(A)

(B) (C)

Chapter V

156

(Previous page) Figure 5.3. Effect of carbon monoxide treatment on neuronal apoptosis. (A)

Representative micrographs of neurons treated or not with 20 μM of glutamate and 10 μM of CO.

Apoptotic hallmarks were analyzed by fluorescent microscopy. Upper panel, for the photos taken with

the filter for phase contrast, middle panel, for Hoechst (white arrows for nuclei with condensed

chromatin) and lower panel, for propidium iodide (white arrows for cells which membrane integrity was

lost). (B) Primary cultures of neuronal cells were pre-treated with 10 µM CO, followed by 24 h of

glutamate (10–30 µM) treatment. Cell viability was assessed by counting cells containing normal nuclei

and plasmatic membrane integrity. For each coverslip, at least 1500 cells were counted. All values are

mean±SD (error bars), n=5; *p< 0.05 compared to control. (C) The effect of 10 µM CO treatment on

Bcl-2 expression was assessed by its mRNA quantification.

3.2. Effect of preconditioning on HI-induced apoptosis in vivo

75 min of hypoxic exposure after carotid occlusion (perinatal model for cerebral

hypoxia-ischemia) reliably induced mild damage in terms of cell death in the

subregions of the hippocampus (Figure 5.4. – A-E), predominantly at the level of

CA2 and CA3 ipsilateral to the CCA occlusion, while the cortex did not show any

detectable damage. The cytoarchitecture was partly lost due to shrinkage relative to

the contralateral side, in which histological morphology was preserved (Figure 5.4. –

A-E). A great number of apoptotic profiles were detected at the level of CA1, CA2/3

and dentate gyrus (DG) after HI. Early stages of the apoptotic cascade were

revealed by the presence of cells with darkly stained nuclei, condensed chromatin

and dense cytoplasm (pyknosis); late stages of apoptosis were also visible in cells

that had DNA fragmentation, and nuclei broken into several discrete chromatin

bodies (karyorrhexis), cytoplasmic disruption, and appearance of apoptotic bodies

(Figure 5.4. – C-E). The density of apoptotic cells was significantly higher in the

hippocampus ipsilateral to the occlusion (ischemic side) compared to the

contralateral (intact) side, both in the HI group and the CO+HI group (Figure 5.4. –

F). However, the density of apoptotic profiles in the ischemic hemisphere was

efficiently reduced (64%) in the CO+HI group compared to the HI group (Figure 5.4.

– F).

Cytotoxic edema, known to contribute to early ischemic damage, may represent

a confounding factor in estimating cell density and, when severe, may lead to an

underestimation of apoptosis. However, this was not the case in our material: no


157

Ch

apte

r V

ischemia-related differences in hippocampal volumes (mm3) between the

hemispheres were seen in any of the groups (Figure 5.4. – G).

Figure 5.4. Carbon monoxide effect in hippocampus after perinatal hypoxia-ischemia – apoptotic

profiles. Whereas contralateral hippocampus displayed a preserved morphology (A) following HI,

diffuse tissue disruption was detected in the hippocampus ipsilateral to the occlusion (B). C-E are

representative pictures of ischemic hippocampus, where diffuse apoptosis was documented; with

peculiar morphological features including pyknotic nuclei (C), indicating early stage of apoptosis,

progressive nuclear fragmentation (D) and karyorrhexis as confirmed by detectable apoptotic bodies

(E). Compared to HI group, the number of apoptotic profiles was significantly lower when animals were

exposed to CO prior to HI (F). All values are mean±SD (error bars); *p< 0.05 compared to Control group

for the corresponding side and **p< 0.05 compared to HI group ischemic hippocampus. (G) For each

group there is no significant difference in cytotoxic edema volume (mm3) between the ipsi- and the

contralateral hippocampus.

(F) (G)

Chapter V

158

Similarly, the volume of ischemic or intact hippocampus did not significantly vary

between groups. Accordingly, active caspase 3 positive cells were predominantly

detected in CA2, CA3 and DG regions of the hippocampus in the HI group (Figure

5.5. – A-C), whereas fewer immunolabeled cells were visible in the CO+HI group

(Figure 5.5. – D-F) and none were detected in CO+SHAM group (Figure 5.5. – G-I).

No significant differences were detected between the HI and CO+HI group with

regard to glial activation and cell proliferation as assessed by S100β and Ki67

immunohistochemistry, respectively (data not shown).

Figure 5.5. Carbon monoxide effect in hippocampus after perinatal hypoxia-ischemia – cleaved

caspase 3 expression. Low (scale bar = 100 µm) magnification CLSM photographs of the

hippocampus of HI (A, B), CO-HI (C, D) and CO sham operated (E, F) rat pups. In blue, DAPI-stained

nuclei; in red, cleaved caspase 3-positive cells. Caspase 3-positive profiles following HI were

particularly frequent in CA1-2 and in the dentate gyrus, and were decreased in number following CO

preconditioning. CO preconditioning alone did not induce caspase 3 activation in sham operated

animals.

