From the Department of Medical Nutrition Karolinska Institutet, Stockholm, Sweden

The pathophysiology of respiratory chain dysfunction

José Pablo Silva

Stockholm 2005

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Handledare: Professor Nils-Göran Larsson

Avdelningen för Medicinsk Näringslära, Karolinska Institutet

Opponent: Professor Robert N. Lightowlers

School of Neurology, Neurobiology and Psychiatry, Medical School, University of Newcastle upon Tyne, Newcastle, United Kingdom

Betygsnämnden: Professor Elzbieta Glaser

Institutionen för biokemi och biophysik, Stockholms Universitet

Professor Anna Wedell

Institutionen för Molekylär Medicin, Karolinska Institutet

Professor Rune Toftgård

Institutionen för biovetenskaper, Karolinska Institutet

José Pablo Silva, 2005 ISBN 91-7140-234-9 Printed by Repro Print AB, Stockholm, Sweden

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To my parents

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Abstract

Mutations of mitochondrial DNA cause a variety of clinical syndromes. It is unclear

whether impaired oxidative phosphorylation on its own is the main cause of pathology or whether other factors such as secondary metabolic alterations, enhanced formation

of reactive oxygen species (ROS) and induction of apoptosis also may contribute to the clinical phenotype. This thesis focuses on several topics relevant to the

pathophysiology of mitochondrial disease, i.e. a mouse model for mitochondrial

diabetes, analyses of ROS formation and cell death in mouse strains with tissue-specific respiratory chain (RC) deficiency and regulation of uncoupling protein (UCP)

activity and RC function by superoxide. We studied the pathogenesis of mitochondrial diabetes by tissue-specific (Cre-

loxP mediated) inactivation of the gene encoding the mitochondrial transcription

factor A (Tfam) in insulin producing pancreatic β-cells. Inactivation of Tfam resulted

in mtDNA depletion, severe RC deficiency and abnormally appearing mitochondria in mutant islets. The β-cell specific Tfam knockout (KO) mice were diabetic with

lowered pancreatic insulin release. Studies of isolated islets showed the lowered

insulin release was caused by altered Ca2+ signaling. The RC deficient pancreatic β-

cells eventually died as concluded from histological studies. This study thus

demonstrates 2 phases in the pathogenesis of mitochondrial diabetes. Impaired glucose-stimulated insulin release is observed at an early stage and is later followed

by β-cell loss. This study provides the first genetic in vivo evidence for a critical role

of the RC in glucose-stimulated insulin release (Paper I).

We next examined whether RC deficiency caused ROS formation and cell death. We found increased cell death in homozygous germline Tfam KO embryos and

in mice with tissue-specific inactivation of Tfam in cardiomyocytes and pyramidal forebrain neurons. Tfam KO embryos showed massive induction of apoptosis at

embryonic day 9.5, Tfam KO cardiomyocytes showed a moderate increase in

apoptosis and Tfam KO in forebrain neurons caused massive cell death. The majority of the neurons affected by the knockout died by necrosis as reflected by absence of

apoptosis markers and presence of an inflammatory reaction in brain sections. We found only a moderate induction of antioxidant defenses in the Tfam KO

cardiomyocytes and almost no induction in Tfam KO neurons. The activities of

several iron-sulphur-cluster enzymes sensitive to ROS damage were essentially

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normal in mtDNA-depleted heart and brain cortex. This result suggests that

mitochondrial ROS production is normal or only moderately increased in tissues with RC deficiency and that any increase in ROS formation is fully compensated for by

induction of antioxidant defenses (Paper II and III). Superoxide has been reported to activate mitochondrial UCPs thus providing a

feedback control to lower generation of superoxide by uncoupling respiration. The

superoxide-effect is expected to significantly affect energy metabolism due to widespread tissue distribution of UCPs. We generated transgenic mice harboring P1

artificial chromosomes with the human SOD2 gene to investigate whether superoxide regulates UCP activity and energy expenditure in vivo. The human SOD2 protein was

ubiquitously expressed and SOD2 enzyme activities showed an overall increase in

transgenic mice. There was a linear correlation between SOD2 enzyme activity and mitochondrial oxidative capacity. Mitochondria with increased SOD2 activity were

also resistant to induction of mitochondrial permeability. However, despite these

obvious effects on mitochondria, SOD2 overexpressing mice exhibited normal UCP activities and adapted normally to cold. They also displayed normal metabolic rates

and no change in mitochondrial mass or mitochondrial gene expression. These results suggest that superoxide does not regulate UCP activity and energy expenditure in vivo

(Paper IV).

Keywords: mitochondrial disease, mitochondrial DNA (mtDNA), mitochondrial transcription factor A (Tfam), Cre-loxP, conditional knockout, reactive oxygen species (ROS), superoxide, uncoupling protein (UCP), mitochondrial bioenergetics, energy expenditure ISBN: 91-7140-234-9

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List of publications

This thesis is based on the following publications, which will be referred to in the text

by their roman numerals.

I Impaired insulin secretion and β-cell loss in tissue-specific knockout mice

with mitochondrial diabetes Silva JP, Koehler M, Graff C, Oldfors A, Magnuson MA, Berggren PO,

Larsson NG.

Nature Genetics 2000, 26 (3): 336-340

II Increased in vivo apoptosis in cells lacking mitochondrial DNA gene expression Wang J, Silva JP, Gustafsson CM, Rustin P, Larsson NG.

Proceedings of the National Academy of Sciences USA 2001, 98 (7): 4038-4043

III Late-onset corticohippocampal neurodepletion attributable to catastrophic failure of oxidative phosphorylation in MILON mice Sörensen L, Ekstrand M, Silva JP, Lindqvist E, Xu B, Rustin P, Olson L,

Larsson NG. The Journal of Neuroscience 2001, 21 (20): 8082-8090

IV Superoxide dismutase 2 overexpression: enhanced mitochondrial tolerance but absence of effect on uncoupling protein activity Silva JP, Shabalina IG, Dufour E, Petrovic N, Backlund EC, Hultenby K, Wibom R, Nedergaard J, Cannon B, Larsson NG.

Manuscript

The original publications were reproduced with permission from the publishers.

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Table of contents Abstract ................................................................................................................... 5

List of publications.................................................................................................. 7

Abbreviations .........................................................................................................10

Background ............................................................................................................11

Mitochondrial Genetics .....................................................................................11

Mitochondria .......................................................................................................11

Evolution of Mitochondria...................................................................................13

Regulation of mitochondrial fusion and fission ....................................................14

Structure, gene content and organization of mammalian mtDNA .........................15

Transcription of mammalian mtDNA...................................................................18

Replication of mammalian mtDNA......................................................................20

Mitochondrial transciption factor A (TFAM).......................................................22

Transmission and segregation of mammalian mtDNA .........................................23

Regulation of mitochondrial gene expression and biogenesis ...............................24

Mitochondrial bioenergetics and ROS formation ............................................26

The respiratory chain ...........................................................................................26

Generation of ROS by the respiratory chain.........................................................32

Antioxidant defense mechanisms .........................................................................34

Mitochondrial uncoupling proteins and their role in energy expenditure

and control of ROS homeostasis ..........................................................................37

Mitochondrial regulation of apoptosis..................................................................39

Free radical theory of aging .................................................................................44

Respiratory chain diseases ................................................................................46

Respiratory chain diseases ...................................................................................46

Mitochondrial diabetes ........................................................................................50

ROS formation in respiratory chain deficient cells ...............................................51

Metabolic alterations and cell death in respiratory chain deficient cells................53

Mouse models for respiratory chain disease .........................................................55

Aims of this study...................................................................................................59

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Comments on methods ...........................................................................................60

Tfam knockout strategy........................................................................................60

Enzyme histochemistry........................................................................................60

Results and Discussion ...........................................................................................62

Pathogenesis studies in a mouse model of mitochondrial diabetes (Paper I) .........62

Apoptosis studies of cells and tissues lacking mtDNA gene expression

(Paper II) .............................................................................................................64

Analysis of the relationship between prolonged respiratory chain deficiency,

ROS formation and cell death in late-onset neurodegeneration mice (Paper III) ...66

Regulation of uncoupling protein activity and mitochondrial bioenergetics

by superoxide (Paper IV) .....................................................................................68

Concluding remarks...............................................................................................74

Acknowledgements.................................................................................................77

References...............................................................................................................79

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Abbreviations AIF Apoptosis-inducing factor ANT Adenine nucleotide translocase bp, Kbp, Mbp base pair, kilo base pair, mega base pair CaMK Calcium/calmodulin-dependent protein kinase COX Cytochrome c oxidase CSB conserved sequence block Da, kDa Dalton, kDa DNA pol γ DNA polymerase γ DFF DNA fragmentation factor ds double-stranded EndoG Endonuclease G FAD flavine-adenine dinucleotide GPx glutathione peroxidase GSH glutathione H2O2 hydrogen peroxide HSP Heavy strand promoter IMS mitochondrial intermembrane space LHON Leber’s hereditary optic neuropathy LSP Light strand promoter MELAS Mitochondrial encephalopathy, lactic acidosis and stroke-like episodes MERFF myoclonic epilepsy and ragged-red fibres mtDNA mitochondrial DNA mtSSB mitochondrial single stranded protein MIM mitochondrial inner membrane MOM mitochondrial outer membrane MM mitochondrial matrix NRF-1/NRF-2 nuclear respiratory factor-1/nuclear respiratory factor-2 NO Nitric Oxide O2

-· superoxide OH· hydroxyl radical PAC P1 artificial chromosome PGC-1α PPARγ coactivator-1α POLRMT human mtRNA polymerase PT permeability transition PTP permeability transition pore Q ubiquinone Q· ubisemiquinone QH2 ubiquinol RC respiratory chain ROS reactive oxygen species SDH Succinate dehydrogenase SOD superoxide dismutase STP Staurosporine ss single stranded TAS termination associated sequence TFAM mitochondrial transcription factor A TCA cycle tricarboxylic acid cycle TNFα Tumour necrosis factor α TUNEL terminal deoxynucleotidyl transferase mediated dUTP nick end labelling UCP uncoupling protein VDAC voltage dependent anion channel

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Background

Mitochondrial Genetics

Mitochondria

Mitochondria exist in most eukaryotic cells and form a dynamic inter-connected,

tubular network in which they constantly divide and fuse. Mitochondria consist of two membranes, the outer and inner membrane separated by the intermembrane space

(IMS). The interior part of the mitochondria is called the mitochondrial matrix (MM).

The mitochondrial inner membrane (MIM) forms invaginations called tubular cristae that extensively increase the surface area of the MIM. The MIM is impermeable to

most molecules. Transport of small molecules across the MIM depends on specifc carriers. The MIM harbors the respiratory chain (RC) and the adenine nucleotide

translocase (ANT) whose main function is to exchange mitochondrial ATP for

cytosolic ADP. The mitochondrial outer membrane (MOM) is permeable to solutes up to 1500Da through the voltage dependent anion channel (VDAC). Most of the

mitochondrial proteins are nucleus-encoded and imported into mitochondria by a mitochondrial protein import machinery. This import machinery sorts the protein to

the corresponding mitochondrial compartment. Mitochondria also have their own

genome (mtDNA) encoding several essential subunits of the respiratory chain (RC). Mitochondria have a central role in energy metabolism (Figure 1). They are the main

cellular site of ATP generation through the process of oxidative phosphorylation whereby electron transport along the RC is coupled to ADP phosphorylation.

Mitochondria harbor the tricarboxylic acid cycle (TCA), a pathway that generates

reducing equivalents, NADH and FADH2 to feed electrons to the RC. The TCA cycle is connected to the cytosolic glycolytic pathway and to the mitochondrial fatty acid β-

oxidation cycle. The TCA cycle is also a source of amino acid precursors and

porphyrin/heme biosyntheses. In specific cell-types, mitochondria are also a site of

steroid biosynthesis and carry out some steps of the gluconeogenesis and urea synthesis pathways. Mitochondria also participate in regulation of cellular Ca2+

homoestasis. The high Ca2+ buffering capacity of mitochondria protects the cell from high cytosolic Ca2+ concentrations. Transient cytosolic Ca2+ peaks can act as a

metabolic signal and activate Ca2+ sensitive enzymes of the TCA cycle. The

mitochondrial intermembrane space harbors several apoptosis-inducing factors that

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become active once released from the mitochondria. Finally, mitochondria are the

main cellular source of reactive oxygen species (ROS) that are produced as a byproduct of respiration and may determine the rate of ageing.

