INCREASE OF BASAL OXIDATIVE STRESS LEVELS AND …

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Theses and Dissertations--Chemistry Chemistry

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INCREASE OF BASAL OXIDATIVE STRESS LEVELS AND INCREASE OF BASAL OXIDATIVE STRESS LEVELS AND

IMPAIRMENT OF HEME OXYGENASE-1/BILIVERDIN REDUCTASE IMPAIRMENT OF HEME OXYGENASE-1/BILIVERDIN REDUCTASE

POST-TRANSLATIONAL MODIFICATION BY THE DEFECT OF POST-TRANSLATIONAL MODIFICATION BY THE DEFECT OF

PARKINSON-RELATED GENE OF PARKINSON-RELATED GENE OF PINK1

Zhaoshu Zhang University of Kentucky, [email protected]

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Zhaoshu Zhang, Student

Dr. D. Allan Butterfield, Major Professor

Dr. Dong-Sheng Yang, Director of Graduate Studies

INCREASE OF BASAL OXIDATIVE STRESS LEVELS AND IMPAIRMENT OF HEME

OXYGENASE-1/BILIVERDIN REDUCTASE POST-TRANSLATIONAL MODIFICATION

BY THE DEFECT OF PARKINSON-RELATED GENE OF PINK1

Copyright © Zhaoshu Zhang 2014

THESIS

A thesis submitted in partial fulfillment of the

requirements for the degree of Master of Science in the

College of Arts and Sciences at the University of Kentucky

By Zhaoshu Zhang

Lexington, Kentucky

Director: Dr. D. Allan Butterfield, Professor of Chemistry

Lexington, Kentucky

2014

ABSTRACT OF THESIS




Parkinson disease (PD) is the most common movement disorder and the second most common neurodegenerative disease. PINK1, PTEN-induced kinase 1, functions as a serine/threonine kinase as well as a protector of mitochondrial function. Mutations in PINK1 gene result in either mitochondria dysfunction or disruption of kinase signaling pathways involved in the pathogenesis of PD.

In this thesis, oxidative stress levels were examined in the brain of PINK1 knockout mice, and also how heme oxygenase-1 and biliverdin reductase are affected in brain of PINK1 knockout mice. In addition, posttranslational modifications are a way to control the behavior of proteins, so posttranslational modifications of the brain of PINK1 knockout mice, including both oxidative modification and phosphorylative modification, were examined.

Keywords: Parkinson disease (PD), PTEN-induced putative kinase 1 (PINK1),

Biliverdin reductase (BVR), Oxidative stress, Posttranslational modification

Zhaoshu Zhang

05/23/2014




By

Zhaoshu Zhang

Dr. D. Allan Butterfield

Director of Thesis

Dr. Dong-Sheng Yang

Director of Graduate

Studies

05/23/2014

Date

To my family

iii

ACKNOWLEDGMENTS

I would like to express my very appreciation to my advisor Dr. D. Allan

Butterfield for his guidance and encouragement during my courses study and

research work. He also provided a great support helping me pursue my life

dream. I also want to thank Rukhsana for all the suggestions and help she gave

me in regards to learning techniques, planning experiments, and interpreting data.

I would like to thank my committee members, Dr. James Geddes and Dr.

Yinan Wei, for choosing to serve on my committee, helping to strengthen my

thesis, and attending my defense. I would like to thank Dr. Büeler for providing all

the samples used in this thesis work.

In addition, I would like to extend my thanks to the past and current group

members, especially Jerry Keeney, Aaron Swomley, Judy Triplett, and Xiaojia

Ren, for their generous help, stimulating discussions, and all the fun we had in

the past three years.

Lastly, I want to thank my parents, for all the love and encouragement

over these years.

iv

TABLE OF CONTENTS

ACKNOWLEDGMENTS ....................................................................................... iii

LIST OF TABLES .................................................................................................vi

LIST OF FIGURES .............................................................................................. vii

Chapter 1. Introduction .................................................................................... 1

Chapter 2. Background ................................................................................... 3

2.1 Oxidative Stress .......................................................................................... 3

2.1.1 Overview of Oxidative Stress ............................................................... 3

2.1.2 Protein carbonylation ........................................................................... 5

2.1.3 Lipid peroxidation ................................................................................. 8

2.1.4 Protein nitration .................................................................................. 10

2.2 Parkinson Disease .................................................................................... 13

2.2.1 Parkinson Disease Overview ............................................................. 13

2.2.2 PINK1 Involvement in PD ................................................................... 14

2.3 Heme oxygenase ...................................................................................... 15

2.4 Biliverdin reductase .................................................................................. 16

2.4.1 Biliverdin reductase Regulation, and Reductase Activity ................... 16

2.4.2 Biliverdin reductase kinase activity, and the role in cell signaling ....... 17

2.5 Hypothesis ................................................................................................ 19

Chapter 3. Materials and Experimental Procedures ...................................... 20

3.1 Chemicals and Materials .......................................................................... 20

3.2 Animals ..................................................................................................... 20

3.3 Sample Preparation .................................................................................. 21

3.4 Bicinchoninic Acid (BCA) Protein Estimation Assay ................................. 21

3.5 Slot Blot Analysis Oxidative Stress ........................................................... 22

3.5.1 Slot Blot Technique ............................................................................ 22

3.6 One-Dimensional SDS-PAGE and Western Blotting ................................ 24

3.7 Identification of Post-translational Protein Modifications Using

Immunoprecipitation Approaches ....................................................................... 26

3.7.1 Identification of Protein Carbonyl Post-translational Modification ....... 29

3.8 Image analysis .......................................................................................... 29

3.9 Statistical analysis .................................................................................... 29

Chapter 4. Results ......................................................................................... 30

4.1 Oxidative stress levels in brain of PINK1 knockout mice compared to age-

matched control .................................................................................................. 30

4.2 Protein carbonyl levels increase in PINK1 knockout mice compared to age-

matched control .................................................................................................. 31

4.3 HNE-bound protein levels increase in brain of PINK1 knockout mice

compared to age-matched control ...................................................................... 32

v

4.4 Protein-3NT levels are unchanged in brain of PINK1 knockout mice


4.5 HO-1/BVR system levels in PINK1 knockout mice compared to age-

matched control .................................................................................................. 34

4.6 HO-1 levels decrease slightly, but not statistically significant in brain of

PINK1 knockout mice compared to age-matched control ................................... 35

4.7 BVR levels increase in brain of PINK1 knockout mice compared to age-

matched control .................................................................................................. 36

4.8 Levels of BVR oxidative post-translational modification in brain of PINK1

knockout mice compared to age-matched control .............................................. 37

4.9 Protein carbonyl levels on BVR levels do not change in brain of PINK1


4.10 Levels of HNE-bound BVR do not change in brain of PINK1 knockout mice


4.11 Levels of 3NT on BVR do not change in brain of PINK1 knockout mice


4.12 BVR phosphorylation post-translational modification levels in PINK1


4.13 BVR phosphoserine levels significantly increase in brain of PINK1


4.14 BVR phosphothreonine levels increase in brain of PINK1 knockout mice


Chapter 5. Discussion ................................................................................... 44

Chapter 6. Conclusion and Future Studies .................................................... 47

Appendix: Data to Supplement Figures .............................................................. 48

References ......................................................................................................... 58

Vita ..................................................................................................................... 63

vi

LIST OF TABLES

Table 2.1 Carbonyl groups modified amino acids ................................................. 6

Table 2.2 HNE adduct of Histidine, Lysine, and Cysteine .................................. 10

vii

LIST OF FIGURES

Figure 2.1 Cleavage of the peptide bond by diamide or α-amidation ................... 7

Figure 2.2 Oxidation of glutamyl residues to form protein carbonyl groups .......... 7

Figure 2.3 Formation of nitric oxide (NO.) by nitric oxide synthase (NOS) ........ 11

Figure 2.4 Formation of 3-nitrotyrosine (3-NT) ................................................... 12

Figure 2.5 Function of BVR as a kinase in the IR/IGF-1 signaling cascade ....... 18

Figure 3.1 Derivatization of protein carbonyls by DNPH .................................... 23

Figure 3.2 1D SDS-PAGE ................................................................................. 25

Figure 3.3 Immunoprecipitation procedure ......................................................... 28

Figure 4.1 Protein carbonyl level in PINK1 knockout mice compared to age-

matched control mice. ........................................................................................ 31

Figure 4.2 HNE-bound protein level in PINK1 knockout mice compared to age-


Figure 4.3 3-NT resident protein level in PINK1 knockout mice compared to age-


Figure 4.4 Heme Oxygenase-1 level in PINK1 knockout mice compared to age-


Figure 4.5 Biliverdin Reductase level in PINK1 knockout mice compared to age-


Figure 4.6 Protein carbonyl of BVR level in PINK1 knockout mice compared to

age-matched control mice. ................................................................................. 38

Figure 4.7 HNE-bound BVR level in PINK1 knockout mice compared to age-


Figure 4.8 3NT-bound BVR level in PINK1 knockout mice compared to age-


Figure 4.9 BVR phosphoserine level in PINK1 knockout mice compared to age-


Figure 4.10 BVR phosphothreonine level in PINK1 knockout mice compared to

age-matched control mice. ................................................................................. 43

1

Chapter 1. Introduction

The studies presented in this thesis gained insight into the role of oxidative

stress, levels of heme oxygenase-1/biliverdin reductase (HO-1/BVR) along with

post-translational modifications of BVR in the brain of PINK1 knockout mice to

explore possible pathogenic mechanisms of Parkinson Disease (PD). Currently,

there are about 6.3 million people worldwide suffered from PD, and this number

will increase to 8.7 to 9.3 million by 2030 according to the report from European

Parkinson’s Disease Association. Therefore, the study of PD associated gene,

PINK1, would be meaningful.