3.3. Effect of carbon monoxide on expression of apoptotic markers

The results of immunoblots for apoptotic markes in total cellular extracts of

ipsilateral (IL) and contralateral (CL) hippocampus or in mitochondria-enriched

fractions are shown in Figure 5.6. CO pre-treatment lead to an increased expression


159

Ch

apte

r V

of anti-apoptotic protein Bcl-2 at 6 and 24 h in both hemispheres compared to the HI

group. At 24 h, Bcl-2 expression among CO pre-treated pups was also higher than

control pups (Figure 5.6. – A). These data are in accordance with cell death

prevention by CO in hippocampus and validate the in vitro results showing higher

levels of Bcl-2 mRNA for CO-treated neurons (Figure 5.3. – C). Therefore, CO

appears to trigger tissue preconditioning and to stimulate cell survival, by changing

gene expression, such as bcl-2. Cytochrome c levels in mitochondria-enriched

fraction of hippocampus of the HI group were lower at 6h than at 24h after the insult

relative to controls. At both 6 and 24 h after HI, CO pre-treated rat pups had higher

levels of cytochrome c than the HI group in mitochondria-enriched fraction of

hippocampus (Figure 5.6. – B). Thus, CO prevented cytochrome c release from

mitochondria, which can limit apoptosome formation and caspase-3 activation.

Indeed, caspase-3 activation was less pronounced in IL hippocampal extracts from

CO+HI group than from HI group at 6 and 24 h, while caspase-3 activation was

similar in CL CO+HI and HI groups (Figure 5.6. – C).

Chapter V

160

Figure 5.6. Effect of carbon monoxide on apoptotic markers in hippocampal extracts after 6 and

24 h of HI – protein expression and sub-cellular localization. Representative immunoblots (upper

panels) and the corresponding quantifications, as relative percentages to the hippocampus from control

rat pups (lower panels). (A) Bcl-2 expression in total hippocampal extracts. All values are mean±SD

(error bars), n=3; *p< 0.05 compared to HI group for the corresponding side and **p< 0.05 compared to

HI group ipsilateral hippocampus. (B) Cytochrome c levels in enriched mitochondrial fraction from

hippocampus, which is an indirect way for measuring cytochrome c release. All values are mean±SD

(error bars), n=3; *p< 0.05 compared to HI group hippocampus for the corresponding side. (C) Caspase-

3 activation in total extracts. All values are mean±SD (error bars), n=3; *p< 0.05 compared to HI group

for the ipsilateral hippocampus.

4. DISCUSSION

Two complementary approaches were applied to demonstrate the

neuroprotective role of carbon monoxide: in vitro excitotoxicity induction in primary


161

Ch

apte

r V

culture of rat cerebellar neurons and an in vivo model of perinatal ischemia in rat

pups. In primary neuronal cultures, 1h of CO pre-treatment had a positive outcome

on the survival of neurons against excitotoxic-induced cell death and increased

expression of the anti-apoptotic gene bcl-2 (Figure 5.3.). Likewise, prior exposure to

CO significantly reduced the number of apoptotic profiles in the hippocampus (by

64%) 24 h following hypoxia-ischemia in rat pups (Figure 5.4.).

Apoptotic factors are up-regulated in early stages of brain development and play

a major role in brain damage following perinatal ischemia because apoptosis is a key

cellular process during CNS development (Hagberg 2004; Ge et al. 2011). Therefore,

apoptotic hallmarks were assessed in vivo. Indeed, CO pre-treatment improved the

cellular anti-apoptotic machinery (Figures 5.5. and 5.6.). In accordance with the in

vitro data, increased Bcl-2 expression was found in hippocampal extracts from CO-

treated pups (Figure 5.6. – A). Furthermore, increase in Bcl-2 expression activates

many anti-apoptotic pathways and indicates that late preconditioning might be

involved in CO neuroprotection (Ness et al. 2006). Accordingly, previous studies

have reported that protein synthesis is required for CO-conferred neuroprotection

(Vieira et al. 2008) and mitochondrial biogenesis stimulation is also involved in CO’s

apoptosis inhibition (Almeida et al. 2012). Thus, CO cytoprotection might be related

to late preconditioning induction, although further studies are necessary.

Nevertheless, early preconditioning responses cannot be excluded from CO’s mode

of action, since ROS signaling is involved in CO-induced cytoprotection (Motterlini

and Otterbein 2010; Queiroga et al. 2010; Queiroga et al. 2011), which presents new

hypotheses for further studies based on cerebral cell anti-oxidant defense.

In hippocampus, higher cytochrome c levels in mitochondria were found with CO

pre-treatment (Figure 5.6. – B). This indicates a reduced release of cytochrome c

from mitochondria, which can be related to the reported direct action of CO on

mitochondrial membrane permeabilization (Queiroga et al. 2010). Still, the increased

cytochrome c sequestration in the mitochondria contributes to the lower levels of

caspase-3 activation found in hippocampus from CO pre-treated pups (Figure 5.6. –

C). Furthermore, in hippocampal CA2, CA3 and DG regions, activated caspase-3

Chapter V

162

positive cell populations decreased when pups were exposed to CO before the

hypoxic-ischemic insult (Figure 5.5.). An active role of CO in cerebral apoptosis

prevention in vivo has been suggested by other authors: (i) HO-1 knockout mice

exhibit a larger volume of tissue damage following injection of NMDA compared to

wild type mice (Ahmad et al. 2006), and (ii) Zeynalov and colleagues report that low

doses of exogenous CO protect against transient or permanent focal ischemia in

adult mice (Zeynalov and Dore 2009; Wang et al. 2011).

Perinatal HI can be partially predicted based on the presence of several risk

factors: signals of distress during intrauterine life and hypoxic-ischemic insults at birth

(Bonifacio et al. 2011). Also, preterm newborns represent a high-risk population for

brain injury due to HI (Arpino et al. 2005). Therefore, preconditioning-based

strategies can become potential therapies for perinatal HI, and CO is a promising

candidate. Other preconditioning-based strategies have also been developed for

perinatal cerebral ischemia. In piglets, 3 h of exposure to 8% oxygen prior to HI insult

induced neuroprotection via up-regulation of hypoxia-inducible factor-1α (HIF-1α)

and vascular endothelial factor (VEGF) (Ara et al. 2011). Similarly, in adult mice,

preconditioning by exposure to hypoxia and isoflurane presented a neuroprotective

effect (McAuliffe et al. 2007). In an experimental model of HI similar to ours (left

carotid artery ligation and 90 minutes of 8% O2), hydrogen has shown to be

neuroprotective by blocking apoptosis (Cai et al. 2008). Finally, lithium pre-treatment

prevented apoptotic and autophagic neuronal cell death in the hippocampus

following neonatal HI (Li et al. 2010).