Figure 1 Mitochondria have a central role in energy metabolism. ADP denotes adenosine diphosphate, ATP adenosine triphosphate, ANT adenosine nucleotide translocator, CACT carnithine-acyl carnitine translocase, CPT-I/-II Carnithine palmitoyltrasnferase-I/-II, CoQ coenzyme Q, carnithine palmitoyltransferase DIC dicarboxylate carrier, TCA tricarboxylic acid cycle, ETF electron transfer-flavoprotein, ETF-DH electron transfer-dehydrogenase, I complex I, II complex II, III, complex III, IV complex IV (DiMauro and Schon, 2003).

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Evolution of Mitochondria

Mitochondria probably originate from a single ancient invasion of an Archea-type

host by a α-proteobacterium-like ancestor over 2 billion years ago. The symbiosis

between the host and the proto-mitochondrial ancestor was probably driven by metabolic requirements. One theory assumes the host was a methanogenic archaean

that associated with a methanotropic proteobacterium to obtain essential compounds,

for example hydrogen (Martin and Muller, 1998), about 2.7 billion years ago under anaerobic conditions well before the rise in atmospheric oxygen tension. A second

theory proposes that symbiosis was driven by the rise in atmospheric oxygen levels caused by photosynthetic cyanobacteria 2.2 billion years ago. Anaerobic life forms

were exposed to the toxicity of oxygen and adopted α-proteobacteria to detoxify

oxygen by respiration. The genome sequence of the α-proteobacterium Rickettsia

prowazeckii is the most mitochondria-like genome. The genome of Rickettsia

prowazeckii has a size of 1.1Mbp and encodes 834 proteins. It contains a complete set of genes for aerobic respiration, ATP production and ATP transport functions

(Andersson et al., 1998). The mitochondrial genome (mtDNA) has the same

fundamental role in all eukaryotes. It encodes a limited number of components of the RC, several ribosomal RNAs and a full or partial complement of tRNAs. MtDNA

exhibits remarkable variation in conformation, size and actual gene content. MtDNA molecules are circular but linear mtDNAs exist as well. Mitochondrial genome sizes

range from less than 6kbp (Plasmodium falciparum) to over 200kbp in land plants.

Contemporary mitochondrial genomes contain 3 to 67 protein-coding genes which is much less than the 834 proteins encoded by the genome of Rickettsia prowazeckii.

The evolution of the mitochondrial proteome displays both, reductive and expansive processes: (i) Many ancestral mitochondrial genes have been lost. The function of

these genes are no longer required because these genes were needed by the α-

proteobacterium as a free-living organism, for example genes for bacterial cell wall

synthesis; (ii) the coding sequences of a small number of mitochondrial proteins have been transferred to the nucleus; (iii) the largest number of mitochondrial proteins have

no bacterial or archeal orthologues and have been adopted by the mitochondrion after

the endosymbiotic event. They are encoded by the nuclear genome and imported into mitochondria (Andersson et al., 2003). Gene transfer from the mitochondrion to the

nucleus is an ongoing process in eukaryotes, for example in angiosperms (Adams and

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Palmer, 2003) and yeast (Thorsness and Fox, 1990). Numerous examples of

mitochondrial pseudogenes have been documented in the nucleus of humans (Woischnik and Moraes, 2002). Furthermore, some nuclear genes appear to derive

from edited mitochondrial transcripts (Nugent and Palmer, 1991) suggesting both, RNA- and DNA–mediated mechanisms that drive genome transfer and reduction.

Nucleic acids could escape from the mitochondrion during mitochondrial

fusion/fission or following damage to mitochondrial membranes (Adams and Palmer, 2003).

Several hypotheses have been proposed why mitochondria retain genes (Adams and Palmer, 2003). Firstly, some highly hydrophobic proteins may be

difficult to import across the mitochondrial membranes and to sort to the correct

location (Popot and de Vitry, 1990). The only two protein genes contained in every completely sequenced mitochondrial genome – cox1 and cob – encode the most

hydrophobic proteins present in mitochondria (Claros et al., 1995). Secondly, mtDNA

encoded protein genes may be toxic in the cytosol (Martin and Schnarrenberger, 1997). However this would only account for the cox1 and cob proteins that have been

retained in all mitochondrial genomes. Thirdly, expression of genes in the mitochondrion may allow for quick and direct regulation by the redox state of the

mitochondrion (Allen, 1993). However, there is no experimental evidence for this

assumption. Fourthly, the use of the non-standard genetic code in the mitochondria prevents further gene transfer to the nucleus. In this context it is noteworthy that

animal mitochondrial protein-coding gene content is almost completely constant at 13 genes (Boore, 1999).

Regulation of mitochondrial fusion and fission

Mitochondria divide, grow and segregate independently from the cell cycle through mitochondrial fission and fusion mechanisms (Osteryoung and Nunnari, 2003). A

group of proteins referred to as dynamin related proteins (DRPs) regulate

mitochondrial fission. DRPs form a group of self-assembling GTPases. The mitochondrial fission DRPs in yeast, Dnm1, and its homolog in higher eukaryotes,

Drp1, self assemble to form punctate structures that associate with the cytosolic face of the MOM at sites of mitochondrial constriction and fission. At least two more

proteins interact with self-assembled Dnm1 structures, the integral MOM protein Fis1

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and Mdv1, a peripheral MOM protein. Fis1 targets assembled Dnm1 structures to the

MOM and an interaction between Fis1 and Mdv1 transmits a signal for mitochondrial membrane fission by Dnm1. Fis1 homologs have been identified in animals and

structural data suggest mouse Fis1 acts as an adaptor molecule similar to yeast Fis1. Mdv1 homologs have not been identified yet in mammals. All components required

for mitochondrial fission to date associate with the outer membrane and it is at present

unclear how fission of the outer and inner membrane is coordinated and what components of the inner membrane are involved in this process (Shaw and Nunnari,

2002). The coordinated fusion of the mitochondrial outer and inner membranes is

regulated by a group of proteins called the fuzzy onions (Fzo)/mitofusin (Mfn) family

of mitochondrial outer membrane GTPases (Shaw and Nunnari, 2002). Yeast cells possess one Fzo-related gene, Fzo-1, that plays a direct role in mitochondrial fusion.

In mouse and humans, two Mfn genes, Mfn1 and Mfn2, have been identified that are

ubiquitously expressed and required for mouse embryonic development (Chen et al., 2003). In yeast, Fzo-1 interacts physically with the integral MOM protein Ugo-1 and

the IMS protein Mgm1 but the exact nature of their interactions and their specific roles in mitochondrial fusion are largely unknown. Mutations of the human

homologue of Mgm1, Opa1, result in autosomal dominant optic atrophy and cause

defects in mitochondrial morphology (Alexander et al., 2000; Delettre et al., 2000). Mitochondrial fission may allow segregation of mitochondria by transmission

of their membranes and genomes without need for de novo synthesis and the exchange of mtDNA molecules between mitochondria. Mitochondrial fission may

also participate in organelle remodelling during apoptosis as suggested by the

colocalization of the pro-apoptotic BAX protein with assembled Drp1 structures on the MOM (Newmeyer and Ferguson-Miller, 2003).

Structure, gene content and organization of mammalian mtDNA

The structure, gene content and organization of mammalian mtDNA is strongly conserved (Fernandez-Silva et al., 2003). Human mtDNA is a double-stranded (ds)

closed-circular molecule of about 16.6kb length (Figure 2). In most cells it represents about 0.5-1% of the total cellular DNA content. MtDNA is thought to be normally

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present as a supercoiled molecule. In yeast, mtDNA is organized as supramolecular

structures called nucleoids. Yeast nucleoids contain several (3-4) mtDNA molecules, about 10-20 different polypeptides (Kaufman et al., 2000; Miyakawa et al., 1987) and

are associated with mitochondrial membranes (Hobbs et al., 2001). Very little is known about the organization of mtDNA molecules in mammalian cells. In most cells

it forms monomeric closed double-stranded circles. In quiescent cultured cells,

catenated structures have been observed. Studies indicate an association between mtDNA and the mitochondrial inner membrane in vertebrate cells (Albring et al.,

1977).

Figure 2 The gene organization in human mitochondrial DNA showing the 13 protein coding genes (ND1-ND6, COXI-III, ATP6 and 8), the 22 tRNA genes (three letter amino acid symbols) and 2 rRNA genes (12S and 16S). The D-loop region controls replication and transcription of mtDNA. OH denotes the origin of H-strand replication and OL the origin of light strand replication (DiMauro and Schon, 2003).

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The two strands of mtDNA are called the heavy (H-) and the light (L-) strand

because they can be separated in density gradients due to their different Guanosine plus Thymidine content. A high proportion of mtDNA molecules in a metabolically

active cell contain a triple-stranded structure called the displacement loop (D-loop) (Figure 3). In the D-loop, a nascent H-strand of about 700-800nt length, called 7S

DNA, is annealed to the parental L-strand. The D-loop is about 1kb long in humans. It

is the major non-coding segment comprising the origin of replication of the H-strand (OH) and the promoters for H- and L-strand transcription. The D-loop also contains

conserved sequences called CSB (conserved sequence blocks) and TAS (termination associated sequences). The D-loop sequence is also the most variable sequence

between different species. The second non-coding region is around 30nt long and

about two-thirds of the entire mtDNA molecule away from the OH. It contains the origin of replication for the L-strand (OL) and is located inside a tRNA cluster.

Figure 3 The D-loop region controls replication and transcription of mtDNA. L denotes the light strand promoter, H1 the Heavy strand promoter 1, H2 the Heavy strand promoter 2, Phe the tRNAPhe gene, and Leu the tRNALeu gene, mtTFA the mitochondrial transciption factor A, TFB1M/TFB2M the mitochondrial transcription factor B1 and B2, mTERF the transcription termination factor, mtRNA pol the mitochondrial RNA polymerase (Fernandez-Silva et al., 2003).

Mammalian mtDNA encodes 37 genes, of these 22 are tRNA genes, 2 are rRNA genes (16S rRNA and 12S rRNA), and 13 are mRNAs encoding critical

subunits of the RC (Figure 2). MtDNA encodes seven subunits of complex I (ND1-

ND6 and ND4L), one subunit of complex III (cytochrome b), three subunits of

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complex IV (COXI, COXII, and COXIII) and two subunits of complex V (ATP 6 and

ATP 8). Of note, complex II is completely nucleus-encoded. The genes are asymmetrically distributed in mtDNA: The H-strand encodes 2 rRNA, 14 tRNAs, 12

mRNAs. The L-strand encodes the remaining 8 tRNAs and 1 mRNA (ND6). Mammalian mtDNA has a very compact gene organization consisting of

closely packed, intron-less genes. All the coding sequences are contiguous to each

other or separated by a few bases. Some of the protein genes overlap (ATP 6 and ATP 8 by 46 nucleotides in humans; ND4 and ND4L by 7 nucleotides in humans). In

several cases part of the termination codons are not encoded in mtDNA but are generated by the posttranscriptional polyadenylation of the corresponding mRNA

(Ojala et al., 1981). The genetic code used for translation of genes encoded by

mtDNA shows differences to the universal genetic code, for example in mammalian mitochondria, UGA specifies tryptophan instead of a termination codon.