As we known, oxidative stress is an imbalance between the levels of

antioxidants and oxidants which contributes to the pathogenesis of

neurodegenerative diseases (Butterfield and Kanski 2001). Normally, antioxidant

system helps to neutralize oxidative insults. However, it could be not sufficient to

compensate the excess amount of oxidative stress when homeostasis in cells is

disrupted. Oxidative damage can be quantified by protein carbonylation, lipid

peroxidation, and protein nitration (Dalle-Donne, Rossi et al. 2003, Dalle-Donne,

Rossi et al. 2006). PINK1 is a serine/threonine protein kinase served as a

protector of mitochondria. Dysfunction of PINK1 protein disrupts mitochondrial

function and results in the formation of oxidative stress (Gautier, Kitada et al.

2008). The HO-1/BVR system plays a key role in the response to cell stress.

Heme oxygenase-1 (HO-1) is an enzyme that converts heme b to biliverdin.

Biliverdin subsequently is converted to bilirubin by biliverdin reductase (BVR).

Bilirubin is considered as a strong antioxidant protecting cells from oxidative

damage (Barone, Di Domenico et al. 2014).

In addition, oxidative translational modification of a specific protein shows

how it responds to oxidative stress, while phosphorylation translational

modification regulates the activity of this protein. This thesis also provides

evidence that the potential antioxidant characteristic of BVR keeps oxidative

modification of BVR back to normal level. On the other hand, increases of BVR

2

phosphoserine and phosphothreonine in the brain of PINK1 knockout mice

conceivably is a compensatory response to the elevated oxidative stress,

potentially activating the reductase function of BVR. Analysis of the PINK1

knockout brain will provide further understanding of PD.

To summarize, this thesis research has used the brain of PINK1 knockout

mice to achieve the following goals:

1. To provide an evidence that oxidative stress plays an important role in

PINK1 deficiency cells.

2. To verify the efficiency of HO-1/BVR antioxidant system in the cells

suffered from oxidative stress.

3. To determine the oxidative/nitrosative stress-induced modification and

phosphoserine and phosphothreonine of BVR.

3

Chapter 2. Background

2.1 Oxidative Stress

2.1.1 Overview of Oxidative Stress

Oxidative stress (OS) is defined as an imbalance between the level of

antioxidants and oxidants favoring the latter when physiological homeostasis is

disrupted. OS has been reported to play a prominent role in the pathogenesis of

neurodegenerative disorders including Parkinson’s disease (PD) (Jenner, Dexter

et al. 1992, Jenner 2003). Brain is one of the most vulnerable organs to oxidative

damage since it has a high consumption of oxygen, high number of mitochondria

that leak superoxide free radicals, and is rich in polyunsaturated fatty acids

(PUFAs) that can be attacked by free radicals easily. Moreover, brain

concentrates metal ions suggesting a strong possibility to contribute to oxidative

stress in age-related neurodegenerative diseases (Bush 2000). Excess iron

levels in the substantia nigra in PD leads to enhanced production of free radicals

and eventually cause oxidative events (Hirsch, Brandel et al. 1991). Low level of

antioxidants in brain is another reason to cause oxidative damage in brain. In PD,

reduced glutathione (GSH) levels are decreased while oxidants make more

negative effects on brain (Sofic, Lange et al. 1992).

Within cells oxidative damage to proteins, lipids and DNA is caused by the

excess production of oxidants even though they serve as second messengers

and activators of signaling cascades with a basal level (Suzuki, Forman et al.

1997, Goldstein, Mahadev et al. 2005). Oxidants can be either free radical or

non-radical molecules and either reactive oxygen species (ROS) or reactive

nitrogen species (RNS). In general, the majority of ROS/RNS are derived from

superoxide anions (O2.-), which are the products of O2 and electron by

transferring electron leaked from the ETC to ground state O2 (reaction 1)

(Turrens 2003). However, ROS/RNS also come from other sources, such as

environmental toxins, ultraviolet light, and cellular processes (Mena, Ortega et al.

2009).

4

O2 + e- O2.- (reaction 1)

Furthermore, O2.- readily reacts with other reactive species when

antioxidants are deficient to produce more detrimental ROS/RNS, such as

hydroxyl (OH.), hydrogen peroxide (H2O2), hypochlorous acid (HOCl) and

nitrogen dioxide (NO2.). Some of superoxide anions can be dismutated to

hydrogen peroxide (H2O2) by superoxide dismutase (SOD) (reaction 2).

Subsequently, newly formed H2O2 will react with excess superoxide anion

(reaction 3). It is noteworthy to indicate that the partially reduced product, OH., is

the most reactive and unstable form of ROS which can also be generated by the

other two pathways (Turrens 2003). One of which is via Fenton reaction using

reduced metal ions, such as iron and copper, released by some metal-binding

proteins. (reaction 4-5) (Berlett and Stadtman 1997, Barnham, Masters et al.

2004).

O2.- SOD H2O2 + H2O (reaction 2)

2H2O2 + 2O2.- OH- + O2 + OH. (reaction 3)

H2O2 + Fe2+ OH- + Fe3+ + OH. (reaction 4)

H2O2 + Cu+ OH- + Cu2+ + OH. (reaction 5)

The third pathway of OH. formation is through the presence of nitric oxide

(NO.) and the hemolysis of ONOOH. The product of reaction 6, peroxynitrite

(ONOO-), is another strong oxidant. This is also a pathway to generate some

prominent RNS (reaction 6-8) (Radi 2004, Goldstein, Lind et al. 2005). A more

detailed mechanism for reaction of NO. with O2.- is shown in section 2.1.4.

NO. + O2.- ONOO- (reaction 6)

ONOO- + H+ ONOOH (reaction 7)

ONOOH NO2. + OH. (reaction 8)

OH., as a secondary oxidizing species, is quite damaging. It is able to

interact with almost any other free radical or nonradical species to cause post

5

translational modifications (PTM) such as protein oxidation, lipid peroxidation. At

the same time, the other product, NO2., is involved in protein nitration. Thereby,

oxidative damage has been amplified. In addition, when NO. is consumed to form

ONOO-, NO. is depleted. However, NO. is important to many physiological

processes including, among others, vasodilation and neurotransmission (Belvisi,

Stretton et al. 1992, Steinberg, Brechtel et al. 1994). As we can see, cells

undergo oxidative stress even if slight overabundance of ROS/RNS. The results

of oxidative damage usually cause protein structural alteration, enzyme inhibition,

and the impairment of regulatory functions.

2.1.2 Protein carbonylation

The presence of protein carbonyl groups in brain proteins is one of the

most common features in neurodegenerative disease. Because protein carbonyl

groups form in early stages of these disorders and are relative stable, these are

commonly used as an index of oxidative stress. There are five pathways to

generate protein carbonyl groups. Carbonyl derivatives are firstly yielded by

oxidation of the side-chains of several amino acids especially for proline, arginine,

lysine, and threonine residues (Table 2.1) (Dalle-Donne, Rossi et al. 2003).

Cleavage of the peptide bond by either diamide or α-amidation pathways is the

second way to form CO double bonds (Figure 2.1) (Berlett and Stadtman 1997,

Stadtman and Levine 2003). Protein carbonyl groups can be generated through

oxidation of glutamyl side chains (Figure 2.2) (Berlett and Stadtman 1997,

Stadtman and Levine 2003). Protein carbonyls can also react for processes of

glycoxidation (Stadtman and Levine 2003). Lastly, protein carbonyl groups result

from protein addition by a product of lipid peroxidation, HNE, (discussed later).

6

Table 2.1 Carbonyl groups modified amino acids

Amino acid Carbonyl derivatives Structure

Proline 2-Pyrrolidone

Arginine Glutamic semialdehyde

Lysine Aminoadipic semialdehyde

Threonine 2-amino-3-keto butyric acid

7

Figure 2.1 Cleavage of the peptide bond by diamide or α-amidation

Figure 2.2 Oxidation of glutamyl residues to form protein carbonyl groups

8

As an oxidative damage, protein carboxylation disrupts tertiary structure of

protein leading to unfolding or conformational changes. The amino acid on

protein become more polar with the introduction of protein carbonyl groups,

thereby exposing hydrophobic residues to the aqueous environment and losing

functions (Dalle-Donne, Aldini et al. 2006). Increased protein carboxylation,

coupled with less efficiency of proteolysis may result in the aggregates of

misfolded proteins associated diseases including PD. Protein carbonyl levels can

be quantified by immunohischemistry after being derivatized with 2,4-

dinitrophenylhydrazine (DNPH) to form the protein hydrazone (Dalle-Donne,

Rossi et al. 2003).