CO may be a useful therapeutic adjunct. In fact, other inhalation therapies have

been introduced in clinics (Robinson et al. 2009), to reduce toxicity resulting from the

metabolization of administered drugs, since drug extraction occurs by exhalation.

The therapeutic application of nitric oxide (NO) as vasodilator in several disease

models and also in injured lungs of premature and newborn babies is now widely

accepted (Bloch et al. 2007). NO is chemically similar to CO. However, unlike CO,

NO reacts rapidly with molecular oxygen and produces peroxynitrite (ONOO-), which

is highly reactive. Likewise, noble gases have also been studied for medical


163

Ch

apte

r V

applications, especially xenon (Sanders et al. 2004). Recently, Ryang and co-

workers (Ryang et al. 2011) described the efficacy of argon in protecting rat brains in

a model of transient middle cerebral artery occlusion. Taken all together, potential

CO-inhaled based therapy has the added value of integrating two critical advantages:

it is chemically inert compared to NO and is an endogenous molecule compared to

noble gases (Bonifacio et al. 2011).

To overcome a possible systemic CO toxic effect, the use of CO-releasing

molecules (CORM’s) has been proposed (Motterlini 2007). These small organic and

organometallic compounds are able to deliver CO in a timely and tissue-specific

manner. This permits a significant reduction in carboxyhaemoglobin formation and

toxicity, which opens novel windows of opportunity for clinical applications. The

biological activities of CORMs include cardioprotection in isolated perfused rat heart,

protection in acute hepatic reperfusion injury in rats, endothelial cells protection

during cold preservation, and injury impairment in the case of HI injury during kidney

transplantation, among others (Motterlini and Otterbein 2010; Stein et al. 2012). Very

recently, CORM-3 was shown to modulate the inflammatory response and to reduce

brain damage in an adult rat model of hemorrhagic stroke (Yabluchanskiy et al.

2012), indicating that CORM-3 can cross the blood brain barrier.

Altogether, in vitro and in vivo approaches have demonstrated that CO initiates a

cascade of events that prevents neuronal apoptosis: (i) increase of Bcl-2 expression,

(ii) prevention of cytochrome c release from mitochondria, (iii) inhibition of caspase-3

activation and (iv) decrease of chromatin condensation. CO administration induced

late preconditioning and limited hippocampal neuronal cell death following cerebral

perinatal ischemia. In conclusion, the biological neuroprotective role of CO, coupled

with the possibility of using CORMs, opens avenues of further research and potential

applications of CO-based therapies in cerebral ischemic models. Moreover, this

innovative approach takes advantage of an endogenous molecule (CO) and

intracellular pathways (preconditioning) for limiting neuronal cell death.

Chapter V

164

5. ACKNOWLEDGMENTS

The authors express their gratitude to João Seixas from Alfama, Portugal, for

measurements of CO in solution; to Marcos Sousa from Instituto de Tecnologia

Química e Biológica, Portugal, for technical support.

6. REFERENCES

Ahlstrom, K., B. Biber, et al. (2009). "Metabolic responses in ischemic myocardium after inhalation of carbon monoxide." Acta Anaesthesiol Scand 53(8): 1036-42.

Ahmad, A. S., H. Zhuang, et al. (2006). "Heme oxygenase-1 protects brain from acute excitotoxicity." Neuroscience 141(4): 1703-8.

Alkan, T., B. Goren, et al. (2008). "Effects of hypoxic preconditioning in antioxidant enzyme activities in hypoxic-ischemic brain damage in immature rats." Turk Neurosurg 18(2): 165-71.

Almeida, S. S., C. S. Queiroga, et al. (2012). "Carbon monoxide modulates apoptosis by reinforcing oxidative metabolism in astrocytes: role of BCL-2." J Biol Chem 287(14): 10761-10770.

Ara, J., S. Fekete, et al. (2011). "Hypoxic-preconditioning induces neuroprotection against hypoxia-ischemia in newborn piglet brain." Neurobiol Dis 43(2): 473-85.

Arpino, C., L. D'Argenzio, et al. (2005). "Brain damage in preterm infants: etiological pathways." Ann Ist Super Sanita 41(2): 229-37.

Biermann, J., W. A. Lagreze, et al. (2012). "Preconditioning with inhalative carbon monoxide protects rat retinal ganglion cells from ischemia/reperfusion injury." Invest Ophthalmol Vis Sci 51(7): 3784-91.

Bloch, K. D., F. Ichinose, et al. (2007). "Inhaled NO as a therapeutic agent." Cardiovasc Res 75(2): 339-48.

Bonifacio, S. L., H. C. Glass, et al. (2011). "A new neurological focus in neonatal intensive care." Nat Rev Neurol 7(9): 485-94.

Cai, J., Z. Kang, et al. (2008). "Hydrogen therapy reduces apoptosis in neonatal hypoxia-ischemia rat model." Neurosci Lett 441(2): 167-72.

Carloni, S., G. Buonocore, et al. (2008). "Protective role of autophagy in neonatal hypoxia-ischemia induced brain injury." Neurobiol Dis 32(3): 329-39.