Transcription of mammalian mtDNA

Transcription of human mtDNA starts at 3 different promoters in the D-loop region (Figure 3), one in the L-strand (Light strand promoter; LSP) and two in the H-strand

(Heavy strand promoter 1 and 2; HSP1 and HSP2) (Bogenhagen et al., 1984; Montoya et al., 1982). Transcription from HSP1 starts 19nt upstream of the tRNAPhe gene and

yields the two rRNAs (12S and 16S rRNA), tRNAPhe and tRNAVal (Figure 2 and 3). Transcription from HSP1 is subject to transcription termination immediately downstream of the 16S rRNA gene inside the gene for tRNALeu (Kruse et al., 1989;

Montoya et al., 1983). Transciption from HSP2 starts close to the 12S rRNA 5’-end

and originates an almost full-genomic length polycistronic transcript of the H-strand that is further processed to release individual mRNAs and tRNAs. Transcription from

HSP2 operates 20 times less frequent compared with transcription from HSP1. The presence of two transcription units, originating at HSP1 and HSP2, suggests a

differential regulation of rRNA versus mRNA transcription (Montoya et al., 1983).

Transcription from LSP results in a single polycistronic full-length genomic transcript from which the eight tRNAs and the ND6 mRNA derive.

HSP1 and LSP have bipartite structure. Both consist of a 15bp consensus sequence surrounding the transcription start site and of an element immediately

upstream of the initiation site (-15 to -39bp) containing a binding site for the

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mitochondrial transcription factor A (TFAM). The third promoter, HSP2, has only

limited similarity to the 15bp consensus sequence and lacks the upstream TFAM binding site. Transcription termination at the 3’end of the 16S rRNA gene (following

initiation at HSP1) is dependent on a specific tridecamer sequence in the tRNALeu gene and a specific protein factor, mTERF (Christianson and Clayton, 1988; Hess et al.,

1991). The termination promoting activity of mTERF is bidirectional (Shang and

Clayton, 1994) and therefore it could also act to terminate L-strand transcription. The primary polycistronic transcripts originating from LSP and HSP2 are

endonucleolytically cleaved (Doersen et al., 1985; Montoya et al., 1983; Ojala et al., 1981). tRNA sequences located between each rRNA and mRNA (Figure 2) probably

act as signal for the processing enzymes after acquiring a cloverleaf structure on the

nascent RNA chain. The 5’ endonucleolytic cleavage is probably mediated by mitochondrial RNAse P (Doersen et al., 1985) or other unidentified RNases.

Polyadenylation of the rRNAs and mRNAs occurs during or immediately after

cleavage of the primary transcript by a mitochondrial poly (A) polymerase (Amalric et al., 1978).

Transcription of human mtDNA requires an organelle-specific RNA polymerase, human mtRNA polymerase (POLRMT). POLRMT is homologous to

phage T7 RNA polymerase (Tiranti et al., 1997). Recombinant POLRMT together

with recombinant TFAM and either recombinant human mitochondrial transcription factor B1 (TFB1M) or B2 (TFB2M) initiate transcription in vitro from an mtDNA

template containing the mitochondrial promoters HSP1 or LSP. TFB1M and TFB2M are not needed for transcription elongation (Falkenberg et al., 2002). TFB2M has at

least two orders of magnitude more activity than TFB1M. TFB1M and TFB2M

interact with POLRMT in a 1:1 ratio suggesting heterodimerization between POLRMT and TFB1M or TFB2M. TFB1M and TFB2M were identified through

database homology searches for the yeast transcription factor Sc-mtTFB. Sc-mtTFB binds to the catalytic subunit of yeast mtRNA polymerase, Rpo41p, to confer specific

promoter binding.

The rRNAs are methylated and contain a short poly(A) tail of 1-10 residues (Dubin et al., 1982). Mitochondrial tRNAs are smaller, have some structural

differences and additional functions in replication and transcription regulation compared to nucleus-encoded tRNAs. Mitochondrial mRNAs start directly at the

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initiation codon or have an extremely short untranslated 5’end (of 1-3nt) and have a

poly(A) tail of about 55 residues immediately after the stop codon. Thus, they lack the typical features of cytosolic mRNAs, for example 5’ and 3’ untranslated regions, 3’

polyadenylation signal or the 5’end cap structure (Montoya et al., 1981).

Replication of mammalian mtDNA

MtDNA replicates independently from the cell cycle phase and from nuclear DNA

replication (Bogenhagen and Clayton, 1977). Two models have been proposed for mtDNA replication: (i) the strand-displacement and (ii) the strand-coupled replication

model. The strand displacement model involves two unidirectional, independent

origins. Replication starts at OH located downstream of the LSP in the D-loop region (Figure 3) and proceeds along the parental L-strand producing a daughter H-strand.

When H-strand replication reaches the OL (Figure 2), the replication site of the L-

strand is exposed and replication of the light strand in the opposite direction starts. H-strand replication is dependent on the generation of an RNA primer by transcription

from the LSP. Consequently, replication of mtDNA is likely coupled to the mitochondrial transcription machinery, consisting of POLRMT, TFAM and the

TFBM factors. The triplicate structure consisting of the newly synthesized RNA

strand upstream of OH, the parental H- and L-strand is called an R-loop. RNA processing activities, probably mediated by RNAse MRP or Endonuclease G (Cote

and Ruiz-Carrillo, 1993), cut the RNA strand generating the mature RNA primer. The mtDNA polymerase (DNA pol γ) starts H-strand replication through extension of the

RNA primer (Xu and Clayton, 1996). The R-loop is processed at the CSB I-III. The

CSB are D-loop sequences of extreme sequence conservation between human, rat and

mouse and are located downstream of the LSP and upstream of OH (Figure 3). A major H-strand replication initiation point is almost always found near CSB I

(Walberg and Clayton, 1983). After replication of the H-strand has started, most of the nascent DNA strand is arrested around the TAS (termination associated sequence)

creating the 7S DNA strand and the characteristic triple stranded structure of the D-

loop. At a much lower frequency however, replication proceeds over the entire length of the genome. The function of the 7S DNA strand and the mechanisms that

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determine whether elongation proceeds beyond TAS are unknown. The initiation for

replication of the L-strand at OL occurs in a small non-coding region that is thought to assume a stable stem-loop structure once it is exposed as a single strand. Initiation of

L-strand replication requires the activity of a specific primase to generate a short RNA primer with 5’ends mapping onto a T-rich portion of the loop in the stem-loop

structure. Once initiation has started, elongation proceeds until completion of L-strand

duplication (Wong and Clayton, 1985). The whole process of replication has been estimated to last 1 h implying a polymerization rate of 270nt/min (Graves et al.,

1998). After completion of the synthesis of both strands, RNA primers must be removed and the gaps filled and ligated. The closed circular mtDNA adopts its tertiary

structure following introduction of supercoils and interaction with DNA binding

proteins. The second model, the strand-coupled replication model, has been proposed to

exist along with the strand-displacement model and corresponds to coupled leading

and lagging strand synthesis of mtDNA (Holt et al., 2000). MtDNA polymerase, DNA pol γ, is composed of a catalytic α-subunit and a

smaller β-subunit. The β-subunit is needed for processivity and primer recognition.

DNA pol γ has a 3’-5’ exonuclease activity in the α-subunit (Longley et al., 1998).

DNA pol γ requires an ATP-dependent mitochondrial helicase for unwinding mtDNA

called Twinkle. Twinkle has a helicase domain that shares homology to the helicase domain of phage T7 primase/helicase. Replication of mtDNA requires mitochondrial

single stranded (ss) binding protein (mtSSB) to stabilize single stranded DNA during

replication and to increase the activity and fidelity of DNA pol γ (Farr et al., 1999;

Hoke et al., 1990). A minimal mammalian mtDNA replisome has been reconstituted consisting of DNA pol γ, mtSSB and Twinkle in recombinant form. In combination,

recombinant DNA pol γ and Twinkle form a processive replication machinery that

can use dsDNA as a template to synthesize ssDNA of about 2 kb. Addition of

recombinant mtSSB stimulates the reaction further generating DNA products of about

16kb. The observed DNA synthesis rate in this in vitro system is 180bp/min corresponding closely to the previously reported in vivo value of 270bp/min. The

functional interaction between Twinkle and DNA pol γ explains why mutations in

these two proteins cause an identical human disease, autosomal dominant external ophtalmoplegia (Korhonen et al., 2004)

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Mitochondrial transciption factor A (TFAM)

TFAM is a nucleus encoded transcription factor of 24kDa. TFAM belongs to a group of small nuclear DNA binding proteins, the nuclear high mobility group box proteins

(HMG box proteins). TFAM contains two high mobility group domains (HMG box)

that confer DNA binding. TFAM has the ability to wrap, unbend and unwind the DNA (Fisher et al., 1992). The C-terminal tail is essential for the activation of

mitochondrial transcription (Dairaghi et al., 1995). TFAM is required for mitochondrial transcription initiation (Falkenberg et al., 2002) and likely for mtDNA

replication because an RNA primer must be synthesized to initiate H-strand

replication. TFAM has a leader peptide sequence for protein import into the mitochondria.

TFAM appears to have a second, structural role besides its function as a transcription initiation factor. It appears that TFAM is regularly spaced bound to the

D-loop probably allowing other transcription factors to interact with their target

sequences (Ghivizzani et al., 1994). TFAM levels exceed the levels that would be expected for a transcription factor with regulatory functions. It has been proposed to

have a histone-like function covering the entire mtDNA molecule (Fisher et al., 1992; Ghivizzani et al., 1994). In this sense, TFAM homologues of yeast, ABF2, and of

Xenopus, wrap entirely the mtDNA molecule (Shen and Bogenhagen, 2001). In

Xenopus, the TFAM homologue has been found to bind to mtDNA as a tetramer (Antoshechkin et al., 1997).

The yeast homologue of TFAM is ABF2p (sc-mtTFA). ABF2p also bends and

introduces negative supercoils in DNA (Fisher et al., 1992) and functions as a histone-like protein with DNA packaging properties (Megraw and Chae, 1993). However,

ABF2p lacks the C-terminal domain of TFAM and has only a minor effect on transcription (Xu and Clayton, 1992). Yeast strains lacking ABF2p lose mtDNA when

grown on glucose indicating that ABF2p has a role in mtDNA maintenance. TFAM

when expressed in an ABF2p deficient yeast strain can complement ABF2p function by partially rescuing the growth and respiratory defects of this strain (Parisi et al.,

1993). The level of TFAM is lower in cells depleted of mtDNA after treatment with

Ethidium bromide and higher in tissues from patients with increased levels of mtDNA

23

(Davis et al., 1996; Larsson et al., 1994). TFAM levels may follow the mtDNA levels

by a feedback regulation or may be more stable in the presence of DNA targets. Heterozygous Tfam KO mice have reduced mtDNA copy number and homozygous

Tfam KO embryos completely lack mtDNA and die at embryonic day 8.5-10.5 (Larsson et al., 1998) demonstrating that Tfam is required for mtDNA maintenance in

vivo. Overexpression of the human TFAM protein upregulates mtDNA copy number

in mice (Ekstrand et al., 2004). TFAM likely controls mtDNA copy number by activating L-strand transcription and primer generation for H-strand replication

(Ekstrand et al., 2004). TFAM may also enhance mtDNA stability by acting as a histone-like protein.

Transmission and segregation of mammalian mtDNA

The mtDNA copy number spans usually a range of 200-5000 in different cell types (Ekstrand et al., 2004)(Bogenhagen and Clayton, 1974; Robin and Wong, 1988; Veltri

et al., 1990). Each mitochondrion contains several copies of mtDNA. In a given cell,

all mtDNA copies are presumably identical, a condition called homoplasmy. Mutations of mtDNA can coexist with wild-type mtDNA and this condition is called

heteroplasmy. As a consequence of this, the fraction of mutated mtDNA molecules has to reach a certain percentage, in order to cause disease. This usually happens

when the fraction of mutated mtDNA is higher than 60-80% (Lightowlers et al.,

1997). The mitochondrial genome is maternally inherited. The few mitochondria

from the sperm cell that enter the oocyte during fertilization are actively eliminated by

a ubiquitin-dependent mechanism (Sutovsky and Schatten, 2000). In some rare cases, paternal mtDNA can escape the mechanism of active elimination and be transmitted

to the muscle tissues of affected patients (Schwartz and Vissing, 2002). The mature oocyte contains 105 mtDNA molecules. The sperm cell has about 102 copies and there

is some evidence that downregulation of TFAM contributes to the observed

downregulation of mtDNA copy number during spermatogenesis (Larsson et al., 1997b; Rantanen et al., 2001). MtDNA undergoes a bottleneck phenomenon during

oogenesis by which a small subset of mtDNA molecules are amplified and transmitted to the offspring (Marchington et al., 1998). There is little mitochondrial proliferation

despite massive cell proliferation between the primordial germ cell in the 3 week-old

24

fetus and the diplotene primary oocyte in the 20 week-old fetus resulting in a

mitochondrial bottleneck (Poulton and Marchington, 2002). The reduction in mtDNA copy number is followed by amplification and selection of mtDNA later during

oogenesis. This phenomenon probably explains the shift towards homoplasmy within one or a few generations. It appears that the number of transmitted mtDNA molecules

(transmission units) is between 1-200 in the female germ line (Howell et al., 2000;

Jenuth et al., 1997). Studies of heteroplasmic mice demonstrate random genetic drift in some

tissues but in others strong, tissue-specific and age-related, directional selection for different mtDNA genotypes (Jenuth et al., 1996; Jenuth et al., 1997). This trait

showed linkage to loci on several chromosomes providing evidence for nuclear

control of mtDNA segregation (Battersby et al., 2003).