2.1.3 Lipid peroxidation

Lipids are the most common components of biological membrane. Lipids

play an important role in fluidity, flexibility, and selective permeability. Even small

changes of lipid may greatly alter the physiological properties of the membrane,

contributing to the pathology of diseases. Lipid peroxidation is one of the

outcomes of oxidative stress and is a source to spread and amplify free radical

reaction (Gutteridge 1995). Among all kinds of lipids, phospholipid is the most

susceptible one to undergo this process not only because of high level of

polyunsaturated fatty acids (PUFAs) but also because of a relative easier

accessibility to free radicals. Generally, lipid peroxidation consists of three stages:

initiation, propagation, and termination. In the initiation stage, allylic hydrogen on

acyl chains of phospholipids is easily abstracted by free radicals such as OH.,

RO., ROO. to form a carbon center lipid radical (L.). The lipid radical

subsequently reacts with oxygen, producing a lipid peroxyl radical (LOO.). This

radical is able to abstract another allylic hydrogen from a different PUFA to

produce a lipid hydroperoxide (LOOH) and a new lipid radical (L.), which

continuously reacts with oxygen molecular. This process is a chain reaction

called propagation. As a result, more and more LOOH are formed. In the

presence of reduced metal ions, LOOH can be reduced to phospholipid alkoxyl

radical (LO.), and then break down to reactive aldehyde products. When the

9

concentration of lipid radical is high enough, the chain reaction stops by collision

of two radicals (reactions 9-12) (Catala 2006, Catala 2009). This complex

process modifies physical properties of membrane function including change in

membrane fluidity, membrane permeability, and membrane protein activity.

LH + X. L. + XH (reaction 9)

L. + O2 LOO. (reaction 10)

LOO. + LH LOOH + L. (reaction 11)

LOO. + LOO. Non-radical + O2 (reaction 12)

4-Hydroxynonenal (HNE), a major aldehyde product of lipid peroxidation,

has been recognized as an indicator of lipid peroxidation. HNE is involved in

apoptosis, damage of proteasomal function, and the alteration of signal

transduction, contributing to neurodegenerative diseases (Kruman, BruceKeller

et al. 1997, Ferrington and Kapphahn 2004). The γ carbon of aldehyde group is

electron deficient so that be able to react with nucleophilic groups such as

sulfhydryl groups, imidazole groups, and amino groups. Therefore, HNE reacts

with cysteine, histidine, and lysine by Michael addition to produce reactive

aldehyde derivatives (Table 2.2) (Esterbauer, Schaur et al. 1991). This is another

way to generate protein carbohydrate derivatives. Protein polarities and

structures are altered because of the products of lipid peroxidation. As a

consequence, this whole process alters lipid-lipid and lipid-protein interactions as

well as membrane functions.

10

Table 2.2 HNE adduct of Histidine, Lysine, and Cysteine

Amino Acid HNE Protein–alkenal Adducts

Histidine

Lysine

Cysteine

2.1.4 Protein nitration

Similar to ROS, protein nitration caused by toxic RNS leads to oxidative

damage to proteins as well. Nitric oxide (NO.) is regarded as an initial source of

this process, which is able to produce other relative reactive intermediates.

Normally, NO. is involved in cell signaling, vasodilation, and immune response

(Drew and Leeuwenburgh 2002). However, excess nitric oxide can participate as

a cytotoxin in cellular. NO. is formed by nitric oxide synthase (NOS) family. NOS

catalyzes the conversion of L-arginine to L-citrulline and forms NO. as a

byproduct (Figure 2.3). The NOS family insists of three distinct isoforms: Ca2+-

11

dependent neuronal NOS (nNOS), Ca2+-dependent endothelial NOS (eNOS),

and Ca2+-independent inducible NOS (iNOS), which play different roles in

different organs (Alderton, Cooper et al. 2001, Drew and Leeuwenburgh 2002).

Figure 2.3 Formation of nitric oxide (NO.) by nitric oxide synthase (NOS)

Mostly, nitric oxide mediates protein nitration on two aspects. It also

triggers S-nitrosylation of reduced cysteine residues. Oxygen that is required in

the S-nitrosylation is converted to superoxide (Gow, Buerk et al. 1997,

Broniowska and Hogg 2012). Most of superoxide in vivo is dismutated by SOD.

However, the reaction of HO-O. with NO. has a larger rate constant than with

SOD. Therefore, NO. competes SOD for HO-O. to form peroxynitrite and nitrogen

dioxide (reaction 13-16) (Radi 2004).

Protein-SH + NO. Protein-S-N.-OH (reaction 13)

Protein-S-N.-OH + O2 Protein-S-NO + HO-O. (reaction 14)

HO-O. SOD H2O2 + O2 (reaction 15)

HO-O. + NO. ONOOH NO2. + OH. (reaction 16)

The most pronounced modification caused by nitric oxide is the formation

of 3-nitrotyrosine (3-NT). Nitric oxide is first converted to a very reactive oxidant

peroxynitrite anion (ONOO-). The latter has been observed to react with CO2 to

form ONOOCOO-, which subsequently breaks down into nitrogen dioxide (NO2.)

and carbonate radical (CO3.-) (reaction 17-18). With the presence of CO3

.-, tyrosyl

radical is generated by losing an electron from tyrosine. Ultimately, NO2. attaches

to tyrosyl radical to yield 3-NT (Figure 2.4). This is a radical-radical reaction,

12

peroxynitrite itself cannot react with tyrosine directly. Site specific nitration of

tyrosine causes steric restriction so that changes the function of protein by

lowering pKa of hydroxyl group and providing steric hindrance, thereby inhibiting

tyrosine phosphorylation (Gow, Duran et al. 1996, Di Stasi, Mallozzi et al. 1999,

Radi 2004). Nitrated mitochondrial proteins contribute to oxidative damage.

Therefore, it is important to measure 3-NT level as an index to evaluate protein

nitration.

NO. + O2.- ONOO- (reaction 17)

ONOO- + CO2 ONOOCOO- NO2. + CO3

. (reaction 18)

Figure 2.4 Formation of 3-nitrotyrosine (3-NT)

13

2.2 Parkinson Disease

2.2.1 Parkinson Disease Overview

PD is the second most common neurodegenerative disease after

Alzheimer disease and the most common movement disorder (Giasson, Duda et

al. 2002, Maragakis and Rothstein 2006). PD is characterized by progressive

loss of dopaminergic neurons in the substantia nigra. The clinical symptoms of

PD include muscle rigidity, tremor, a slowing of physical movement

(bradykinesia), even a loss of physical movement (akinesia) (Fahn 2003,

Thomas and Beal 2007). PD usually affects people over the age of 50 with a

current estimation of 1.5 million in the United States. PD can be either sporadic

or familial. Although most cases of PD are the sporadic form, studies of genes

linked to rare familial disease help people better understand the mechanism of

PD and provide the targets for therapeutic intervention.

Several genes, including α-synuclein, parkin, DJ-1, UCHL-1, PINK1 and

LRRK2, have been clearly identified to be associated with PD in both sporadic

and familial forms. All these five genes play prominent roles in protecting neurons

from mitochondrial dysfunction, oxidative damage, abnormal protein

accumulation and protein phosphorylation (Thomas and Beal 2007). α-synuclein

is a presynaptic protein to play an important role in neurotransmission (Yavich,

Tanila et al. 2004). Mutations or oxidation of α-synuclein enhance α-synuclein

aggregation. The abnormal protein aggregation, termed Lewy bodies (LBs), is

the most typical hallmark of PD brain (Spillantini, Crowther et al. 1998). Parkin

functions as an E3 ubiquitin protein ligase catalyzing transfer of ubiquitin to

cellular proteins for subsequent proteolysis and protecting dopamine neurons

(Walden and Martinez-Torres 2012). DJ-1, known as an antioxidant, is able to

serve as a scavenger of reactive oxygen species (ROS) and an activator of

glutamyl cysteine ligase. Moreover, α-synuclein aggregation and subsequent

death can be blocked by DJ-1 (Shendelman, Jonason et al. 2004, Zhou and

Freed 2005). UCHL-1 plays an important role in the ubiquitin proteasome

pathway. It helps to hydrolyze peptide ubiquitin bonds and generate ubiquitin

14

monomers for re-use. The I93M mutation of UCHL-1 was first discovered to

cause PD, whereas S18Y may reduce risk for PD by inhibiting ligase activity

(Healy, Abou-Sleiman et al. 2004). Both PINK1 and LRRK2 are mitochondrial

serine/threonine protein kinases, protecting mitochondrial function. The

mitochondrion, a critical target in PD, is impaired by mutations of any of above

genes, which might cause dysfunction in maintenance and survival of

dopaminergic neurons (Beal 2007).

Disrupted mitochondrial function is likely to decrease oxidative

phosphorylation and decrease complex I activity in PD, resulting in ROS

formation, oxidative stress, and loss of ATP production. Increased oxidative

stress can induce overload function of the ubiquitin proteasomal system (UPS),

ultimately leading to irreversible cellular damage and death. In parallel, loss of

the mitochondrial membrane potential occurs. This leads to opening of the

mitochondrial permeability transition pore (mPTP), releasing of cytochrome c,

and activation of mitochondrial-dependent apoptosis (Abeliovich 2007). Growing

evidence shows that mitochondrial deficiency and oxidative stress play prominent

roles in PD pathogenesis.