165

Ch

apte

r V



Fitting, S., R. M. Booze, et al. (2008). "Differential long-term neurotoxicity of HIV-1 proteins in the rat hippocampal formation: a design-based stereological study." Hippocampus 18(2): 135-47.



Ge, W., Y. W. Chen, et al. (2011). "Overlapping functions of microRNAs in control of apoptosis during Drosophila embryogenesis." Cell Death Differ.

Gidday, J. M. (2006). "Cerebral preconditioning and ischaemic tolerance." Nat Rev Neurosci 7(6): 437-48.

Gunn, A. J. and L. Bennet (2008). "Brain cooling for preterm infants." Clin Perinatol 35(4): 735-48, vi-vii.

Hagberg, H. (2004). "Mitochondrial impairment in the developing brain after hypoxia-ischemia." J Bioenerg Biomembr 36(4): 369-73.

Heazell, A. E. and J. F. Froen (2008). "Methods of fetal movement counting and the detection of fetal compromise." J Obstet Gynaecol 28(2): 147-54.


Kroemer, G. (2003). "Mitochondrial control of apoptosis: an introduction." Biochem Biophys Res Commun 304(3): 433-5.

Levene, H. (1960). "Robust tests for equality of variances." Stanford University Press: 278-292.

Levine, S. (1960). "Anoxic-ischemic encephalopathy in rats." Am J Pathol 36: 1-17.

Li, Q., H. Li, et al. (2010). "Lithium reduces apoptosis and autophagy after neonatal hypoxia-ischemia." Cell Death Dis 1: e56.

McAuliffe, J. J., B. Joseph, et al. (2007). "Isoflurane-delayed preconditioning reduces immediate mortality and improves striatal function in adult mice after neonatal hypoxia-ischemia." Anesth Analg 104(5): 1066-77, tables of contents.

Chapter V

166

Motterlini, R. (2007). "Carbon monoxide-releasing molecules (CO-RMs): vasodilatory, anti-ischaemic and anti-inflammatory activities." Biochem Soc Trans 35(Pt 5): 1142-6.



Nakao, A., D. J. Kaczorowski, et al. (2010). "Amelioration of rat cardiac cold ischemia/reperfusion injury with inhaled hydrogen or carbon monoxide, or both." J Heart Lung Transplant 29(5): 544-53.

Ness, J. M., C. A. Harvey, et al. (2006). "Selective involvement of BH3-only Bcl-2 family members Bim and Bad in neonatal hypoxia-ischemia." Brain Res 1099(1): 150-9.

Otterbein, L. E., A. May, et al. (2005). "Carbon monoxide increases macrophage bacterial clearance through Toll-like receptor (TLR)4 expression." Cell Mol Biol (Noisy-le-grand) 51(5): 433-40.

Queiroga, C. S., A. S. Almeida, et al. (2011). "Carbon monoxide prevents hepatic mitochondrial membrane permeabilization." BMC Cell Biol 12: 10.

Queiroga, C. S., A. S. Almeida, et al. (2010). "Glutathionylation of adenine nucleotide translocase induced by carbon monoxide prevents mitochondrial membrane permeabilization and apoptosis." J Biol Chem 285(22): 17077-88.

Rice, J. E., 3rd, R. C. Vannucci, et al. (1981). "The influence of immaturity on hypoxic-ischemic brain damage in the rat." Ann Neurol 9(2): 131-41.

Robinson, B. R., K. P. Athota, et al. (2009). "Inhalational therapies for the ICU." Curr Opin Crit Care 15(1): 1-9.

Ryang, Y. M., A. V. Fahlenkamp, et al. (2011). "Neuroprotective effects of argon in an in vivo model of transient middle cerebral artery occlusion in rats." Crit Care Med 39(6): 1448-53.

Sanders, R. D., D. Ma, et al. (2004). "Xenon: elemental anaesthesia in clinical practice." Br Med Bull 71: 115-35.

Schousboe, I., O. M. Larsson, et al. (1989). "Development of homospecific activity of GABA-transaminase in the mouse cerebral cortex and cerebellum and in neurons cultured from these brain areas." Int J Dev Neurosci 7(1): 115-21.

Shapiro, S. S. and M. B. Wilk (1965). "An analysis of variance test for normality (complete samples)." Biometrika 52: 591-611.


167

Ch

apte

r V

Stein, A. B., R. Bolli, et al. (2012). "Carbon monoxide induces a late preconditioning-mimetic cardioprotective and antiapoptotic milieu in the myocardium." J Mol Cell Cardiol 52(1): 228-36.

Stenzel-Poore, M. P., S. L. Stevens, et al. (2007). "Preconditioning reprograms the response to ischemic injury and primes the emergence of unique endogenous neuroprotective phenotypes: a speculative synthesis." Stroke 38(2 Suppl): 680-5.

Taylor, R. C., S. P. Cullen, et al. (2008). "Apoptosis: controlled demolition at the cellular level." Nat Rev Mol Cell Biol 9(3): 231-41.


Vannucci, R. C., J. R. Connor, et al. (1999). "Rat model of perinatal hypoxic-ischemic brain damage." J Neurosci Res 55(2): 158-63.

Vannucci, R. C. and S. J. Vannucci (1997). "A model of perinatal hypoxic-ischemic brain damage." Ann N Y Acad Sci 835: 234-49.

Vannucci SJ, H. H. (2004). "Hypoxia-ischemia in the immature brain." J Exp Biol.: 3149-3154.



Wideroe, M., O. Olsen, et al. (2009). "Manganese-enhanced magnetic resonance imaging of hypoxic-ischemic brain injury in the neonatal rat." Neuroimage 45(3): 880-90.