Regulation of mitochondrial gene expression and biogenesis

Different cell-types have a rather constant mtDNA copy number (Moraes, 2001) and

the ratio between mtDNA copy number and total DNA is cell-type specific (Shay et al., 1990). The mtDNA copy number in mammalian cells harboring wild-type

mtDNA, mtDNA with partial deletions or mtDNA with partial duplications is inversely proportional to the size of the respective mtDNA suggesting that cells

maintain a constant mass of mtDNA rather than a constant copy number of

mitochondrial genomes (Tang et al., 2000). The above study also reported a correlation between mtDNA copy number and the levels of mtDNA-encoded RNA

and polypeptides indicating that mtDNA copy number affects mtDNA expression

(Tang et al., 2000). Genetic studies have addressed the role of transcription and replication factors in the regulation of mtDNA copy number. Homozygous Tfam KO

mice display severe mtDNA depletion demonstrating the importance of Tfam for mtDNA maintenance (Larsson et al., 1998). Overexpression of human TFAM in

transgenic mice upregulates mtDNA copy number without affecting RC function and

mitochondrial mass, suggesting that regulation of mtDNA copy number can be dissociated from regulation of mtDNA expression and mitochondrial biogenesis

(Ekstrand et al., 2004). Studies regarding the regulation of mtDNA transcription have been carried out in isolated mitochondria and have revealed some degree of

autonomous regulation. External signals, for example a change in ATP concentration

25

or treatment with thyroid hormone activate mtDNA transcription. This may be

relevant in the local (subcellular) tuning of mitochondria (Enriquez et al., 1999a; Enriquez et al., 1999b; Enriquez et al., 1996). However, no evidence has been

provided yet that thyroid hormone receptors can use the mitochondrial transcriptional machinery to direct mitochondrial gene expression.

The vast majority of proteins required for mitochondrial biogenesis are

nucleus-encoded including: (i) most of the about 100 subunits of the RC; (ii) the metabolic enzymes needed for fatty acid β-oxidation, the TCA cycle, the biosyntheses

of certain amino acids and of heme; (iii) mitochondrial protein import and assembly

factors; (iv) factors of the mitochondrial replication, transcription and translation

machinery. These genes are under transcriptional control of the nuclear respiratory factors-1 (NRF-1) and -2 (NRF-2), thyroide hormone T3, stimulation protein 1 (Sp1),

and others (Kelly and Scarpulla, 2004). NRF-1 and -2 regulate the expression of most nucleus-encoded subunits of all respiratory complexes, mtDNA transcription and

replication factors, mitochondrial enzymes of the heme biosynthetic pathway,

mitochondrial protein import and assembly factors, and ion channels of the MOM. The expression of the human TFAM and TFB1M/TFB2M genes was found to rely on

functional NRF-1 and NRF-2 consensus sites in their promoters suggesting a regulatory link between nuclear and mitochondrial gene expression in mitochondrial

biogenesis (Kelly and Scarpulla, 2004; Virbasius and Scarpulla, 1994). However, this

contrasts with the presence of NRF-2 but absence of obvious NRF-1 sites in the human TFB1M/TFB2M promoters and rodent Tfam and Tfb1m/Tfb2m promoters

(Larsson et al., 1997a; Rantanen et al., 2001) (Rantanen et al., 2003). The transcriptional coactivator PPARγ coactivator-1α (PGC-1α) was cloned

in a yeast two hybrid screen for brown adipose-specific factors that interacted with the

adipogenic nuclear receptor PPARγ (Puigserver et al., 1998) and has proven to be a

key regulator of mitochondrial biogenesis in various tissues. Forced expression of

PGC-1α in adipogenic and myogenic mammalian cell lines induces the expression of

NRF-1, NRF-2 and Tfam. PGC-1α can interact directly with NRF-1 to activate the

mouse Tfam gene promoter (Wu et al., 1999). Overexpression of PGC-1α in primary

cardiac myocytes and in hearts of transgenic mice upregulates the expression of genes

involved in mitochondrial fatty acid β-oxidation (Lehman et al., 2000). Forced

expression of PGC-1α in skeletal muscle of transgenic mice drives mitochondrial

26

proliferation and the formation of mitochondrial-rich type I, oxidative (slow-twitch)

muscle fibres (Lin et al., 2002). PGC-1α interacts with a variety of nuclear-receptors

and non-nuclear receptors (Kelly and Scarpulla, 2004). Several upstream signaling events activate PGC-1α and mitochondrial biogenesis in a tissue-specific manner.

Upon cold exposure, the β-adrenergic/c-AMP pathway activates PGC1α- and UCP1-

expression in brown adipose tissue (Puigserver et al., 1998). Exercise stimulates PGC-

1α gene expression in skeletal muscle. Forced overexpression of

Calcium/calmodulin-dependent protein kinase (CaMK) or of PGC-1α in skeletal

muscle activates mitochondrial biogenesis (Lin et al., 2002; Wu et al., 2002).

Overexpression of CaMK and calcineurin A induces PGC-1α expression (Handschin

et al., 2003; Wu et al., 2002) suggesting that CaMK and calcineurin A are upstream of PGC-1α. Nitric oxide (NO) can activate PGC-1α and mitochondrial biogenesis in

adipocytes and HeLa cells. The NO effect is dependent on cGMP activation and does

not result from inhibition of oxidative phosphorylation by NO (Nisoli et al., 2003).

Mitochondrial bioenergetics and ROS formation

The respiratory chain

The RC consists of five distinct complexes (Complex I-V) that are bound to the mitochondrial inner membrane. Electrons are fed to Complex I or II and transferred to

Complex III and IV. During the passage of electrons a proton gradient across the inner membrane is built up by proton translocation at Complex I, III and IV. The

proton gradient is then used by Complex V to synthesize ATP from ADP and

phosphate (Figure 4). This general mechanistic principle of oxidative phosphorylation explaining the coupling between respiration and ATP synthesis in mitochondria is

referred to as the chemiosmotic theory and was proposed by Peter Mitchell in 1961

(Mitchell, 1961). Recent observations suggest that the different respiratory complexes are organized in supercomplexes (Schagger and Pfeiffer, 2000). The crystal structures

of Complex II-V have been elucidated to some extent and important mechanistic conclusions have been drawn (Saraste, 1999).

27

Figure 4 The respiratory chain consists of 5 complexes, designated Complex I-V. Complex II does not translocate protons and is completely nucleus-encoded. Reprinted with permission from (Saraste, 1999). © AAAS. Complex I (NADH:ubiquinone oxidoreductase) is the largest of all complexes, consisting of at least 46 subunits, one flavin mononucleotide (FMN), seven or eight

different iron-sulfur centers, covalently bound lipids and at least 3 bound ubiquinol molecules. Seven subunits (ND1-ND6, ND4L) are encoded by mtDNA. Two

electrons from NADH enter Complex I. The electrons are transferred along an

electron transport chain consisting of FMN, iron-sulfur centers and bound ubiquinones to a mobile ubiquinone (Q) that takes up electrons to yield ubiquinol

(QH2) (Stryer, 1995). The flow of 2 electrons from NADH to ubiquinone leads to the pumping of 4 protons from the mitochondrial matrix side to the IMS. Electron

microscopy (EM) studies demonstrated that Complex I has an L-shaped structure with

two major domains separated by a thin collar (Grigorieff, 1998; Guenebaut et al., 1998).

Complex II, succinate:ubiquinone reductase (SQR) or succinate dehydrogenase (SDH), is completely nucleus-encoded and a component of the TCA

cycle. It catalyzes the conversion of succinate to fumarate using FAD (flavine-adenine

dinucleotide) as a cofactor. This reaction yields FADH2. FADH2 donates 2 electrons that are transferred to Q along several iron-sulfur centers. Mammalian mitochondrial

and many bacterial SQR are composed of two hydrophilic subunits, a flavoprotein

(SdhA) and iron-sulfur protein (SdhB), and two hydrophobic membrane anchor subunits, SdhC and SdhD (Figure 6). The crystal structure of E.coli SQR has recently

been determined (Yankovskaya et al., 2003). SQR is composed of a trimer. Each monomer consists of one SdhA, SdhB, SdhC and SdhD subunit. The SdhA subunit

contains the FAD cofactor and the substrate-binding site. The SdhB subunit contains

28

three iron-sulfur clusters for electron transfer from the substrate-binding site to the

ubiquinone-binding site. The SdhC and SdhD subunits form a membrane-bound cytochrome b containing the ubiquinone-binding site that is sandwiched between the

SdhC and SdhD subunits (Figure 5). The residues in the ubiquinone-binding site are strictly conserved among human, mouse and E.coli SQR. Importantly, mutations of

the residues in the ubiquinone-binding site are associated with hereditary

paraganglioma (Astuti et al., 2001a; Astuti et al., 2001b) and premature ageing in C.

elegans mev-1 mutant (Ishii et al., 1998). The observed increase in ROS levels in the

C. elegans mev-1 mutant is explained by leakage of electrons that are released from succinate but not accepted by ubiquinone because of the dysfunctional binding site.

Figure 5 Three-dimensional structure of E.coli SQR. SQR is a trimer viewed parallel to the plasma membrane (top left), viewed from the cytoplasm normal to the plasma membrane (top right) or viewed as a monomer parallel to the membrane (bottom). One SQR monomer is composed of 2 hydrophilic subunits, SdhA and SdhB, and 2 hydrophobic subunits, SdhC and SdhD that are buried in the plasma membrane and provide the ubiquinone binding site. Reprinted with permission from (Yankovskaya et al., 2003). © AAAS.

29

Similarly, it has been proposed that mutations of the SdhB, SdhC and SdhD subunits

associated with hereditary paraganglioma cause ROS formation and thereby predispose to cancer (Rustin et al., 2002).

Complex III (cytochrome bc1) delivers electrons from QH2 to cytochrome c. The transfer of electrons is coupled to the transfer of protons across the inner

membrane by the so-called Q cycle (Figure 6). The mammalian Complex III is a

dimer and each monomer contains eleven subunits but only three of them carry the redox centers for electron transfer. These key subunits are: (i) cytochrome b, the only

mtDNA encoded subunit of Complex III; (ii) a membrane-anchored iron-sulphur protein (ISP) carrying a Rieske-type center (Fe2S2); and (iii) a membrane anchored

cytochrome c1. There are two active sites in Complex III (Iwata et al., 1998; Xia et al.,

1997; Zhang et al., 1998b): one for oxidation of QH2 and release of protons on the outer surface of the membrane, Qo; and one for the reduction of Q coupled to the

uptake of protons from the inner side of the membrane, Qi. In the first step of the Q

cycle, one ubiquinol donates one electron to the to the Rieske-type iron-sulphur center and one electron to the bL heme group of the cytochrome b subunit. The donation of

both electrons is coupled to the release of two protons to the intermembrane space at the Qo site. The first electron passes from the Rieske-type iron-sulphur center to the

heme c group of cytochrome c1 and from there to cytochrome c. The second electron

passes from the bL heme to the bH heme group of cytochrome b and is then transferred back to a new incoming ubiquinone molecule at the Qi site of cytochrome b. A

subsequent Q cycle provides a second electron at the Qi site to reduce ubisemiquinone (Q·) to QH2 that can again enter the Q cycle.