2.2.2 PINK1 Involvement in PD

PINK1 (PARK6), PTEN-induced kinase 1, was identified in 2004 as the

second most genetic cause of recessively inherited early-onset parkinsonism

(Gandhi, Muqit et al. 2006, Ibanez, Lesage et al. 2006). PINK1 functions as a

serine/threonine kinase as well as a protector of mitochondrial function. The C-

terminal domain of PINK1 mediates kinase function while the N-terminal

mitochondrial targeting motif helps to maintain mitochondrial function and

morphology. PINK1 is localized in the mitochondria membrane, can be found

throughout the body, and has a function in the whole brain not only in the

substantia nigra (Gandhi, Muqit et al. 2006). In brief, PINK1 protects neurons in

four pathways. Firstly, PINK1 phosphorylates TRAP1. Phosphorylated TRAP1 in

turn blocks generation of mitochondrial ROS and prevents release of cytochrome

15

c from the mitochondrial intermembrane space into the cytoplasm (Plun-Favreau

and Hardy 2008). Secondly, PINK1 enhances phosphorylation of the

mitochondrial protease HtrA2. Activated HtrA2 then increases protease activity

and enhances resistance of cells to mitochondrial stress. Both these pathways

demonstrate a role for the kinase in maintenance of mitochondrial function and

prevention of stress-induced apoptosis. Thirdly, PINK1 is also able to interact

with Parkin and protect the mitochondria by either PI3-kinase/Akt pathway or

IKK/NF-κB signaling pathway. Parkin acts downstream of PINK1 indicating that

two genes might share a conserved genetic pathway. Overexpression of Parkin

can protect against the effect of PINK1 mutation. Lastly, PINK1 enhances the

activity of anti-apoptotic protein Bcl-2 to reduce apoptosis (Abeliovich 2007, Plun-

Favreau, Klupsch et al. 2007, Mills, Sim et al. 2008). Mutations in PINK1 gene

result in either mitochondria dysfunction or disruption of kinase signaling

pathways involved in the pathogenesis of PD. PINK1-deficient cells trigger

intrinsic mitochondrial apoptosis as stated in the last section. Mutations of PINK1

also alter mitochondrial integrity and proliferation. Proliferation of mitochondria

may occur to compensate for dysfunctional mitochondria induced by oxidative

stress to produce more ATP needed for cell survival. However increased

numbers of mitochondria will in turn cause extra ROS, which means more

oxidative damage to cells (Wood-Kaczmar, Gandhi et al. 2008). In the current

thesis research, PINK1 knockout mice were used as a model to investigate PD.

2.3 Heme oxygenase

Heme oxygenase (HO) is an enzyme responsible for degradation of heme

b to biliverdin, ferrous ion, and carbon monoxide (CO) (reaction 19). Heme b

comes from heme-containing proteins such as hemoglobin, myoglobin,

cyclooxygenase, etc. Biliverdin is a potential antioxidant which is immediately

converted to bilirubin by biliverdin reductase. Bilirubin has been known as a

powerful antioxidant. On the other hand, ferrous ion and CO would be toxic at

high concentrations. Accumulation of ferrous ion would lead to Fenton chemistry

16

as mentioned in Section 2.1.1, even though some of ferrous ion can be

sequestered by ferritin. Therefore, excess of ferrous ion and CO may contribute

the pathology of Parkinson disease. Generally, heme oxygenase has two

isoforms: Heme oxygenase-1 (HO-1) and Heme oxygenase-2 (HO-2). HO-1 is an

inducible isoform that is upregulated by transcription factors in response to

inducers. Stress inducers can be numerous, such as oxidants, heavy metals,

hormones, etc. HO-2 is a constitutive isoform that is usually expressed at a low

level under physiological conditions. In addition, HO-1 has been observed to be

beneficial for learning ability (Chen, Huang et al. 2013).

(reaction 19)

2.4 Biliverdin reductase

2.4.1 Biliverdin reductase Regulation, and Reductase Activity

As mentioned above, biliverdin reductase (BVR) firstly was recognized as

a reductase that converts biliverdin to bilirubin (reaction 20). NADPH participates

this reaction under basic pH, whereas NADH is used under acidic conditions.

Bilirubin is the final product from heme degradation which is an antioxidant

capable of ROS scavenge. As a response to the increase level of heme

degradation and HO-1 expression, the conversion of biliverdin to bilirubin would

increase subsequently. However, organisms are able to balance these changes.

Extra bilirubin inhibits BVR activity to accumulate biliverdin. The negative

feedback inhibits HO-1 activity, leading HO-1/BVR system to return to normal

(Kutty and Maines 1981, Kutty and Maines 1984).

(reaction 20)

17

2.4.2 Biliverdin reductase kinase activity, and the role in cell signaling

Biliverdin reductase recently has been also identified as one of a rare

group of dual-specificity kinases that has the ability to autophosphorylate, or

phosphorylate serine/threonine and tyrosine residues (Hunter and Cooper 1985).

Different mammalian species share more than eighty percent of BVR gene

sequence (Maines 2005). Traditionally, the sequence consists of catalytic domain

and regulatory domain. In the recent years, human biliverdin reductase (hBVR)

has been studied extensively. BVR is identified to have potential functions in the

insulin signaling pathway. Insulin signaling pathway starts with insulin receptor

(IR) which is a protein tyrosine kinase consisting of α-subunit and β-subunit

connected by disulfide bonds. The α subunit has a binding site to insulin or

insulin growth factor 1 (IGF-1). Insulin/IGF-1 attaches to the α-subunit of IR and

tyrosine phosphorylation of β-subunit activates IR. BVR is activated by

phosphorylation. In the insulin signaling pathway, BVR is upregulated by insulin

receptor tyrosine kinase and negatively affects glucose uptake. The subsequent

phosphorylated BVR effectively competes with insulin receptor kinase for

serine/threonine phosphorylation of insulin receptor substrate 1 (IRS-1) (Lerner-

Marmarosh, Shen et al. 2005, Maines 2005). Then two major arms of signaling

pathways are activated: mitogen-activated protein kinase (MAPK) and PI3K

cascades (Figure 2.5). Except for kinase acitivity, hBVR also functions as a

transcriptional factor in insulin signaling pathway (Maines 2007).

18

Figure 2.5 Function of BVR as a kinase in the IR/IGF-1 signaling cascade

19

2.5 Hypothesis

Since oxidative stress contributes to neurodegenerative disease and

PINK1 knockout mice is a model of Parkinson’s disease, there will be an

increase level of oxidative stress observed in the brain of PINK1 knockout mice.

The increased level of oxidative stress is able to induce overexpression of heme

oxygenase 1 and biliverdin reductase with aid of the transcription factor Nrf2.

With the enhanced expression of BVR, increased level of biliverdin will result

which scavenges ROS/RNS. Therefore, the oxidative posttranslational

modification of BVR can return to normal. Additionally, biliverdin reductase

phosphoserine and phosphothreonine in the brain of PINK1 knockout mice may

be increased responding to the elevated oxidative stress.

20

Chapter 3. Materials and Experimental Procedures

3.1 Chemicals and Materials

2, 4-Dinitrophenylhydrazine (DNPH), primary Rb x DNP antibody, re-blot

plus strong antibody stripping solution were purchased from Chemicon

(Temecula, CA). Protease inhibitors, primary anti-biliverdin reductase and anti-

nitrotyrosine antibodies, secondary anti-rabbit IgG alkaline phosphatase and anti-

tubulin antibodies were purchased from Sigma-Aldrich (St. Louis, MO). Primary

anti-HNE antibody was purchased from Alpha Diagnostic (San Antonio, TX).

Primary anti-phosphothreonine, anti-phosphoserine antibodies were obtained

from Invitrogen (Camarillo, CA). Secondary anti-rabbit IgG horseradish

peroxidase, cydye3 and cydye5 were purchased from GE healthcare (Pittsburgh,

PA). BCA protein estimation assay kit, BCIP/NBT color development substrate

(5-bromo-4-chloro-3-indolyl-phosphate/nitro blue tetrazolium) were purchased

from Pierce (Rockford, IL). Polyacrylamide gels, Precision plus proteinTM all blue

standards, XT MES electrophoresis running buffer, nitrocellulose membranes,

and ClarityTM western ECL substrate were got from Bio-Rad (Hercules, CA).

3.2 Animals

In this work, whole brain samples of PINK1 knockout mice (male, n=6) and

age-matched control mice (male, n=6) were obtained from our collaborator, Dr.

Hans Büeler, University of Kentucky. All mice were mixed background (C57BL/6

x 129/Sv). Generation of PINK1 knockout mice was described in a previous

publication (Akundi, Huang et al. 2011). Knockout and control mice were

euthanized at six months old. Brain tissues were isolated immediately and stored

at -80°C until use.