Yabluchanskiy, A., P. Sawle, et al. (2012). "CORM-3, a carbon monoxide-releasing molecule, alters the inflammatory response and reduces brain damage in a rat model of hemorrhagic stroke*." Crit Care Med 40(2): 544-52.



Zuckerbraun, B. S., T. R. Billiar, et al. (2003). "Carbon monoxide protects against liver failure through nitric oxide-induced heme oxygenase 1." J Exp Med 198(11): 1707-16.

Chapter V

168

“Nothing in the universe is more mysterious than the inner workings of the human

mind.”

The Teaching Company (2008)

VI DISCUSSION AND CONCLUSIONS

Cláudia S.F. Queiroga has written the whole chapter, based on the referred bibliography and her own results described in chapters II to V.

DISCUSSION AND CONCLUSIONS

171

Ch

apte

r V

I

CONTENTS

1. Discussion ........................................................................................................... 171

1.1. Mitochondria as target for anti-apoptotic strategies ..................................... 175

1.2. Astroglial active role in therapy .................................................................... 176

1.3. Preconditioning as an efficient future therapeutic strategy .......................... 177

1.4. Carbon monoxide in a therapeutic context .................................................. 178

2. Looking ahead ..................................................................................................... 180

3. Conclusion .......................................................................................................... 182

4. References .......................................................................................................... 182

Chapter VI

172

Ch

apte

r V

I

1. DISCUSSION

Perinatal complications are a serious clinical problem, in particular hypoxic-

ischemic encephalopathy (HIE), which is caused by birth asphyxia or uterine and

fetal blood flow interruption (Wintermark 2011). HIE corresponds to 23% of all

neonatal deaths worldwide, being one of the top 20 leading causes of burden of

disease in all age groups (World Health Organization). The social impact is both

sociological and economical.

Progress in neonatology contributed to the reduction of mortality rates,

nevertheless, there is still a high neurological morbidity related to HIE accounting for

significant disability (Bonifacio et al. 2011). Physical consequences include cerebral

palsy, mental retardation, learning disabilities, epilepsy, deafness and blindness

(Robertson et al. 2009). Thus, disclosure of the mechanisms involved in neonatal

brain injury is crucial for improving long-term neurological status and developing

potential therapeutic strategies.

It is well known that in cerebral HIE the damage is initiated during the insult

(hypoxia and ischemia), being perpetuated and magnified during post-resuscitative

period (reperfusion). The main cellular consequences are excitotoxicity, oxidative

stress, metabolic failure, inflammation and apoptosis (Robertson et al. 2009). The

post-reperfusion phase of brain lesion is the main target for neuroprotective

interventions (Wintermark 2011).

The standard treatment in severe perinatal hypoxia-ischemia and reperfusion

(HIR) includes resuscitation with high levels of O2 (hyperoxia) and hypothermia.

However, hyperoxia can have severe consequences such as increased oxidative

stress and brain damage (Sola et al. 2008). On the opposite, therapeutic

hypothermia presents very promising results (Yenari et al. 2012). Still, the time

window for its application is very narrow and the knowledge on the cellular

mechanisms is limited. Thus, there is an increasing interest in the development of

novel therapeutic strategies and biological targets in perinatal HIR.


173

Carbon monoxide (CO) protective actions include anti-inflammation, anti-

proliferation, anti-apoptotic in several models (Table 1.5., Chapter I). Still, in brain

environment its protection was poorly explored. This thesis explores the possible

application of CO as an anti-apoptotic agent. As described in Figure 1.12. (Chapter

I), the general objective is the evaluation of CO’s role in the pathological model of

cerebral ischemia. The strategy aimed at disclosing the main cellular players in

different cerebral models, with increasing complexity (subcellular – cellular – organ),

by posing different but complementary questions. Each study contributed for an

increasing knowledge about the endogenous molecules and their pathways

associated with CO protective mechanisms. The main achievements (summarized in

Figure 6.1.) contribute to open another avenue of therapeutic opportunities for CO.

Chapter VI

174

Ch

apte

r V

I

Figure 6.1. Main achievements of this PhD thesis. Several cerebral ischemia were adopted to study carbon monoxide (CO) effect. At the mitochondrial level a myriad of subcellular events lead to mitochondrial membrane permeabilization prevention (Chapter II) and mitochondrial metabolism reinforcement and the Bcl-2 role (Chapter III). At the cellular level, CO prevents astrocytic apoptosis (Chapter II and III) and stimulates the non-cell autonomous effect to confer neuroprotection (Chapter IV). At the systemic level, low doses of CO confer neuronal apoptosis prevention after perinatal hypoxia-ischemia (Chapter V).


175

1.1. Mitochondria as target for anti-apoptotic strategies

Mitochondrial dysfunction is related to the propagation of acute (stroke) and

chronic (as Alzheimer and Parkinson Disease) neurodegenerative disorders (Lezi et

al. 2012). In particular, mitochondria assume relevance in perinatal ischemia since

apoptotic factors are up-regulated in immature stages of the brain, since apoptosis is

a crucial cell process during cerebral development (Tsujimoto et al. 2006; Galluzzi et

al. 2007).

Mitochondria can be considered as the convergence point within many cellular

processes: modulation of cell death pathways, regulation of metabolic network and

controlling the balance between these two major cellular events. Thus, proposing

mitochondria as central organelle in the PC-involved mechanisms is reasonable.

Several scientific evidences support a key role for mitochondria in PC, namely:

reactive oxygen species (ROS) generation for further signalling, modulation of

potassium channels located in the inner mitochondrial membrane, modulation of

oxidative phosphorylation process and mitochondrial membrane permeabilisation

(Busija et al. 2008; Correia et al. 2011).