Complex IV (cytochrome c oxidase) contains 13 subunits (Figure 7). The three

major subunits are encoded for by mtDNA (COXI-III) and form the functional core of complex IV. This core is surrounded by 10 smaller nucleus-encoded subunits. Subunit

II receives electrons from cytochrome c. These electrons are first transferred to cytochrome a in subunit I and then to the bimetallic cytochrome a3/CuB active site in

subunit I. Two hydrophilic channels, called D and K, connect the active site to the

aequeous phase of the mitochondrial matrix. The reduction of oxygen at the active site in subunit I is linked to the translocation of four protons, two for the reduction of

30

Figure 6 Structure of the bovine mitochondrial cytochrome bc1 and the Q cycle. Cytochrome bc1 is a dimer and each monomer consists of 11 subunits (left). The functional core of the enzyme consists of three subunits: cytochrome b, Rieske ISP and cytochrome c1 (middle). In the Q cycle, bifurcation of electron transfer occurs at the Qo site. One electron is transferred to the Fe2S2 center of Rieske ISP and from there to cytochrome c1. The second electron is transferred to the heme bL and from there to the heme bH of cytochrome b and delivered back to a new incoming ubiquinone at the Qi site. Reprinted with permission from (Saraste, 1999). © AAAS.

Figure 7 Structure of the bovine cytochrome c oxidase. The functional core of the enzyme consists of subunits I-III (COXI-III) that are mtDNA encoded. Subunits I and II contain the metal centers and the active site (cytochrome a3/CuB) resides in subunit I. Protons that are used for reducing oxygen to water or pumped to the IMS are transferred through two channels, D and K, from the matrix side. Reprinted with permission from (Saraste, 1999). © AAAS.

31

oxygen into water, and two for release into the intermembrane space (Tsukihara et al.,

1995; Tsukihara et al., 1996; Yoshikawa et al., 1998). Complex V (F1F0 ATPase) synthesizes ATP using a proton-motive force

across the inner mitochondrial membrane, but it can also hydrolyze ATP to pump protons against an electrochemical gradient. The bovine enzyme appears to contain 16

subunits (Lutter et al., 1993). It consists of a membrane bound part containing a

proton channel, called Fo, and of a catalytic part located in the matrix, called F1, which contains an ATP synthesizing or hydrolyzing activity (Figure 8). MtDNA encodes 2

subunits of the Fo part. The Fo and F1 parts are connected by two parallel structures, referred to as the “rotor” and the “stator” (Elston et al., 1998). The F1 part can be

detached from the F0 part and acts as a soluble ATPase. The F1 part consists of 5

subunits (α, β, γ, δ, ε). The β subunit contains the catalytic site and there are three

active sites per one F1F0 ATPase because each F1 part contains three β subunits. Each

catalytic site passes through a cycle of open (unbound) state, loose (bound ADP and phosphate) state and tight (tightly bound ATP) state. The formation of ATP requires

energy for substrate binding and ATP release but not for the phosphorylation reaction

itself. The crystal structure of the F1 part of the bovine F1F0 ATPase indicates that it operates as a rotational catalyst (Abrahams et al., 1994). A central structure inside the

Figure 8 Structure of ATP synthase. The F1 part corresponds to the crystallized bovine ATP synthase (Abrahams et al., 1994) and the remaining parts to the crystal structure of the bacterial ATP synthase (Lutter et al., 1993). A dodecamer of subunit c, the ε and γ subunits form the rotor, while subunits b, d and δ form the stator. The active sites are present in the β subunits. Reprinted with permission from (Saraste, 1999). © AAAS.

32

F1 ATPase rotates in 120o steps during catalysis (Noji et al., 1997; Sabbert et al.,

1996; Yasuda et al., 1998). The membrane bound Fo part consists of three subunits called a, b and c. The subunit c forms a dodecamer and is thought to constitute the

rotor together with the γ and ε subunits of the F1 part (Figure 8). The subunit a and b

form the stator together with the δ subunit of the F1 part. The mechanism how ATP

synthesis is coupled to proton transfer across the inner membrane has not been

clarified yet. The current model proposes that proton movement through the interface between a-subunit and the subunit c dodecamer (Figure 8) causes the rotor and stator

to move in opposite directions thereby causing a torque. The subsequent release of free energy by the torque may allow substrate binding and release of ATP,

respectively (Elston et al., 1998; Wang and Oster, 1998). Complex V has recently

been ascribed a role in the biogenesis of the mitochondrial inner membrane (Paumard et al., 2002).

Generation of ROS by the respiratory chain

O2-· is constantly produced in respiring mitochondria at a rate of about 1-3% of all

oxygen consumed (Boveris and Chance, 1973). O2-· dismutates spontaneously to H2O2

and this reaction is accelerated by superoxide dismutase (SOD). H2O2 can react with reduced transition metals, for example with Fe2+ in the Fenton reaction (Fe2+ + H2O2

-> Fe3+ + OH + OH·), and give rise to the highly reactive hydroxyl radical (OH·). O2-·,

H2O2 and OH· are collectively referred to as reactive oxygen species (ROS). It is difficult to determine the exact site of O2

-· generation because O2-· has a

short lifetime due to its rapid conversion to H2O2 by SOD or because it immediately reacts with lipids of the mitochondrial inner membrane. H2O2 is more stable than O2

-·

and can diffuse through the membrane lipid bilayer out of the mitochondrion. Most of

the O2-· is probably produced at the matrix side of the inner mitochondrial membrane

because O2-· generation is only found in submitochondrial particles, which are inside

out with respect to mitochondria while in intact mitochondria only H2O2 is detectable extramitochondrially. However, a recent study using spin traps revealed O2

-·

formation in mitoplasts (mitochondria devoid of the outer membrane) (Han et al.,

2001). O2

-· generation depends on whether mitochondria are actively respiring (low

O2-· production) or the RC is highly reduced (high O2

-· production). The rate of O2-·

33

generation increases when electron flow slows down or the concentration of oxygen

increases. The proton gradient built up during respiration is dissipated through complex V to synthesize ATP. In the absence of complex V activity, for example

following inhibition by oligomycin, the proton gradient builds up massively increasing the mitochondrial membrane potential and causing electron flow to slow

down and the RC to become more reduced. This results in increased steady-state

concentration of O2-· (Boveris et al., 1972). Inhibitors of the RC result in O2

-· generation due to increased reduction of the carriers upstream of the site of inhibition

and have revealed specific sites of O2-· formation within complex I, II, and III. In

complex III, O2-· is formed when the electron flow between the bL and bH heme of

cytochrome b is blocked, for example by antimycin A (Figure 9). Reoxidation of

ubisemiquinone (Q·) to ubiquinone (Q) at the outer side of the membrane and reduction of Q· to ubiquinol (QH2) at the inner side of the membrane are inhibited,

leading to increased steady-state concentrations of Q· (Figure 9). O2-· formation by

Figure 9 The Q cycle in complex III. Ubiquinone (Q) is reduced to ubiquinol (QH2) either by electron transfer from Complex I or Complex II or by electrons transferred from cytochrome b of complex III. QH2 releases one electron to the Rieske ISP, cytochrome c1 (c1) and cytochrome c (c). The second electron is released at Qo from a ubisemiquinone (Q·) and transfers to the heme groups of cytochrome b to reduce a new Q at the Qi site. Superoxide is probably produced at the two sites, Qo and Qi, but the contribution of each site has not been clearly determined yet, Antimycin A blocks electron transfer between bL and bH heme of cytochrome b.

34

antimycin A is blocked when electron flow between the Rieske protein and

cytochrome c1 or oxygen is inhibited, for example by myxathiazol or cyanide, indicating that O2

-· formation depends on generation of Q· by the Q-cycle (Figure 9)

(Trumpower, 1990; Turrens, 2003; Turrens et al., 1985). In Complex I, O2-·

production likely occurs in one of the iron-sulphur clusters, either N1 or N2 upstream of the binding site for ubiquinone and rotenone (Genova et al., 2001; Kushnareva et

al., 2002). In Complex II, mutations affecting the ubiquinone binding site result in

electron leakage and increased O2-· formation (Yankovskaya et al., 2003). Reduced

FAD was also identified as an electron donor to oxygen in reconstituted complex II

(Zhang et al., 1998a).

Antioxidant defense mechanisms

Antioxidant defenses are classified into non-enzymatic and enzymatic defenses. The

non-enzymatic defenses are compounds that reduce oxidizing agents, for example the lipid soluble vitamin E and ubiquinone, or the water-soluble vitamin C, glutathione,

uric acid and ceruloplasmin (Halliwell and Gutteridge, 1989).

The superoxide dismutases (SOD) are the first line of enzymatic antioxidant defense. SOD catalyze the reaction O2

-· + O2-· —> H2O2 + O2. All members of the

SOD familiy utilize different transition metals at their active sites. There are three

different SOD isoforms (Fridovich, 1995). SOD1 (Cu/ZnSOD) is expressed in the cytosol and in the mitochondrial intermembrane space (Okado-Matsumoto and

Fridovich, 2001). SOD2 (MnSOD) is expressed in the mitochondrial matrix and is closely related to the bacterial MnSOD. The expression of SOD2 is induced by agents

that cause oxidative stress, for example radiation and hyperoxia through oxidative

activation of the transcription factor NFkB and by various cytokines, for example TNFα , IL-1 and IFN-γ (Li and Karin, 1999). SOD2 is transported into the

mitochondrial matrix where it assembles into an active homo-tetramer (Fridovich, 1995). Hydrogen peroxide, the product of the dismutation reaction is reduced to water

and oxygen mainly by glutathione peroxidases and to a lesser extent by catalases. Glutathione peroxidases utilize glutathione (GSH) as a reductant according to the

following reaction: H2O2 + 2 GSH —> 2H2O + GSSG. There are four GPx isoforms.

The major GPx isoform, GPx1, is expressed in mitochondria and cytosol of most tissues (Esposito et al., 2000; Frampton et al., 1987). GPx2 is found in plasma

35

(Maddipati and Marnett, 1987), GPx3 in gastrointestinal tract, i.e. liver, intestine and

colon (Chu et al., 1993; Chu and Esworthy, 1995), and GPx4 (phospholipid GPx) in the mitochondria of testis and brain (Arai et al., 1999; Esposito et al., 2000).

Glutathione is a tri-peptide consisting of L-γ-glutamyl-L-cysteinyl-glycine. Each GSH

molecule provides one reducing equivalent to the conversion of H2O2 into water and oxygen by the sulfhydril moiety of the cysteine residue leading to the formation of

oxidized GSH in form of the disulfide–bonded compound GS-SG. The enzyme

glutathione reductase utilizes NADPH to re-reduce one molecule of GS-SG into two molecules of GSH: GS-SG + 2NADPH —> 2GSH + 2 NADP+.

Catalases are almost exclusively found in peroxisomes to remove H2O2 formed

during β-oxidation of long chain fatty acids. Catalases have only been detected in

heart mitochondria but not in other mitochondria (Radi et al., 1991). Mitochondrial uncoupling may provide another line of anti-oxidant defense

(Skulachev, 1996). Uncoupling decreases the mitochondrial membrane potential, leading to increased respiration, oxidation of the RC and lowered O2

-· formation. A

threshold membrane potential exists below which no ROS is formed (Korshunov et

al., 1997). In rat hepatocytes, the futile cycle of proton pumping and proton leak may account for 20-25% of respiration. In perfused rat muscle, this value is increased to

35% in contracting and 50% in resting muscle. Mitochondrial uncoupling proteins (UCP) have been proposed to regulate O2

-· formation and there is evidence that O2-·

directly activates UCP (Brand, 2000).