21

3.3 Sample Preparation

Brain samples were homogenized using a glass mortar and pestle in

isolation buffer (pH 7.4, 300-500 µL for each sample) containing 0.32M sucrose,

2mM EDTA, 2mM EGTA, 20mM HEPES, 0.2mM PMSF, leupeptin (4µg/ml),

pepstatin (4µg/ml), aprotinin (5µg/ml), and phosphatase inhibitor cocktail (1:100).

After homogenization, tissues were transferred into a set of Eppendorf tubes.

Aliquot samples (200 µL) to another Eppendorf tube and each sample was

sonicated with a Fisher 550 sonic Dismembrator (Fischer Scientific, Pittsburgh,

PA) for 20s at 20% power. Then another 100 µL of isolation buffer was added.

Samples were centrifuged 10 min at 3000rpm, 4°C using a Hettich Mikro 22R

Microcentrifuge (Hettich, Beverly, MA) and all the supernatants were collected for

one-dimensional SDS-PAGE processing. Supernatants (100 µL) were transferred

into another set of tubes following the addition of 200 µL of isolation buffer, and

the tissues were saved for slot blotting. Sample concentrations were determined

by the Bicinchoninic Acid (BCA) protein estimation assay. All the samples were

stored at -80°C.

3.4 Bicinchoninic Acid (BCA) Protein Estimation Assay

All the protein concentrations in this study were determined using the BCA

protein estimation assay. This assay couples the reduction of Cu2+ to Cu1+ by

protein in an alkaline medium with the colorimetric detection of Cu1+ by BCA. The

BCA assay reagent is the mixture of reagent A (sodium carbonate, sodium

bicarbonate, bicinchoninic acid and sodium tartrate in 0.1 M sodium hydroxide)

and reagent B (4% cupric sulfate pentahydrate) in the ratio (49:1) freshly made

before the experiment. In the former reaction, which known as the Biuret reaction,

Cu2+ ions from reagent B are reduced by the presence of four amino acids

(cysteine, cystine, tryptophan and tyrosine) and peptide bonds (reaction 1). The

resultant Cu1+ ions subsequently react with two BCA molecules from reagent A to

generate purple colored complexes which exhibit a strong linear absorbance at

22

562 nm (reaction 2). Protein concentration is directly proportional to the intensity

of absorbance.

Proteins (peptide bonds & amino acids) + Cu2+ Cu1+ (reaction 1)

Cu1+ + 2 Bicinchoninic Acid (BCA) BCA-Cu1+ complex (purple-colored)

(reaction 2)

Each sample (2 µL) was pipetted along with in duplicate gradient

concentrations of standards, bovine serum albumin (BSA) into a 96-well

microplate (Evergreen Scientific, Los Angeles, CA); samples and standards were

diluted with the BCA assay reagent to make a final volume to 100 µL in each well;

the microplate was incubated for 15 min at 37 °C and gently rocked before

reading the absorbance in a μ-Quant UV plate reader (Bio-Tek, Winooski, VT).

Finally, the protein concentration follows was calculated using the Lambert-Beer

law.

3.5 Slot Blot Analysis Oxidative Stress

3.5.1 Slot Blot Technique

Slot blot, a microfraction of the dot blot technique, is a useful tool for the

detection of global levels of a protein modification such as protein carbonyl (PC)

or 3-nitrotyrosine (3-NT) with very small amount of samples required (< 1 µg).

Protein samples firstly are dissolved in SDS containing buffer, whereas PC later

needs to be incubated with DNPH before loading in duplicate on the

nitrocellulose membrane under vacuum. SDS imparts negative charges to

proteins. Therefore, positively charged nitrocellulose membrane is able to bind

proteins, but any uncharged materials will pass through the pores of

nitrocellulose membrane. The membrane is removed from the apparatus

followed by blocking with BSA and probing with specific primary and secondary

antibodies corresponding to a specific protein modification. With the colorimetric

development, spots are visualized and can be quantitated based on the intensity

using Image Lab software.

23

3.5.1.1 Detection of Protein-Bound Carbonyls

Protein carbonyls are a global index of protein oxidation. Carbonyl groups

of protein except for the primary chain peptide bond carbonyls are derivatized by

DNPH to form hydrazones (Figure 3.1). The protein-bound 2,4-

dinitrophenylhydrazone can be attached to the primary antibody reorganizing the

hydrazone adduct. A secondary antibody coupled to alkaline phosphatase is

attached to the primary antibody, and the amount of primary antibody bound is

finally detected by BCIP/NBT color development substrate (5-bromo-4-chloro-3-

indolyl-phosphate/nitro blue tetrazolium).

Figure 3.1 Derivatization of protein carbonyls by DNPH

Each brain sample (5 µL) was derivatized at room temperature by

incubation with 5 µL of 12% SDS and 10 µL of 10mM DNPH solution in 2N HCl

for 20 min. The samples were then neutralized by adding 7.5 µL of neutralization

buffer. Derivatized samples were diluted with PBS to make a final concentration

of 1 µg/mL. Diluted samples (250 µL/well) were loaded in duplicate onto a

nitrocellulose membrane under vacuum pressure. Membrane was then incubated

in a blocking solution containing 750 mg BSA per 25 mL wash blot (35.2g NaCl,

1.77g NaH2PO4, 9.61g Na2HPO4, 1.5mL TWEEN, diluted to 4L with DI water) for

one and a half h to prevent non-specific binding of antibodies. Next the

membrane was incubated with a 1:100 dilution of rabbit polyclonal anti-DNP

primary antibody for 2 h. Blot was then rinsed three times, 5 min each in wash

blot, and subsequently incubated with 1:8000 dilution of anti-rabbit IgG-alkaline

24

phosphatase secondary antibody for exactly 1 h. After another three times, 5, 10,

10 min, respectively, washes, the membrane was colorimetrically developed and

dried overnight. The membrane was scanned on the second day using

ChemiDocTM MP Imaging System (Bio-Rad, Hercules, CA) and the bands were

quantified using Image Lab software.

3.6 One-Dimensional SDS-PAGE and Western Blotting

One-dimensional sodium dodecyl sulfate polyacrylamide gel

electrophoresis (1D SDS-PAGE) is used to separate proteins based on their size,

shape and molecular weight. Prior to running the gel, 75 µg of each sample were

suspended in DI water and 4X SDS-sample buffer (5 µL) composed of 0.5 M Tris

(pH 6.8), 40% glycerol, 8% SDS, 20% β-mercaptoethanol, and 0.01%

bromophenol blue to make final volume 25 µL. SDS is able to coat proteins and

impart negative charges on proteins so that samples can migrate from the

negatively charged anode to the positively charged cathode under the influence

of a current. Samples were then denatured in boiling water for 5 min and cooled

immediately on ice before electrophoresis. Precision Plus Protein TM All Blue

Standards (5 µL) and the prepared samples were loaded in separate lanes on

12% Bis-Tris polyacrylamide gel using 1X XT MES as running buffer. The electric

field was initially set at 80 V for the first 8 min and increased to 120 V until the

blue line reached the bottom of the gel. Sample proteins were separated through

gel electrophoresis. The relative migration rate (Mr) depends on the resistance of

gel. Generally, larger proteins have higher resistance with lower Mr compared to

smaller proteins, though shape (i.e. cylindrical shaped) proteins migrate slower

than similar MW proteins of spherical shape.

25

Figure 3.2 1D SDS-PAGE

Western blotting is used to immobilize proteins onto the nitrocellulose

membranes. The membranes can be easily incubated with antibodies of interests

to measure specific proteins or post-translational modifications of specific

proteins. In this study, this technique has been extensively used for the detection

of HNE-bound protein, protein-resident 3-NT, the expression of HO-1 and BVR,

and oxidative or phosphorylation post-translational modifications of BVR.

Through running a semi-dry Transfer Cell System (BioRad, Hercules, CA) at 1.0

A for 30 min, negatively charged proteins were transferred from the gel to the

positively charged nitrocellulose membrane. During transfer, the gel sandwich

was made from two thick filter papers, a nitrocellulose membrane, and the gel

was submerged in transfer buffer containing Tris-HCl, glycine, and methanol.

Methanol is present to prevent the gel from swelling. The membrane was then

incubated in a blocking solution for one and a half hours. Protein levels were

evaluated via incubating with primary antibodies of interest overnight. After three

5 min washes, samples were incubated with secondary antibodies for exactly 1 h.

The membrane was washed three times again, and finally developed using

26

immunofluorescence or chemiluminescent techniques. The intensities of bands

were quantified by Image Lab analysis software.

1:1000 dilutions of primary anti-heme oxygenase-1 and anti-biliverdin

reductase antibodies, 1:5000 dilution of primary anti-HNE polyclonal antibody,

and 1:2000 dilution of rabbit polyclonal anti-3-NT primary antibody were used to

measure HO-1, BVR, protein-bound HNE, and protein-resident 3-NT levels,

respectively. Because immunofluorescence development was adopted, the blots

were then incubated with Cy5 anti-rabbit secondary antibody for 1 h in dark. For

loading controls, the blots were probed with primary anti-Tubulin antibody and

secondary Cy3 anti-mouse antibody.