In mitochondria, O2.- production occurs close to superoxidase dismutase (SOD),

where it is rapidly converted to H2O2 (a much less reactive molecule), which can

initiate subsequent signalling (Zuckerbraun et al. 2007). Carbon monoxide (CO)

increases mitochondrial ROS production (Chapter II), triggering downstream events

towards organelle resistance to damage. By inducing glutathionylation of adenine

nucleotide translocase (ANT), CO prevents ANT pore-forming conformation and

mitochondrial membrane permeabilization, avoiding apoptosis activation (Chapter

II). Moreover, CO modifies cytochrome c oxidase (COX) activity (Chapter III),

reinforces oxidative phosphorylation (Chapter III) and increases mitochondrial

biogenesis (Chapter III). In addition, CO increases Bcl-2-COX interaction within

mitochondria (Chapter III). In conclusion, CO seems to act largely at mitochondrial

level (Queiroga et al. 2012) with two different functions: (i) preventing MMP and the

release of pro-apoptotic factors (Chapter II and V) and (ii) improving oxidative

metabolism (Chapter III).

Chapter VI

176

Ch

apte

r V

I

1.2. Astroglial active role in therapy

Brain is a dynamic and interconnected network of cells and information. Most of

the current therapies lack this integrative feature and target one cell type, a single

cell process or pathway. CO, with its PC mode of action, multiplies the cellular

targets. Furthermore, the PC cellular state is not restricted to the target cell,

presenting a non-cell autonomous effect (Chapter IV).

In the neuroscience field the uncertainty regarding mechanisms and order of

events on neurodegenerative disorders, limits the development of novel and more

efficient therapies. The neurodegenerative processes with irreversible neuronal loss

can also occur due to astroglial abnormal function that is no longer able to assure

correct neuronal survival (Allaman et al. 2011). Thus, along with mitochondrial

dysfunction, astrocytic dysfunction is a transversal event in many neuronal diseases

representing an attractive therapeutic target. Therefore, herein two strategies were

pursuit: (i) directly targeting astrocytes (Chapter II and III) and (ii) targeting neurons

through a non-cell autonomous effect via astrocytes (Chapter IV). In Chapter II and

III, it was demonstrated that CO prevents astrocytic cell death, thus one can

speculate that efficient functioning of these glial cells will maintain cerebral

homeostasis and stimulate neuroprotection. In Chapter IV, the premise was to target

astrocytes, in a non-cell autonomous way, for conferring neuronal protection.

Furthermore, purinergic molecules are exerting the signaling task. Therefore, CO

pre-treatment takes advantage from the astrocytic targeting but also from the

purinergic therapeutic potentialities. Indeed, aligned with described evidences

(Burnstock 2009), purinergic therapeutic solutions are being investigated for

treatment of disorders in several systems as gut, kidney, lung, among others, but not

in the brain (Burnstock 2012). Chapter IV conveys another possible purinergic

application, which is, in this case, stimulated by CO. However, further studies should

be performed to confirm these observations.

The contribution of the different purinergic receptors activation or inhibition to

the propagation of the injury is surrounded by contradictory evidences. In Chapter

IV, A1 receptor was excluded as intervenient in CO-induced modulation of astrocyte-


177

to-neuron communication. Moreover, A2A receptor activation seems to have a central

role (Chapter IV). An interesting fact is that in the literature, opposite evidences were

found. A1 receptor is usually associated to neuroprotective effects in stroke models,

probably due to the inhibitory effect in the brain during hypoxia (Thauerer et al.

2012). A2A receptor blockade results in the restriction of the neuronal damage and

neurological deficits in the brain of adult animals (Cunha 2005).

Further experimental evidences are needed, since the results of Chapter IV are

only from functional experiments with long-term readouts. Nevertheless, one may

speculate that our contradictory results are related to the mode of action of CO-

induced preconditioning.

In summary, targeting astrocytes, inducing PC by CO action is not only

protecting the astroglial cells (Chapter II and III). It is also stimulating the non-cell

autonomous communication between astrocytes and neurons, taking advantage from

already available protective glial pathways, challenging the system towards

protection (Chapter IV).

1.3. Preconditioning as an efficient future therapeutic strategy

Since the late 90’s it is reported by the clinicians that brain stroke patients with

prior transient ischemic attacks (TIA) develop less brain injury than patients with no

previous TIA, demonstrating a natural form of preconditioning (Kitagawa 2012). The

possibility to induce tolerance in patients in whom ischemic events can be anticipated

is very attractive. In order to safely implement PC as a therapeutic strategy, it is

important to dissect precisely the mechanism(s) of action of mediators and pathways

involved, also for the correct choice of the preconditioning stimuli. Due to cross and

remote tolerance features of PC, it is possible to induce ischemic tolerance in the

brain with a different stimuli and by inducing PC in another organ. Already tested in

patients is the cross tolerance induced by limb ischemia. Brain preconditioning can

be achieved by interrupting the blood flow for a short period of time in the limb

(Dirnagl et al. 2009; Kitagawa 2012). The induction of PC as therapeutic strategy has

Chapter VI

178

Ch

apte

r V

I

another advantage: the usage of endogenous resources allows a rapid response

(early PC) with very well-tolerated players. A late PC response can be initiated, with

gene expression. Thus, PC allows the system to react according to the noxious

stimuli, to the dose and period of time of application, taking advantage of the

endogenous mechanisms.