Damage repair and removal systems have been documented for oxidized proteins (Davies, 2000). Cysteine residues can form a disulfide bond on the same

protein or a disulfide crosslink between two different proteins upon oxidation. The re-reduction is performed by disulfide reductases. Oxidized proteins can be also be

recognized by proteases and completely degraded into amino acids. In the cytoplasm

and the nucleus of eukaryotic cells the oxidized proteins can be recognized and degraded by the proteasome complex. In mammalian mitochondria, there is a separate

set of proteases that conduct the degradation of oxidized proteins, for example LON

protease (Bota and Davies, 2002). Membrane phospholipids undergo lipid peroxidation upon oxidative stress

(Halliwell and Gutteridge, 1989). Lipid peroxidation is the result of a chain reaction: It is initiated by extraction of a hydrogen atom from an unsaturated fatty acyl, for

36

example by OH· (O2-· is not reactive enough to induce lipid peroxidation by itself)

giving rise to a lipid radical (L·). L· then reacts with oxygen producing a lipid peroxy radical (LOO·). LOO· can further propagate the peroxidation chain reaction by

extracting a hydrogen atom from another unsaturated fatty acid. The lipid hydroperoxides (LOOH) can easily decompose into other reactive species.

Peroxidized membranes become rigid, loose selective permeability, and can even

loose their integrity. Water-soluble lipid peroxidation products, such as dialdehydes, can diffuse from membranes into other subcellular compartments and cause protein

aggregation leading to formation of the age pigment lipofuscin. Lipid peroxidation products can also impair enzyme function or may form DNA adducts and induce

mutations. Oxidized lipid bilayers become more susceptible to the action of

phospholipases, which hydrolyze the phospholipid glycerol backbone to release a free fatty acid hydroperoxide. Fatty acid hydroperoxides are detoxified by glutathione

peroxidases reducing them to their corresponding hydroxy fatty acid (Davies, 2000).

Oxidative damage of DNA interferes with DNA replication and transcription. The extent of oxidative DNA damage has been estimated to be as high as 1 base

modification per 130.000 bases in nuclear DNA. Damage to mtDNA is estimated to be even higher at 1 per 8.000 bases. Oxidants can give rise to strand breaks (single

and double) by damaging the phosphodiester backbone, sister chromatid exchange,

DNA-DNA and DNA-protein crosslinks, and base modifications. The base modifications can result in dissociation of the base from the DNA double strand,

creating so called apurinic/apyrimidinic sites. Several DNA repair enzyme systems exist that can repair oxidatively damaged DNA (Davies, 2000).

Adaptive responses of cells to oxidative stress involves transient growth-arrest

to enable DNA damage repair and expression of factors involved in damage removal/repair and antioxidant defenses, for example catalase, GPx and SOD2,

mediated by several transcription factors (Shull et al., 1991) (Davies, 2000). Cells exposed to oxidative stress may ultimately undergo apoptosis or

necrosis. The mechanism involves activation of the mitochondrial permeability

transition pore and cytochrome c release (see below).

37

Mitochondrial uncoupling proteins and their role in energy expenditure and control of ROS homeostasis

The mitochondrial uncoupling proteins (UCP) belong to the large family of anion carriers of the mitochondrial inner membrane containing six transmembrane domains

(Ricquier and Bouillaud, 2000). The first identified uncoupling protein, UCP1, is

specifically expressed in brown adipocytes. Based on cDNA sequence similarity, two homologues of UCP1 were identified and named UCP2 (Fleury et al., 1997) and

UCP3 (Boss et al., 1997; Gong et al., 1997; Vidal-Puig et al., 1997). UCP2 and UCP3 share, respectively, 72 and 57% amino acid identity with UCP1. Of note, UCP2 and

UCP3 are adjacent genes on human chromosome 11 and mouse chromosome 7. UCP2

mRNA is found almost ubiquitously, but the protein has been identified only in some organs and cell types including spleen, lung, stomach, brain, kidney, thymocytes and

pancreatic β-cells (Pecqueur et al., 2001; Zhang et al., 2001). UCP3 is mainly

expressed in skeletal muscle. UCP1 KO mice are cold sensitive but not obese and an extension of these studies demonstrated that UCP1 is the only effector of adaptive

non-shivering thermogenesis to cold exposure (Enerback et al., 1997; Golozoubova et

al., 2001). UCP1 creates a proton leak across the inner membrane of brown fat mitochondria to dissipate the electrochemical proton gradient into heat. Brown

adipose tissue (BAT) is a particular form of adipose tissue required for thermogenesis

in infants at birth or rodents exposed to cold. Brown adipocytes differ from white adipocytes by direct sympathetic innervation, a central nucleus, and numerous

mitochondria. Sympathetic innervation of brown adipocytes induces lipolysis, respiration and thermogenesis, accompanied by a marked increase in blood flow and

heat conductance to other organs by the blood stream. UCP1 is abundantly expressed

in brown adipocytes accounting for up to 4% of the total and 8% of the mitochondrial protein content upon cold acclimation. UCP1 expression is induced by the

Norepinephrine/cAMP-pathway leading to the activation of cAMP response elements

(CRE) in the UCP1 promoter. Various transcription factors, i.e. PPARα, PPARγ, and

thyroid hormone T3 bind to a complex enhancer region upstream of the UCP1 promoter and can stimulate UCP1 expression by co-activation with PGC-1α, but the

precise roles of these factors in the regulation of UCP1 expression have not been clarified yet (Cannon and Nedergaard, 2004). Reconstituted UCP1 exhibits fatty-acid

dependent proton translocating activity (Klingenberg et al., 2001). The proton

38

translocation activity of native UCP1 is stimulated by fatty acids and inhibited by

purine nucleotides, such as GDP, in a competitive manner (Shabalina et al., 2004). Of note, fatty acids are obligatory for uncoupling. There are currently two competing

models explaining the proton location mechanism of UCP. In the first model, UCP1 is a pure proton carrier (Klingenberg and Echtay, 2001). In the second model, UCP1 is

an anionic fatty acid exporter: protonated fatty acids move from the external to the

internal lipid layer of the mitochondrial inner membrane by a flip-flop mechanism, release a proton into the matrix and then return as an anionic fatty acid to the cytosol

by UCP-mediated export (Garlid et al., 1998). UCP2 and UCP3 were proposed to work as uncouplers similar to UCP1.

Polymorphic markers encompassing the UCP2-UCP3 locus show genetic linkage to

the resting metabolic rate (Bouchard et al., 1997). Similarly, polymorphisms in the coding region of the UCP2 gene are linked to the metabolic rate at sleep (Walder et

al., 1998). However, homozygous UCP2 and UCP3 KO mice have normal metabolic

rates (Arsenijevic et al., 2000; Vidal-Puig et al., 2000) but compound homozygous UCP2/UCP3 KO mice have not been analyzed yet due to linkage of the UCP2 and

UCP3 genes. UCP2 and UCP3 KO mice show signs indicative of increased ROS production (Arsenijevic et al., 2000; Vidal-Puig et al., 2000) supporting the

hypothesis that UCP2 and UCP3 function as uncouplers to lower mitochondrial ROS

formation. Consistent with this hypothesis, skeletal muscle mitochondria from UCP3 KO mice show improved respiratory control ratios (Vidal-Puig et al., 2000) and

enhanced in vivo ATP production rates (Cline et al., 2001) indicative of improved coupling. UCP2 and UCP3 have been reconstituted into liposomes and both exhibit

proton translocation activity that requires the presence of fatty acids and is inhibited

by GDP (Jaburek et al., 1999; Jezek et al., 2004). A recent report demonstrated superoxide–mediated activation of UCP1, UCP2 and UCP3. This mechanism was

proposed as a feedback control to lower excessive O2-· production by uncoupling

(Echtay et al., 2002b). Superoxide–mediated activation of UCP is fatty-acid

dependent and inhibited by purine nucleotides (Echtay et al., 2002b). The superoxide-

activation of UCP2 occurs from the matrix side (Echtay et al., 2002a). This finding has potentially important consequences for energy metabolism due to the widespread

tissue distribution of UCPs and their suggested role as uncouplers. The hypothesis that O2

-· activates UCPs however is not universally accepted (Couplan et al., 2002).

39

The experiments leading to the conclusion that superoxide activates UCP were

conducted on isolated mitochondria exposed to supraphysiological levels of exogenous superoxide (Echtay et al., 2002a; Echtay et al., 2002b) and only one study

that used forced, adenovirus-mediated SOD2 overexpression in islet cells has indicated that superoxide-stimulated UCP-mediated uncoupling may also occur in

vivo (Krauss et al., 2003).

High fat diet of human subjects does not change mitochondrial coupling despite clear upregulation of UCP3 protein content in muscle (Hesselink et al.,

2003a). It has been proposed that UCP3 functions as an anionic fatty acid exporter to prevent accumulation of free fatty acids inside the mitochondria that cannot enter the

mitochondrial fatty acid β-oxidation cycle. Accumulation of anionic free fatty acids

causes mitochondrial dysfunction, a phenomenon called lipotoxicity. This role of

UCP3 is consistent with: (i) upregulation of UCP3 expression by conditions increasing circulating FFA (fasting, high fat diet, diabetes, and obesity); (ii) an

inverse relationship between mitochondrial fat oxidation capacity and UCP3 protein

levels (low fat oxidation capacity in untrained individuals correlates with high UCP3 protein content; high fat oxidation capacity in trained individuals correlates with low

UCP3 protein levels) (Hesselink et al., 2003b).

Mitochondrial regulation of apoptosis

Apoptosis is a distinct genetic and biochemical pathway required for normal

embryonic development and maintenance of normal tissue homeostasis. Apoptosis was initially defined as a morphological entity. The morphological hallmarks of

apoptosis are cell shrinkage, condensation and fragmentation of the cell at later stages

followed by phagocytosis of the cell remnants by neighbouring cells. Apoptosis is an active process that does not cause inflammation. In contrast to apoptosis, necrosis is a

passive process caused by massive cellular injury. The cell swells because it is unable to maintain ion homeostasis, leading to rupture and release of intracellular contents

causing inflammation (Kerr et al., 1972). Mitochondria are key regulators of apoptosis

because many factors activating apoptosis are kept in the mitochondrial intermembrane space (IMS) and become active when released into the cytosol. These

pro-apoptotic factors include cytochrome c, apoptosis-inducing factor (AIF), SMAC/DIABLO, and Endonuclease G (Endo G) among others. Release of pro-

40

apoptotic factors from the mitochondrial IMS is regulated to prevent accidental

activation of apoptosis. The pro-apoptotic factors are impermeable to the mitochondrial outer membrane. At least three distinct release mechanisms have been

proposed: (i) The existence of a proteinaceous permeability transition pore (PTP) has

been postulated at contact sites between the inner and the outer mitochondrial

membrane (Figure 10) (Susin et al., 1998). The PTP is proposingly formed by the adenine nucleotide translocator (ANT) and cyclophyllin D at the inner membrane

associated with VDAC and the peripheral benzodiazepine receptor in the MOM. Sustained opening of the PTP results in osmotic equilibration of ions between matrix

and cytosol, dissipation of the mitochondrial inner membrane potential and

mitochondrial swelling, a biophysical state called permeability transition (PT). The MOM ruptures because the MOM cannot expand as much as the highly invaginated

MIM. Several triggers of PT have been described, notably lowered mitochondrial

membrane potential, Ca2+, fatty acids, ROS hyperproduction, NAD(P)H depletion, Nitric oxide (NO) and ATP depletion. Cyclosporin blocks opening of the PTP, while

Figure 10 A putative model of the mitochondrial permeability transition pore (Green and Reed, 1998).

atractyloside activates PTP opening. Interactions between BCL-2 protein family members, BAX and BCL-2 (see below), modulate the opening of the postulated PTP

pore. However, gene knockout studies do not support a requirement for ANT, one of

the postulated components of the PTP, for cells to undergo PT (Kokoszka et al., 2004). Furthermore, the importance of the PTP has been questionend in the majority

41

of cell deaths because protein release can often occur in the absence of any of the

hallmarks of PT (Bossy-Wetzel et al., 1998; Newmeyer and Ferguson-Miller, 2003). (ii) Members of the BCL2-family form channels in the MOM that allow

passage of large macromolecules (Danial and Korsmeyer, 2004). The proteins of the BCL-2 family are structurally classified according to the presence of four conserved

regions called the BCL-2 homology (BH) 1 - 4 domains. Anti-apoptotic BCL-2

family proteins (BCL-2, BCL-xL, A1 and BCL-W) contain all BH1-4 domains. Pro-apoptotic BCL-2 family proteins can be subdivided into members containing the BH1

- 3 domains (BAX, BAK) or the BH3 domain only (BID, BAD, BIM, Noxa, Puma). Cell line studies showed that the pro-apoptotic BCL-2 family members, BAX and

BAK, present as inactive monomers in unstimulated cells homo-oligomerize to form

channels that allow passage of cytochrome c (Desagher et al., 1999; Wei et al., 2000). Homo-oligomerization of recombinant monomeric BAX requires recombinant

protease-activated BID (tBID) in the absence of any other protein to permeabilize

membrane vesicles, allowing passage of extremely large macromolecules (Kuwana et al., 2002). This model accounts for the fact that most of the cytochrome c release

clearly occurs prior to swelling of the mitochondria, and that BAX/BAK compound homozygous KO cells are resistant to most stimuli that activate mitochondrial

induction of apoptosis (Lindsten et al., 2000; Wei et al., 2001a). Anti-apoptotic BCL-

2 family members, BCL-2 and BCL-xL, bind and sequester pro-apoptotic BCL-2 family members by interaction via their BH3 domain (Cheng et al., 2001; Oltvai et al.,

1993). Accordingly, the ratio of anti- to pro-apoptotic molecules is one determinant of susceptibility to apoptosis induction (Oltvai et al., 1993).