3.7 Identification of Post-translational Protein Modifications Using

Immunoprecipitation Approaches

Immunoprecipitation (IP) is the technique used in to isolate a particular

protein from samples and quantify post-translational modifications of this

particular protein. The first step of IP is to incubate 250 µg of protein with the IP

buffer (0.05% NP-40, 4 µg/mL leupeptin, 4 µg/mL pepstatin, and 5 µg/mL

aprotinin in PBS; pH 8) shaking 30 min at 4 °C. In order to prevent non-specific

binding, samples were pre-cleaned with washed protein A/G-agarose beads (50

µL per sample) for 1 h at 4 °C. This procedure leas to removal of IgGs in the

samples. Supernatants were transferred to clean Eppendorf tubes after a 5 min

centrifugation in a Hettich Mikro 22 R microcentrifuge (Hettich, Beverly, MA) at

2500 rpm, 4 °C. Samples were incubated overnight with the antibody of interest.

In this study, anti-biliverdin reductase antibody was used to isolate biliverdin

reductase. Insoluble antigen-antibody-protein A/G complex was formed by the

addition of washed protein A/G-agarose beads and incubating for 1 h at 4 °C.

Subsequently the immunocomplex was centrifuged and the pellet was washed

five times with IP buffer (500 µL each time). Continuous shaking was used during

all incubation and washing steps. The pellet was suspended in DI water (20 µL)

27

and 4X SDS-sample buffer (5 µL) followed by denaturation in boiling water for 5

min and cooled immediately on ice. The supernatants containing a particular

protein were ready for 1D-PAGE and Western blotting (see sections 3.6).

As described above, the nitrocellulose membrane was incubated with

primary anti-3NT and anti-HNE antibodies, respectively, followed by the

incubation of anti-rabbit IgG alkaline phosphatase secondary antibody to detect

the levels of specific protein oxidative modifications. Blots were developed with

BCIP/NBT color development substrate.

For analysis of phosphoserine and phospohthreonine beads, the

nitrocellulose membrane was incubated with primary anti-phosphoserine and

anti-phosphothreonine antibodies, respectively, followed by the incubation of

secondary anti-rabbit IgG horseradish peroxidase to assess the levels of specific

protein phosphorylation modifications. Blots were developed by

chemiluminescence techniques using ClarityTM Western ECL substrate. For

loading controls, the blots were first stripped by stripping solution. After washing

to remove the stripping solution, the blots were probed with primary anti-biliverdin

reductase antibody.

28

① Samples in IP buffer

② Add antibody to form

antibody/antigen complexes

③ Add protein A/G-agarose beads

④ Wash antigen/antibody/

(protein A/G-agarose beads)

complexes

⑤ Discard supernatant

1D gel & WB

Figure 3.3 Immunoprecipitation procedure

29

3.7.1 Identification of Protein Carbonyl Post-translational Modification

As for the detection of protein carbonyl modification of specific protein,

post-electrophoretic derivatization was used. The nitrocellulose membrane got

from immunoprecipitation and Western blotting was dried completely before

derivatization. In day 2, the membrane was first incubated in a solution consisting

of 80% TBS and 20% methanol for 5 min. The solution was changed to 2N HCl

and incubate for 5 min. The membrane was then incubated with 0.5mM 2,4-

dinitrophenylhydrazine (DNPH) in 2N HCl for exactly 5 min. After three 5 min

washes with 2N HCl and five 5 min washes with 50% methanol, the membrane

was then incubated with blocking solution. During all the incubation and washing

steps, continued shaking was applied at room temperature (Conrad, Talent et al.

2000). The following steps for membrane development were similar to the

detection of protein-bound carbonyls (see section 3.5.1.1).

3.8 Image analysis

All the blots were scanned using a ChemiDocTM MP Imaging System (Bio-

Rad, Hercules, CA) and quantified using Image Lab software. In order to prevent

slight difference in loading, bands intensities were normalized by the intensities

of loading control.

3.9 Statistical analysis

A Student’s t-test was used to compare the levels of oxidative stress,

expression of HO-1 and BVR, oxidative post-translational modifications of BVR,

and phosphorylation post-translational modifications of BVR in PINK1 knockout

mice and wild-type mice. P-values less than 0.05 were accepted as significant

different.

30

Chapter 4. Results

4.1 Oxidative stress levels in brain of PINK1 knockout mice compared to

age-matched control

PINK1-deficient brain has elevated oxidative stress. Protein carbonyls,

HNE, 3NT, as the widespread indicators of oxidative stress, were used to index

the effects of PINK1 knockout in brain.

31

4.2 Protein carbonyl levels increase in PINK1 knockout mice compared

to age-matched control

Figure 4.1 shows protein carbonyl levels in the brains of PINK1 knockout

mice and age-matched control mice. The level of protein carbonyls was found to

be significantly increased in PINK1 knockout mice compared to age-matched

controls (P<0.002). This increase in protein carbonyl is approximately 13% higher

than control.

Figure 4.1 Protein carbonyl level in PINK1 knockout mice compared to age-

matched control mice.

PC level was measured using slot blot. Data are shown as % control. All the

values are expressed as mean ± SEM, wild type n=6, PINK1 knockout n=6. *P <

0.002.

32

4.3 HNE-bound protein levels increase in brain of PINK1 knockout mice

compared to age-matched control

Figure 4.2 shows HNE-modified protein levels in the brains of PINK1

knockout mice and age-matched control mice. The level of HNE-bound protein

was found to be significantly increased in PINK1 knockout mice compared to

age-matched controls (P<0.02). This increase in protein-bound HNE is

approximately 14% higher than control.

Figure 4.2 HNE-bound protein level in PINK1 knockout mice compared to age-


HNE level was measured using western blot technique. Tubulin was used as a

loading control to normalize the intensity of HNE-bound protein bands. Data are

shown as % control. All the values are expressed as mean ± SEM, wild type n=6,

PINK1 knockout n=6. *P < 0.02.

33

4.4 Protein-3NT levels are unchanged in brain of PINK1 knockout mice

compared to age-matched control

Figure 4.3 shows 3-Nitrotyrosine levels in the brains of PINK1 knockout

mice and age-matched control mice. The level of 3-NT was not found to be

significantly changed in PINK1 knockout mice compared to age-matched controls.

Figure 4.3 3-NT resident protein level in PINK1 knockout mice compared to age-


3-NT level was measured using slot blot. Data are shown as % control. All the

values are expressed as mean ± SEM, wild type n=6, PINK1 knockout n=6. No

significant differences were observed for PINK1 knockout mice.

34

4.5 HO-1/BVR system levels in PINK1 knockout mice compared to age-

matched control

We previously have shown that PINK1 knockout causes increased

oxidative stress in brain. However, HO-1/BVR is an antioxidant system defense

against ROS or RNS stress. Therefore, we wanted to determine the effect of

oxidative stress on HO-1/BVR system. We measured the levels of HO-1 and

BVR in the brains of wild type mice and PINK1 knockout mice, respectively.

35

4.6 HO-1 levels decrease slightly, but not statistically significant in brain

of PINK1 knockout mice compared to age-matched control

Figure 4.4 shows heme oxygenase-1 levels in the brains of PINK1

knockout mice and age-matched control mice. The level of HO-1 was not found

to be significantly changed in brain of PINK1 knockout mice compared to age-

matched controls.

HO-1

Tubulin

WT1 KO1 WT2 KO2

Figure 4.4 Heme Oxygenase-1 level in PINK1 knockout mice compared to age-


HO-1 level was measured using western blot technique. Tubulin was used as a

loading control to normalize the intensity of HO-1 bands. Data are shown as %

control. All the values are expressed as mean ± SEM, wild type n=6, PINK1

knockout n=6. No significant differences were observed for PINK1 knockout mice.

36

4.7 BVR levels increase in brain of PINK1 knockout mice compared to

age-matched control

Figure 4.5 shows biliverdin reductase levels in the brains of PINK1

knockout mice and age-matched control mice. The level of BVR was found to be

significantly increased in PINK1 knockout mice compared to age-matched

controls (P<0.03). This increase in BVR is approximately 14% higher than control.

BVR

Tubulin

WT1 KO1 WT2 KO2

Figure 4.5 Biliverdin Reductase level in PINK1 knockout mice compared to age-


BVR level was measured using western blot technique. Tubulin was used as a

loading control to normalize the intensity of BVR bands. Data are shown as %

control. All the values are expressed as mean ± SEM, wild type n=6, PINK1

knockout n=6 Data are shown as % control. *P < 0.03.

37

4.8 Levels of BVR oxidative post-translational modification in brain of

PINK1 knockout mice compared to age-matched control

In PINK1 knockout mice, heme oxygenase-1 levels did not change while

biliverdin reductase levels increased indicating that BVR plays a more important

role in response to oxidative stress. Therefore, we determined levels of BVR

oxidative post-translational modification.

38

4.9 Protein carbonyl levels on BVR levels do not change in brain of

PINK1 knockout mice compared to age-matched control

Figure 4.6 shows protein carbonyl levels of biliverdin reductase in the

brains of PINK1 knockout mice and age-matched control mice. No apparent

differences in the intensity of PC of BVR was found between PINK1 knockout

mice and age-matched controls.