CO emerges as a very promising PC agent: in low doses is safe, it is an

endogenous molecule and is well-tolerated by the organism, impairs apoptotic

damage and increases pro-survival machinery (Chapters II, III, IV and V). In this

thesis it is shown the CO protection in a pathological model of HIR. Nevertheless,

due to the transversal key role of mitochondria and astrocytes in many disorders, as

well as the large range of PC applications, one may consider that this knowledge can

be extrapolated to several other disease studies. CO is a cytoprotective factor by

inducing cellular PC, and mitochondria appear as the main cellular targets. In

addition, CO is an endogenous gaseoustransmitter, which is physiologically

generated in response to several types of stress. In conclusion, CO-induced PC

combines the limitation of brain injury (the classical drugs) and the initiation of

protective pathways.

PC strategy, as explored in Chapter V, is a very useful and powerful tool for

perinatal ischemia, since these episodes can be partially predicted. The hypothesis

of using CO as PC-inducing agent to limit brain damage resultant from perinatal HI

was successful confirmed in an in vivo model (Chapter V). These results (i) confirm

the data obtained with the in vitro models used (Chapters II, III and IV) and (ii)

strengthen the safety and efficacy of CO clinical application.

1.4. Carbon monoxide in a therapeutic context

As demonstrated throughout the previous chapters (Chapters II-V), CO is a PC

agent. It instructs the cell to initiate pro-survival pathways in a cooperative, concerted

and synchronized manner. Moreover, CO triggers a consistent cellular response,

having different time phases and molecular effectors, since post-translational

modifications to protein expression (Table 6.1.).


179

Table 6.1. This thesis contribution in carbon monoxide downstream molecular effectors, its

modifications and time window of action.

Early events (3h) Late events

↑ ROS (Chapter II) ↑ GSSG (Chapter II) ANT glutathionylation (Chapter II) ↑ COX activity (Chapter III) ↑ mitochondrial biogenesis (Chapter III) ↑ ATP concentration (Chapter III) ↑ ATP release from astrocytes (Chapter IV)

↓Lac/gluc ratio (Chapter III) ↑ Bcl-2-COX interaction (Chapter III) ↑ Bcl-2 expression (Chapter III and V) ↑ ATP concentration (Chapter III) A2A receptors activation (Chapter IV) TrK B receptors activation (Chapter IV) ↓ cytochrome c release from mitochondria (Chapter V) ↓ active caspase-3 (Chapter V)

Unlike most drugs, CO does not act only when it is present in the system.

Instead, CO exerts an initiating role, by activating endogenous pathways. Even after

being expelled by the organism, the CO-induced cellular protection is still exerted.

The use of CO for therapeutic purposes presents two main advantages: (i) it is an

endogenous product and the organism is fully adapted to it and (ii) CO is not

metabolized and reversibly binds to its molecular targets, which becomes the

pharmacokinetic much simpler.

Despite the biological functions associated to CO in vivo and the existence of

several proteins capable for binding CO in vitro, it is still a matter of discussion the

actual physiological target of CO. Probably CO will target many proteins and the

already described effects are the result of concerted and myriad actions. Or, one

may consider that CO biological activity might depend on two main factors: period of

CO exposure and gas concentration, giving rise to distinct targets and responses.

Furthermore, different CO sources increase the system complexity and do not

facilitate data comparison. For instance, CO can be applied by different modes: (i)

one single burst of CO (CO-saturated solutions fast gas diffusion), (ii) gas exposure

(continuously application during the period of exposure), and (iii) CO-releasing

molecules (CORM’s). Depending on the used CORM and its specific molecular

characteristics, these molecules can be slow or fast CO releasers or can differently

Chapter VI

180

Ch

apte

r V

I

respond to tissues or to a physiological situation, such as an increase on oxidative

stress.

As explored in Chapter I, section 6.3., CORM’s are promising molecules,

increasing the potential of CO for clinical application, overcoming systemic toxicity

issues. The main disadvantages may be the blood-brain barrier crossing and the

metabolization of the chemical structure after the CO release.

Another possible way to use CO as PC agent is to induce its endogenous

production, by heme-oxygenase (HO) induction, especially HO-1, as mentioned in

section 6.1., in the Chapter I.

2. LOOKING AHEAD

This thesis presents several indications for CO potential therapeutic

applications. From subcellular to organ level, several questions were disclosed.

However, other questions appeared and several studies can be performed to depict

further the role of CO, in particular:

At subcellular level:

a) It is well known the CO’s affinity to heme proteins with transition

metals in the prosthetic group. Several studies indicates CO cellular

targets, however, CO molecular target remains an open and complex

subject to answer, since CO is a gas with transitory binding properties.

Reports have shown that CO binds to COX, soluble guanlyl cyclase

(sGC) and nitric oxidase synthase (NOS). Also, CO binds to globin

proteins as hemoglobin, myoglobin and neuroglobin. Globins, as COX,

are O2-binding heme-proteins, thus CO is competing directly with O2.

Thus, several studies can be performed addressing the question of CO

cellular target, as well as its dependence on the present O2

concentrations.

b) CO binding to the heme centre of neuroglobin leads to conformational

changes and cellular signalling (Nienhaus et al. 2007). Additionally,

neuroglobin may be able to reduce small amounts of cytochrome c


181

leaking from damaging mitochondria (Nienhaus and Nienhaus 2007).