(iii) BCL-2 family proteins interact with VDAC in the mitochondrial outer

membrane to release pro-apoptotic factors from the IMS. VDAC reconstituted into liposomes mediates passage of cytochrome c. In this model, BAX and BAK stimulate

opening of the VDAC while BCL-xL stimulates VDAC closure (Shimizu et al., 1999). However, genetic proof for this model is lacking and genetic studies indicate

that one of the three VDAC isoforms, VDAC-2, binds BAK and inhibits BAK

oligomerization and BAK-mediated apoptosis (Cheng et al., 2003). The release of cytochrome c is rapid and the extent almost complete

(Goldstein et al., 2000). The minor fraction of the total cytochrome c, about 20%, is located in the very narrow IMS adjunct to the MOM, whereas most of the cytochrome

42

c (about 80%) is stored within involutions of the MIM, so called tubular cristae which

is a highly sequestered compartment separated from the more narrow IMS through narrow cristae junction. In one EM study, tBID induced striking remodeling of the

tubular cristae, mobilizing the stores of cytochrome c (Scorrano et al., 2002). The redistribution was accompanied by a transient opening of the PTP but not by swelling

of the mitochondria. In the opening state of the PTP solutes of up to 1500Da are

permeable (Bernardi et al., 1999). Transient PTP opening thus remodels tubular cristae and redistributes the cytochrome c pool.

Upon release from the IMS, cytochrome c binds to Apaf-1 and this complex recruits pro-caspase 9 in the presence of ATP to form the apoptosome (Li et al., 1997;

Liu et al., 1996). The binding of cytocrome c and ATP to Apaf-1 results in a

conformational change of Apaf-1 that enables Apaf-1 to bind six pro-caspase 9 molecules (Acehan et al., 2002). Caspases are specific proteases that activate effectors

of the apoptotic programme (Shi, 2002). Caspases are usually present as inactive

zymogens (pro-caspase) possessing a large and small subunit. They cleave specific motifs possessing an aspartate. The cleavage depends on a cysteine and occurs at the

aspartate (Asp) site, therefore the name “caspase”. Cleavage of internal specific Asp residues releases one large and one small subunit that dimerize to form the active site

of the enzyme. Caspases can be divided into two groups, initiator and effector

caspases. Initiator caspases are usually upstream caspases that possess the ability of autocatalytic activation. Downstream effector caspases need initiator caspases for

their activation. The formation of the apoptosome enables autolytic activation of pro-caspase 9 into active caspase 9. The binding of six pro-caspase 9 molecules to one

Apaf-1 molecule allows for an amplification of the initial apoptotic signal. Activated

caspase 9 in turn activates the effector caspases 3 and 7. Effector caspases cleave structural components, for example actin and nuclear lamin, and DNA fragmentation

factor 45 (DFF45). Cleavage of DFF45 by caspase 3 releases the DFF45-DFF40 dimer. DFF40 subsequently degrades chromosomes into nucleosomal fragments

(Enari et al., 1998; Liu et al., 1997). Mitochondria also release AIF (Susin et al.,

1999) and EndoG (Li et al., 2001). AIF and Endo G translocate to the nucleus and induce caspase-independent large-scale DNA fragmentation. Mitochondria also

release SMAC/DIABLO, an inhibitor of the inhibitor of apoptosis proteins (IAP) that block effector caspases (Chai et al., 2000; Verhagen et al., 2000).

43

Apoptosis can be triggered by the activation of cell death receptors on the

plasma membrane and can involve mitochondrial release of cytochrome c. The binding of Fas ligand to Fas (receptor for Fas ligand) or of TNFα to the TNFα

receptor, results in homo-oligomerization of the receptor, binding of adaptor proteins

to the oligomerized receptors and recruitment of pro-caspase 8, an intiator caspase, to the receptor complex (Baud and Karin, 2001) leading to autocatalytic activation of

caspase 8. Activated caspase 8 can directly cleave caspase 3 in so-called type-I cells,

for example in immune cells. In so called type-II cells, for example in hepatocytes, a mitochondrial amplification step is required for efficient activation of caspase 3 by

cell death receptors (Scaffidi et al., 1998). In type- II cells, activated caspase 8 cleaves

BID yielding tBID that induces cytochrome c release (Li et al., 1998; Luo et al., 1998) by mediating oligomerization of BAK (Wei et al., 2001a) or BAX (Desagher et al.,

1999). Ca2+ can trigger induction of mitochondrial PT and promote apoptosis or

necrosis. IP3-mediated Ca2+ release from the endoplasmic reticulum (ER) can cause

PT and apoptosis (Szalai et al., 1999). The Ca2+ flow between the ER and mitochondria is facilitated by close contact sites (Rizzuto et al., 1998). BCL-2 reduces

the amount of Ca2+ releasable from the ER and thus prevents mitochondrial Ca2+ overload (Vanden Abeele et al., 2002). Massive cytosolic Ca2+ overflow can cause

necrosis following a wide variety of cellular injuries, for example following

excitotoxic stimulation of neurons. It has been demonstrated that glutamate-induced neuronal cell death by stimulation of NMDA receptors requires mitochondrial Ca2+

uptake (Stout et al., 1998). Glutamate induces necrosis or apoptosis of neurons in culture dependent on mitochondrial function as reflected by the mitochondrial

membrane potential and cellular ATP content (Ankarcrona et al., 1995). The

intracellular ATP level determines the cell death fate by apoptosis or necrosis (Eguchi et al., 1997; Leist et al., 1997). Inhibition of oxidative phosphorylation with

oligomycin reduces staurosporine (STP) and FAS-stimulated apoptosis of T-lymphocytes and leads to enhanced necrosis (Eguchi et al., 1997; Leist et al., 1997).

An ATP depeletion of more than 50-70% is required to shift the cell death modus

towards necrosis (Leist et al., 1997). Addition of glucose to cells pretreated with oligomycin changes the mode of cell death from necrosis to apoptosis indicating that

44

glycolytic ATP production may allow execution of apoptosis in cells devoid of

oxidative phosphorylation (Leist et al., 1997).

Free radical theory of aging

The free radical theory of ageing proposes that mitochondrial ROS generation

determines the rate of ageing (Sohal and Weindruch, 1996). This is supposed to result from an imbalance between prooxidants and antioxidants leading to an age-dependent

accumulation of oxidative cell-damage that exceeds the capacity of cellular repair systems. This theory is based on the following observations: (i) Variations in

longevity among different species inversely correlate with the rates of mitochondrial

generation of superoxide and hydrogen peroxide; (ii) Restriction of caloric intake lowers steady-state levels of oxidative stress and damage, retards age-associated

changes, and extends the maximum life-span in mammals (Sohal and Weindruch, 1996). ROS may damage subunits of the RC, membrane lipids causing proton leak,

and enzymes of the TCA cycle leading to lowered energetic efficiency, cell

dysfunction and cell death through activation of the PTP (Figure 11) (Wallace, 1992; Wallace, 2001). It is assumed that chronic ROS formation increases the mutant

mtDNA load over time leading to synthesis of defective RC subunits, which in turn increase ROS formation and the mutant mtDNA load further (Figure 11). Correlative

Figure 11 Free radical theory of ageing. Chronic mitochondrial ROS formation damages the RC and increases the mtDNA mutant load leading to progressive decline of oxidative phosphorylation, and further increases in ROS formation and mtDNA mutations with age. The final outcome is cell dysfunction and cell death.

45

evidence suggests a causative link between ROS and accumulation of mtDNA

mutations over time. Prolonged exposure to oxidative stress causes more extensive and persistent damage to mtDNA than to nuclear DNA (Yakes and Van Houten,

1997). The levels of an oxidative DNA modification marker were 10-fold increased in mtDNA compared to nuclear DNA (Bowling et al., 1993). Several reasons have been

proposed to contribute to this selective vulnerability: (i) the lack of histones that

protect from oxidative DNA damage; (ii) the lack of an efficient repair system in mitochondria; (iii) the compact gene organization of mtDNA increasing the likelihood

that a gene is mutated by ROS; (iv) the nearby location of mtDNA to the mitochondrial inner membrane, the major site of ROS production (Shigenaga et al.,

1994). MtDNA point mutations and mtDNA deletions have been reported to increase

with age (Larsson et al., 1990; Michikawa et al., 1999; Wang et al., 2001b). Complex I- IV deficiency, isolated or in combination was reported along with oxidative damage

to mtDNA in the brains of patients with age-associated neurodegeneration, i.e.

Parkinson disease, Alzheimer disease, amyotrophic lateral sclerosis, and Huntington disease (Kirkinezos and Moraes, 2001). However, a causative relationship between

ROS and the accumulation of mtDNA mutations remains to be demonstrated. A proof reading deficient mtDNA polymerase γ knockin mouse that accumulates somatic

mtDNA mutations over time develops a premature ageing phenotype (Trifunovic et

al., 2004). In C. elegans, mutations have been documented that confer increased

resistance against ROS and significant prolongation of lifespan: (i) clock-1 mutants that cannot synthesize ubiquinone and accumulate the biosynthetic precursor

demethoxyubiquinone, a more potent anti-oxidant, that can replace Q functionally, at least in part (Lakowski and Hekimi, 1996); (ii) Isp-1 mutants with a mutation in the

Rieske iron sulphur protein of Complex III leading to low oxygen consumption (Feng

et al., 2001). EUK-8 and EUK-134, antioxidant compounds that act simultaneously as SOD and Catalase mimetic increased lifespan in C. elegans (Melov et al., 2000).

Simultaneous overexpression of SOD1 and Catalase prolonged lifespan in Drosophila

melanogaster (Orr and Sohal, 1994).

46

Respiratory chain diseases

Respiratory chain diseases

Diseases of the respiratory chain (RC) have an incidence of at least 1 in 10’000 live births (Chinnery and Turnbull, 2001). RC diseases are caused by mutations in the

nuclear or the mitochondrial genome. MtDNA mutations include point mutations in

protein coding genes, tRNA genes, rRNA genes or deletions that usually encompass at least one tRNA gene (Smeitink et al., 2001). Nuclear mutations involve protein

subunits of the RC, factors required for the assembly of respiratory complexes and for maintenance of mtDNA. Furthermore there are several nuclear mutations affecting

proteins that are important to preserve mitochondrial homoestasis and indirectly affect

the RC, for example components of the mitochondrial protein import machinery. The largest group of patients with a respiratory chain disease display only an

enzymatically verified deficiency but no identified mutation (Graff et al., 2002). The

same clinical features can be caused by various mutations in mtDNA or nuclear DNA. Conversely, the same genetic defect in mtDNA can lead to different clinical

manifestations. The segregation of mutant mtDNA is one key determinant of this variability. It has been demonstrated the percentage of heteroplasmy varies between

different individuals, tissues and cells within a patient (Chinnery et al., 2000;

Chinnery et al., 1999). However, heteroplasmy is not always sufficient to explain this clinical variability: Firstly, in numerous cases the percentage of mutant mtDNA does

not correlate with the clinical phenotype (Zhou et al., 1997). Secondly, symptoms may develop only in specific tissues even when the mtDNA mutation is homoplasmic

in all tissues. Thirdly, nuclear mutations affecting one oxidative phosphorylation

complex can also cause variable phenotypes or involve selective tissues (DiMauro and Schon, 2003) (Rossignol et al., 2003).