CO-BVR

BVR

WT1 KO1 WT2 KO2

Figure 4.6 Protein carbonyl of BVR level in PINK1 knockout mice compared to

age-matched control mice.

PC level was measured using immunoprecipitation. BVR was used as a loading

control to normalize the intensity of protein carbonyl on BVR bands. Data are


PINK1 knockout n=6. No significant differences were observed for PINK1

knockout mice.

39

4.10 Levels of HNE-bound BVR do not change in brain of PINK1 knockout

mice compared to age-matched control

Figure 4.7 illustrates HNE-bound biliverdin reductase levels in the brains

of PINK1 knockout mice and age-matched control mice. No apparent differences

in the intensity of HNE-bound BVR was found between PINK1 knockout mice

and age-matched controls.

HNE-BVR

BVR

WT1 KO1 WT2 KO2

Figure 4.7 HNE-bound BVR level in PINK1 knockout mice compared to age-


HNE level was measured employing immunoprecipitation. BVR was used as a

loading control to normalize the intensity of HNE-bound BVR bands. Data are


PINK1 knockout n=6. No significant differences were observed for PINK1

knockout mice.

40

4.11 Levels of 3NT on BVR do not change in brain of PINK1 knockout


Figure 4.8 shows 3-NT of biliverdin reductase levels in the brains of PINK1

knockout mice and age-matched control mice. 3-NT of BVR did not show any

change between PINK1 knockout mice and age-matched controls.

3NT-BVR

BVR

WT1 KO1 WT2 KO2

Figure 4.8 3NT-bound BVR level in PINK1 knockout mice compared to age-


3-NT level was measured employing immunoprecipitation. BVR was used as a

loading control to normalize the intensity of 3NT on BVR bands. Data are shown

as % control. All the values are expressed as mean ± SEM, wild type n=3, PINK1

knockout n=3. No significant differences were observed for PINK1 knockout mice.

41

4.12 BVR phosphorylation post-translational modification levels in PINK1

knockout mice compared to age-matched control

Biliverdin reductase is an antioxidant enzyme as well as a serine/threonine

kinase. As Section 4.3 shows, oxidative post-translational modification of BVR

was inhibited, conceivably by its antioxidant function, i.e. by production of

bilirubin. In this section, we wanted to investigate whether the increase level of

BVR induce the change of BVR phosphorylation post-translational modification

levels.

42

4.13 BVR phosphoserine levels significantly increase in brain of PINK1

knockout mice compared to age-matched control

Figure 4.9 shows biliverdin reductase phosphoserine levels in the brains

of PINK1 knockout mice and age-matched control mice. The level of BVR

phosphoserine was found to be significantly increased in PINK1 knockout mice

compared to age-matched controls (P<0.03). This increase in BVR

phosphoserine is approximately 90% higher than control.

Ser-BVR

BVR

WT1 KO1 WT2 KO2

Figure 4.9 BVR phosphoserine level in PINK1 knockout mice compared to age-


BVR phosphoserine level was measured using immunoprecipitation. BVR was

used as a loading control to normalize the intensity of BVR phosphoserine bands.

Data are shown as % control. All the values are expressed as mean ± SEM, wild

type n=3, PINK1 knockout n=3. *P < 0.005.

43

4.14 BVR phosphothreonine levels increase in brain of PINK1 knockout


Figure 4.10 shows biliverdin reductase phosphothreonine levels in the

brains of PINK1 knockout mice and age-matched control mice. The level of BVR

phosphothreonine was found to be significantly increased in PINK1 knockout

mice compared to age-matched controls (P<0.05). This increase in BVR

phosphothreonine is approximately 31% higher than control.

Thr-BVR

BVR

WT1 KO1 WT2 KO2

Figure 4.10 BVR phosphothreonine level in PINK1 knockout mice compared to

age-matched control mice.

BVR phosphothreonine level was measured using immunoprecipitation. BVR

was used as a loading control to normalize the intensity of BVR

phosphothreonine bands. Data are shown as % control. All the values are

expressed as mean ± SEM, wild type n=3, PINK1 knockout n=3. *P < 0.05.

44

Chapter 5. Discussion

PINK1 is a gene that is linked to autosomal recessive familial Parkinson’s

disease (Varcin, Bentea et al. 2012). PINK1 functions as a serine/threonine

kinase as well as a protector of mitochondrial function. Mutation of the PINK1

gene contributes to the loss of mitochondrial protection against oxidative stress

(Moore, West et al. 2005). In fact, mitochondrial dysfunction plays a central role

in the pathogenesis of PD (Yao, Gandhi et al. 2011). Because of the function of

PINK1 protein, we expected two aspects of differences, oxidation and

phosphorylation, resulting from the loss of PINK1. This thesis research

investigated the effect of the defect of PINK1 on oxidative stress and heme

oxygenase-1/biliverdin reductase post-translational modifications. PINK1

knockout mouse is a genetic model of PD that used to study oxidative stress in

this thesis.

The dysfunction of mitochondria leads to ROS and oxidative stress. In PD,

oxidative stress contributes to the neurodegeneration (Andersen 2004). PINK1,

as a PD-related gene, may cause oxidative stress as well if it is mutated.

Oxidative stress contributes to the production of protein carbonyls, HNE, and 3-

NT. In this study, significant increased levels of protein carbonyls and HNE were

observed in PINK1 knockout mice compared to control mice. These results are

consistent with our hypothesis that oxidative stress is elevated in the brain of

PINK1 knockout mice. In contrast, however, previous studies reported

unchanged levels of protein carbonyls and HNE-bound proteins in different parts

of the brain of PINK1 knockout mice at 24 months (Gautier, Kitada et al. 2008). It

is possible that the time for sample homogenization in this work differs from the

one reported in the literature. Longer preparation time would increase free

radicals in the samples. Additionally, the level of 3-NT bound protein given by

present data did not show any apparent change.

In order to prevent cellular impairment, antioxidant systems have been

considered to respond to the up-regulated oxidative stress. The heme

oxygenase-1/Biliverdin reductase system is such an example. Biliverdin

45

reductase reduces biliverdin, the product of heme degraded by heme oxygenase-

1, to the antioxidant bilirubin. To determine if the increase in oxidative stress

levels observed in PINK1 knockout mice were related to the HO-1/BVR system,

HO-1 and BVR protein expression were measured, respectively. It is unclear how

the HO-1/BVR system functions in Parkinson’s disease to date, even though it

has been studied well in Alzheimer disease (Barone, Di Domenico et al. 2011,

Barone, Di Domenico et al. 2011, Barone, Di Domenico et al. 2014). The current

results, for the first time, report that the level of BVR was significantly increased

in PINK1 knockout mice. The increased of BVR rather than HO-1 indicates that

the terminal product of this system, bilirubin, becomes more efficient to scavenge

ROS. Recent studies showed the conversion from heme to biliverdin by heme

oxygenase-1 is not always neuro-protective. A product of this reaction, iron (II),

can cause free radicals by Fenton chemistry and subsequently initiate oxidative

stress (Gutteridge, Maidt et al. 1990). Moreover, HO-1 level in the substantia

nigra neurons of PD and control cases are similar (Schipper 2000). PINK1

knockout, as a model of PD, presents similar results as in our study. However, a

significant increase in BVR protein level promotes its ability to keep cellular

homeostasis.

Furthermore, we chose to investigate the post-translational modifications

of BVR for the following experiments because of its elevated ability to combat

oxidative stress as shown in this thesis research. Previous work leads to the

hypothesis that oxidative modification of BVR may decrease. The current results

obtained from immunoprecipitation technique showed similar levels of protein

carbonyls, HNE, and 3-NT of BVR in both PINK1 knockout mice and controls

mice. These results are in accordance with the hypothesis that high levels of

BVR are efficient to keep oxidative modification of BVR back to normal level.

Another novel finding of this thesis research is the change of

phosphorylation modification of BVR in PINK1 knockout mice. Since PINK1

serves as a serine/threonine kinase, phosphorylation of BVR may be changed if

PINK1 gene is deficient. On the other hand, BVR is capable of

46

autophosphorylation, and phosphorylation is important for its activation (Salim,

Brown-Kipphut et al. 2001). Serine and threonine are the targets for BVR

phosphorylation. The data presented in this research demonstrated significant

increased BVR phosphoserine and phosphothreonine in PINK1 knockout mice

brain with respect to controls. These results provided another reasonable

explanation for the previous work showing up-regulation of BVR activated by the

increased phosphorylation. Based on Fabio Di Domenico’s work (Di Domenico,

Perluigi et al. 2013), we may speculate that activated BVR together with

increased production of antioxidant is able to keep nitric oxide levels under

control. Therefore, even elevated NOS levels result from PINK1 knockout will not

cause higher nitrosative stress levels. However, further studies are needed to

confirm this hypothesis. Interestingly, these results, in contrast, show increased

BVR phosphorylation together with deficient PINK1 kinase. A possible

interpretation is that PINK1 protein, as a kinase, does not regulate BVR

phosphorylation directly. This notion will be the subject of future studies from our

laboratory.