Thus, CO influence on neuroglobin protective functions is an

interesting focus of interest.

c) As described in section 3.3., Chapter I, autophagy is now considered

to be a protective cellular pathway. Several cross-talks have been

described between apoptosis and autophagy. Due to CO’s anti-

apoptotic nature, CO effect on the apoptotic-autophagic balance

nodes may also be considered.

d) Mitochondria are the powerhouse of the cell and the gatekeepers for

cell balance between cell death and survival pathways. Although many

data have been reported in this thesis, more can be learnt from the

CO-targetting mitochondria effect. In particular, CO influence on

mitochondrial population and mitochondrial quality (mitochondrial

biogenesis vs autophagy of mitochondria – mitophagy).

At cellular level: the purinergic signalling presents several aspects that are still

unexplored. Complementary experiments regarding genetic evidences

concerning the receptor expression/activity and downstream events should be

performed. In addition, other described factors/pathways already known in

neuron-astroglia communication can also be studied, such as erythropoietin.

At organ level:

a) Different CO administration conditions can be tested (timings and

number of CO exposures, doses of CO inhaled).

b) A persuasive and attractive alternative to CO gas exposure is the

application of CORM’s. Although the existence of several publications

demonstrating CORM’s relevance, its application still need to be better

explored (concentrations, timing of administration and chosen CORM).

c) In addition, one may consider of future interest the exploitation of the

organ cross-tolerance. By applying CO in another organ than brain,

Chapter VI

182

Ch

apte

r V

I

brain tolerance can still be induced. This has the advantage of a more

controlled administration and overcomes the problem of the blood-

brain barrier permeability (important in the case of the CORM’s).

Another area of interest for future studies can be the evaluation of the

HO/endogenous CO axis. Focusing on, not only its induction to produce more CO to

exert beneficial outcomes, but also on the generation of scientific knowledge. The

elucidation of cellular and biochemical pathways can contribute for the potential

identification of new biomarkers in human samples of perinatal cerebral ischemia.

In a translational medical research perspective, HO activity and its importance

in perinatal cerebral ischemia can be explored by using human samples.

3. CONCLUSION

There is not just a unique pathway for the CO-induced endogenous protection;

brain tolerance is the result of a fundamental and complex cellular change in

response to injury, shifting the outcome to cell survival.

In conclusion, this thesis has proven that CO has potential as a novel

therapeutic agent. CO limits brain injury following cerebral ischemia, by stimulating

PC and targeting astrocytes and mitochondria.

Additionally, several cellular players were disclosed, together with the order and

time-window of the events (early vs late, Figure 6.1.). This knowledge has the

potential to be used in the development of novel clinical therapies and therefore have

a tremendous range of applications in the field of neuroprotection.

4. REFERENCES

Allaman, I., M. Belanger, et al. (2011). "Astrocyte-neuron metabolic relationships: for better and for worse." Trends Neurosci 34(2): 76-87.

Bonifacio, S. L., H. C. Glass, et al. (2011). "A new neurological focus in neonatal intensive care." Nat Rev Neurol 7(9): 485-94.

Burnstock, G. (2009). "Purinergic signalling: past, present and future." Braz J Med Biol Res 42(1): 3-8.


183

Burnstock, G. (2012). "Purinergic signalling: Its unpopular beginning, its acceptance and its exciting future." Bioessays 34(3): 218-25.


Correia, S. C., S. Cardoso, et al. (2011). "New insights into the mechanisms of mitochondrial preconditioning-triggered neuroprotection." Curr Pharm Des 17(31): 3381-9.

Cunha, R. A. (2005). "Neuroprotection by adenosine in the brain: From A(1) receptor activation to A (2A) receptor blockade." Purinergic Signal 1(2): 111-34.



Kitagawa, K. (2012). "Ischemic tolerance in the brain: endogenous adaptive machinery against ischemic stress." J Neurosci Res 90(5): 1043-54.

Lezi, E. and R. H. Swerdlow (2012). "Mitochondria in neurodegeneration." Adv Exp Med Biol 942: 269-86.

Nienhaus, K. and G. U. Nienhaus (2007). "Searching for neuroglobin's role in the brain." IUBMB Life 59(8-9): 490-7.

Pugliese, A. M., S. Latini, et al. (2003). "Brief, repeated, oxygen-glucose deprivation episodes protect neurotransmission from a longer ischemic episode in the in vitro hippocampus: role of adenosine receptors." Br J Pharmacol 140(2): 305-14.

Queiroga, C. S., A. S. Almeida, et al. (2012). "Carbon monoxide targeting mitochondria." Biochem Res Int 2012: 749845.

Robertson, C. L., S. Scafidi, et al. (2009). "Mitochondrial mechanisms of cell death and neuroprotection in pediatric ischemic and traumatic brain injury." Exp Neurol 218(2): 371-80.

Sola, A., Y. P. Saldeno, et al. (2008). "Clinical practices in neonatal oxygenation: where have we failed? What can we do?" J Perinatol 28 Suppl 1: S28-34.

Tang, Y., E. Pacary, et al. (2006). "Effect of hypoxic preconditioning on brain genomic response before and following ischemia in the adult mouse: identification of potential neuroprotective candidates for stroke." Neurobiol Dis 21(1): 18-28.

Thauerer, B., S. Zur Nedden, et al. (2012). "Purine nucleosides: endogenous neuroprotectants in hypoxic brain." J Neurochem 121(3): 329-42.

Chapter VI

184

Ch

apte

r V

I


Wintermark, P. (2011). "Current controversies in newer therapies to treat birth asphyxia." Int J Pediatr 2011: 848413.

Yenari, M. A. and H. S. Han (2012). "Neuroprotective mechanisms of hypothermia in brain ischaemia." Nat Rev Neurosci 13(4): 267-78.


Documents

By Cláudia S. F. Queiroga · iii Foreword The present thesis dissertation is the result of more than four years of research at the Animal Cell Technology Unit of Instituto de Tecnologia