47

Chapter 6. Conclusion and Future Studies

In conclusion, the work presented in this thesis showed the presence of

oxidative stress along with the alternation of BVR post-translational modification

in the brain of PINK1 knockout mice. Specifically, we examined levels of protein

carbonyls, HNE-bound proteins, and nitrated proteins. The results established a

relationship between oxidative stress and the mice brain with PINK1

abnormalities. The notion that antioxidant system makes a response to oxidative

stress has been demonstrated by the up-regulated levels of BVR. With regard to

BVR post-translational modification, we observed an unchanged level of

oxidative translational modification along with significant increase in Ser-residue

and Thr-residue phosphorylation, which demonstrated antioxidant and

autophosphorylation abilities of BVR in the brain of PINK1 knockout mice. We

first reported the characterization of HO-1/BVR system in PINK1 knockout mice

which has been only studied with respect to Alzheimer disease to date, providing

a new perspective of treatment of Parkinson’s disease.

Based on the results in this thesis, we may be concern about the following

experiments for future studies:

1. BVR has been found involved in insulin signaling pathway. It is a substrate

for insulin receptor as well as a kinase for insulin receptor kinase,

regulating MAPK and PI3K cascades. The findings of up-regulated levels

of BVR and alternation of phosphorylation of BVR in PINK1 knockout mice

may indicate its possible role in insulin receptor system. Therefore, it will

be meaningful to investigate whether cell signal function of BVR changed

in PINK1 deficiency cells.

2. Determine the level of nitric oxide synthase, which verifies the speculation

that activated BVR keeps protein 3-NT levels in normal.

3. Determine whether PINK1 protein, as a kinase, regulates BVR

phosphorylation directly or not.

48

Appendix: Data to Supplement Figures

Figure 4.1 Protein carbonyl levels

Wildtype Knockout 6823135 8908625 6275115 8637365 7829250 7157315 6759170 7260220 7151485 8443655 8013335 8376885

Average 7141915 8130678

%Control 95.54 124.74 87.86 120.94 109.62 100.22 94.64 101.66 100.13 118.23 112.20 117.29

Average 100.00 113.84 Std Dev 8.54 9.44

SEM 3.49 3.85

49

Figure 4.2 Protein-bound HNE levels

Wildtype Knockout

HNE 34907999 40400364

37492364 39958416

35837672 32791518

34825302 37018140

34725652 36654780

37958856 42221274

Tubulin 46031927 41935731

40305920 35982408

36999128 34531368

40106885 38904120

39598381 37568073

44750475 39060360

HNE/Tubulin 0.758 0.963

0.93 1.11

0.969 0.95

0.868 0.952

0.877 0.976

0.848 1.081

Average 0.875 1.005

%Control 86.66 110.09

106.3 126.9

110.68 108.51

99.22 108.73

100.21 111.49

96.93 123.52

Average 100 114.87

Std Dev 8.27 8.15

SEM 3.38 3.33

50

Figure 4.3 Protein-3NT levels

Wildtype Knockout

3-NT 45019512 46365264

51666336 51932592

52212384 43072632

50711544 45826776

48658968 46948320

50685984 44181144

Tubulin 39980162 36624280

35791371 39364325

36876224 35965853

39662546 40425693

43543962 39682489

41674556 43997800

3-NT/Tubulin 1.13 1.27

1.44 1.32

1.42 1.2

1.28 1.13

1.12 1.18

1.22 1

Average 1.27 1.18

%Control 88.92 99.97

114 104.18

111.81 94.58

100.97 89.52

88.25 93.43

96.05 79.3

Average 100 93.5

Std Dev 11.07 8.66

SEM 4.52 3.53

51

Figure 4.4 Heme Oxygenase-1 levels

Wildtype Knockout

HO-1 1139502 1021488

1351896 1002456

1119924 1119300

1309776 1273584

1327404 1217424

1110330 994578

Tubulin 53901542 46671750

41292636 44621192

41881779 41300625

44186552 45388112

50727465 49461742

48209025 51953671

HO-1/Tubulin

2.11E-02 2.19E-02

3.27E-02 2.25E-02

2.67E-02 2.71E-02

2.96E-02 2.81E-02

2.62E-02 2.46E-02

2.30E-02 1.91E-02

Average 2.66E-02 2.39E-02

%Control 79.54 82.35

123.19 84.53

100.61 101.97

111.53 105.58

98.46 92.61

86.66 72.03

Average 100 89.85

Std Dev 15.94 12.68

SEM 6.51 5.18

52

Figure 4.5 Biliverdin Reductase levels

Wildtype Knockout

BVR 520191 672543

610650 709389

672543 630936

637146 650739

583326 718428

650118

Tubulin 47594448 43322240

42192780 43832000

42309280 42683120

49431120 39930480

44433732 43680000

41476400

BVR/Tubulin 1.09E-02 1.55E-02

1.45E-02 1.62E-02

1.59E-02 1.48E-02

1.29E-02 1.63E-02

1.31E-02 1.64E-02

1.57E-02

Average 1.38E-02 1.58E-02

%Control 79.02 112.24

104.64 117.01

114.92 106.87

93.19 117.82

94.91 118.91

113.32

Average 100 114.57

Std Dev 13.67 5

SEM 5.58 2.04

53

Figure 4.6 Protein carbonyl on BVR levels

Wildtype Knockout

PC 16029778 21038308

12708059 14937260

15736610 20837193

18416659 18730121

13408129 17738197

14260185 20781713

BVR 401147 1207716

387543 656469

521683 528238

750063 397195

371792 715616

370215 698915

PC/BVR 39.96 17.42

32.79 22.75

30.17 39.45

24.55 47.16

36.06 24.79

38.52 29.73

Average 33.68 30.22

%Control 118.66 51.73

97.38 67.57

89.58 117.14

72.91 140.03

107.09 73.61

114.38 88.3

Average 100 89.73

Std Dev 17.06 33.1

SEM 6.96 13.51

54

Figure 4.7 Levels HNE-bound BVR

Wildtype Knockout

HNE 14336397 17455541

16564065 22703803

17595701 16705104

15458559 13785034

12427050 14810996

15956206 17532588

BVR 10769700 15236925

14331360 20543925

17611853 18666900

12054189 13601967

8988501 8221908

12228438 11267703

HNE/BVR 1.33 1.15

1.16 1.11

1 0.89

1.28 1.01

1.38 1.16

1.3 1.56

Average 1.24 1.15

%Control 107.12 92.19

93.01 88.93

80.4 72.02

103.2 81.56

111.26 93.06

105.01 125.22

Average 100 92.16

Std Dev 11.37 18.01

SEM 4.64 7.35

55

Figure 4.8 Levels of 3NT on BVR

Wildtype Knockout

3NT 3018525 4353600

4056150 5238450

5465925 5299520

BVR 10769700 15236925

14331360 20543925

17611853 18666900

3NT/BVR 2.80E-01 2.86E-01

2.83E-01 2.55E-01

3.10E-01 2.84E-01

Average 2.91E-01 2.75E-01

%Control 96.24 98.11

97.19 87.56

106.57 97.49

Average 100 94.39

Std Dev 5.71 5.92

SEM 3.3 3.42

56

Figure 4.9 BVR phosphoserine levels

Wildtype Knockout

Ser 20574264 13354948

22601488 17261880

20813664 11428044

BVR 12768528 3753604

11819556 6156662

10791844 3550182

Ser/BVR 1.61 3.56

1.91 2.8

1.93 3.22

Average 1.82 3.19

%Control 88.66 195.77

105.22 154.27

106.12 177.12

Average 100 175.72

Std Dev 9.83 20.78

SEM 5.68 12

57

Figure 4.10 BVR phosphothreonine levels

Wildtype Knockout

Thr 19667625 24868690

21120890 22653820

23647960 23021740

BVR 10683512 11333476

13345992 10113440

17053968 12339876

Thr/BVR 1.84 2.19

1.58 2.24

1.39 1.87

Average 1.6 2.1

%Control 114.82 136.85

98.7 139.7

86.48 116.36

Average 100 130.97

Std Dev 14.21 12.74

SEM 8.2 7.35

58

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Vita

Zhaoshu Zhang was born in Jiangsu, China. She obtained her bachelor’s

degree in Pharmacy from China Pharmaceutical University in June 2011. She

came to the University of Kentucky and enrolled for her graduate studies in

August 2011. Zhaoshu completed her thesis work under the guidance of Dr. D.

Allan Butterfield.

Publications:

S.A. Farr; J.L. Harris; R. Sultana; Z. Zhang; M.L. Niehoff; T.L. Platt; M.P. Murphy;

J.E. Morley; V. Kumar; D.A. Butterfield, “Antisense Oligonucleotide Against GSK-

3β in Brain of SAMP8 Mice Improves Learning and Memory and Decreases

Oxidative Stress: Involvement of Transcription Factor Nrf2 and Implications for

Alzheimer Disease,” Free Radical Biology & Medicine. (2014)

A.M. Swomley; S. Förster; J.T. Keeney; J. Triplett; Z. Zhang; R. Sultana; D.A.

Butterfield, “Amyloid β-Peptide and Oxidative Stress in Alzheimer Disease:

Evidence Based on Proteomics,” Biochem Biophys Acta--Molecular Basis of

Disease. (2013)

Documents

INCREASE OF BASAL OXIDATIVE STRESS LEVELS AND …