The level of heteroplasmy that causes disease is called the phenotypic threshold (Rossignol et al., 2003). The phenotypic threshold depends on the type of

the mutation and can be approximated to >90% for most mutations in protein coding

genes, 65%-90% for tRNA gene mutations and 60% for deletions (Hayashi et al., 1991). The phenotypic threshold effect is based on an excess of wild-type mtDNA,

mRNA, tRNA, and active RC complexes compared to what is needed for normal respiration. This reserve provides a safety margin against deleterious mutations. The

47

phenotypic threshold is the sum total of thresholds at the level of transcription,

translation, enzyme activity and respiration (Rossignol et al., 2003). The steady-state transcript level of mtDNA encoded subunits appears to

correlate with the mtDNA copy number (D'Aurelio et al., 2001; Tang et al., 2000). Heteroplasmic point mutations of protein coding genes give rise to proportionate

amounts of mutated mRNAs (Bai et al., 2000; D'Aurelio et al., 2001), while

heteroplasmic mtDNA deletions result in proportionate reductions in mRNA levels of the deleted genes (Hayashi et al., 1991). The mutant tRNAs do not necessarily rise

proportionately to the level of heteroplasmy (Chomyn et al., 2000), for example the A3243G tRNALeu MELAS mutation may affect transcription or be subject to

degradation (Jacobs, 2003). A selective upregulation of transcription of wild-type

mtDNA over mutant mtDNA has not been observed in heteroplasmic cells (Bai et al., 2000; D'Aurelio et al., 2001) (Hayashi et al., 1991). This implies that the

transcriptional threshold is determined by (i) the ratio of mutant to wild-type mtDNA

or (ii) by the number of wild-type mtDNA copies. The relationship between mtDNA copy number and steady state-transcript levels may however not be straightforward as

there are indications for posttranscriptional regulation in mitochondria (Gagliardi et al., 2004). Pathogenic mtDNA mutations or deletions impair translation of wild-type

subunits, when the fraction of mutated mRNA (Bai et al., 2000), tRNA (Boulet et al.,

1992) or mtDNA deletions (Hayashi et al., 1991) exceeds 40-50%. Again the translational threshold may be influenced by posttranscriptional regulation (Gagliardi

et al., 2004). Pathogenic mutations in both mtDNA and nuclear DNA can impair the activity of the respective respiratory complex by (i) decreasing the total amount of

assembled active enzyme complexes and/or (ii) by changing the intrinsic kinetic

properties of the complex. Significant reductions in the protein levels of different subunits may not result in lowered enzyme activity (Spelbrink et al., 1994; Triepels et

al., 2001). In cell lines, a decrease in respiration has been reported following an 85% reduction in Complex I activity (Barrientos and Moraes, 1999), or a 40-80% reduction

in Complex IV activity (Davey et al., 1998; Villani and Attardi, 1997), dependent on

the cell lines and experimental conditions used. The respiratory threshold can reflect an excess of active respiratory chain complexes, activation of inactive complexes

(Grivennikova et al., 2001), recruitment and integration of inactive complexes into respiratory super-complexes (Schagger and Pfeiffer, 2000), or biochemical regulation

48

of the kinetic properties of the complex, for example cAMP-mediated

phosphorylation of Complex I (Rossignol et al., 2003). The respiratory threshold for a given complex may also relate to inhibition of the metabolic flux upstream of the

enzymatic deficiency leading to variations in the concentrations of intermediary metabolites (Rossignol et al., 1999). The transcriptional, translational and respiratory

thresholds are clearly tissue-specific (Rossignol et al., 1999) and may explain some of

the phenotypic differences caused by mtDNA mutations. Treatment of mitochondrial disease may therefore aim at modulating these threshold (Taylor et al., 2001).

MtDNA mutations in protein coding genes and tRNA genes cause multi-systemic disorders, for example MELAS (mitochondrial encephalomyopathy, lactic

acidosis and stroke like episodes) and MERFF (myoclonic epilepsy and ragged red

fibres), or present as organ-specific disease, for example mitochondrial diabetes, mitochondrial cardiomyopathy, mitochondrial myopathy, mitochondrial deafness,

Leigh syndrome and LHON (Lebers hereditary optic neuropathy).

LHON is usually caused by mutations of the ND1, ND4 and ND6 subunits of Complex I. The mutations are usually homoplasmic. The penetrance is low and an

additional X-linked factor has been suggested. Affected young adult patients display a bilateral optic neuropathy with degenerating retinal ganglion cells.

Leigh syndrome is a histopathological entity characterized by bilateral

necrosis of the striatum that usually affects infants. Leigh syndrome is caused by mitochondrial or nuclear mutations. The most common mitochondrial mutation

involves the ATPase 6 subunit (T8993C). Another mutation at the same site of the ATPase 6 subunit (T8993G) causes a milder phenotype known as the NARP

syndrome (neurogenic weakness, ataxia and retinitis pigmentosa) with adult onset.

The threshold level to develop symptoms has been found at 80-90% in blood for the T8993C mutation and 60-70% in blood for the T8993G mutation (White et al., 1999).

The most documented mutation leading to MELAS is the A3243G mutation of the tRNALeu gene, while MERFF most commonly associates with the A8344G

tRNALys gene mutation. The MELAS and MERFF mutations are usually

heteroplasmic. In MERFF, symptoms develop above a mutant load of 90% in muscle (Chinnery et al., 1997). Mutations of tRNA genes usually produce a variety of

structural and functional defects that result in impaired aminoacylation, impaired pre-tRNA processing, loss of modifications in the anticodon leading to mistranslations

49

and lowered translational activity (Yasukawa et al., 2001; Yasukawa et al., 2000)

(Jacobs, 2003). MtDNA deletions are most commonly associated with multi-systemic

disorders. MtDNA deletions arise spontaneously, probably somewhere between oogenesis and early embryogenesis, and are rarely transmitted. They are always

heteroplasmic and include at least one tRNA gene. The most common deletion (called

“common deletion”) is found in one third of all patients carrying mtDNA deletions. Large-scale mtDNA deletions encompassing several tRNA genes cause Pearsson or

Kearns-Sayre syndrome. Pearson syndrome has an onset early in childhood and is characterized by severe bone marrow deficiency and exocrine pancreatic dysfunction.

Children can recover from Pearsson syndrome but patients are at risk to develop

Kearns-Sayre Syndrome later in life, characterized by ophthalmoplegia, ptosis, retinal degeneration, ataxia, and heart block. An increased level of mutated mtDNA has been

found over time in muscle of patients with Kearns-Sayre Syndrome probably

representing a mechanism for disease progression (Larsson et al., 1990). Nuclear mutations cause at most 10% of all known respiratory chain disorders,

but they probably represent the majority of all cases with deficient respiratory chain function (Graff et al., 2002). Nuclear gene mutations affect: (i) subunits of the RC, so

far identified only in Complex I and II, that can result in Leigh-syndrome or

leukodystrophy. Complex II mutations are also associated with hereditary paragangliomas and pheochromocytomas (DiMauro and Schon, 2003; Smeitink et al.,

2001); (ii) assembly factors of respiratory chain complexes, so far identified for complex III and IV, and associated with Leigh syndrome (de Lonlay et al., 2001)

(Tiranti et al., 1998; Zhu et al., 1998); (iii) proteins regulating the stability of mtDNA

leading to mtDNA depletion or mtDNA deletions, for example mitochondrial thymidine kinase 2 (Saada et al., 2001), thymidine phosphorylase (Nishino et al.,

1999), adenine nucleotide translocator 1 (Kaukonen et al., 2000), DNA pol γ

(Lamantea et al., 2002) and Twinkle (Spelbrink et al., 2001). The affected proteins are required to maintain mitochondrial nucleotide pools or to replicate mtDNA. The

clinical hallmark of most of these diseases is opthalmoplegia.

50

Mitochondrial diabetes

MtDNA mutations cause approximately 0.5-1% of all types of diabetes mellitus. At

least 42 different mtDNA mutations have been associated with diabetes (Mathews and Berdanier, 1998), for example mtDNA rearrangements such as deletions associated

with partial mtDNA duplications (Ballinger et al., 1992; Rotig et al., 1992), or

mtDNA triplications (Dunbar et al., 1993). The most common mutation reported in patients with mitochondrial diabetes is the mitochondrial A3243G tRNALeu gene

mutation (Maassen et al., 2004). The majority of these patients become clinically manifest at the age of 35-40 years. Hearing impairment precedes in most cases the

onset of clinically manifest diabetes by several years. The A3243G tRNALeu mutation

is generally heteroplasmic and present in all the tissues but the heteroplasmy levels seem to be highest in tissues with low mitogenic activity, for example in muscle.

However, additional studies did not identify insulin resistance as a common pathogenic factor in most carriers of the A3243G tRNALeu mutation, although insulin

resistance has been reported in some cases. Increased hepatic glucose production may

represent another mechanism leading to hyperglycemia but no data are at present available regarding hepatic glucose production in carriers of the A3243G tRNALeu

mutation. Diabetic patients with the A3243G tRNALeu mutation exhibit impaired pancreatic insulin secretion in response to glucose stimulation (Velho et al., 1996).

Pancreatic mtDNA deficient β-cell lines show lowered glucose-stimulated insulin

release (Kennedy et al., 1998; Soejima et al., 1996; Tsuruzoe et al., 1998). Loss of

mtDNA gene expression by the A3243G tRNALeu mutation is expected to impair oxidative phosphorylation. Lowered ATP levels are thought to block the stimulus-

secretion coupling, whereby blood glucose stimulates insulin secretion by the

pancreatic β-cell (Maechler and Wollheim, 2001) . The stimulus-secretion coupling is

initiated by glucose uptake through the Glucose-2 transporter. Glucose enters the glycolytic pathway and is metabolized to pyruvate yielding ATP. Pyruvate enters the

mitochondria and stimulates oxidative phosphorylation leading to further ATP synthesis. The increase in cytosolic ATP originating from both sources, glycolysis

and oxidative phosphorylation, results in closure of ATP dependent K+ channels,

depolarization of the plasma membrane, opening of voltage dependent L-type Ca2+ channels and Ca2+ influx. Cytosolic Ca2+ triggers exocytosis of insulin containing

51

granules located near the plasma membrane. Impaired oxidative phosphorylation may

thus inhibit closure of ATP dependent K+ channels and pancreatic insulin-secretion. Some studies indicate that other molecular processes contribute to

mitochondrial dysfunction. Firstly, it has been reported that high levels of the A3243G tRNALeu mutation can cause severe mitochondrial dysfunction without

significant reduction in mitochondrial protein synthesis (Janssen et al., 1999).

Secondly, studies of transmitochondrial cell lines (cells repopulated with donor-derived wild-type or mutant mitochondria) containing the A3243G tRNALeu mutation

indicate a high threshold level for a reduction in oxygen consumption and oxidative phosphorylation at 80-90% mutant mtDNA versus wild type mtDNA (Chomyn et al.,

1992). These high levels of heteroplasmy are not frequently found in diabetic patients

with the A3243G tRNALeu mutation. In situ characterization of islets of diabetic patients with the A3243G tRNALeu

mutation revealed severely deficient cytochrome c oxidase (COX) activity and a

decreased number of pancreatic β–cells. The heteroplasmy levels of micropunched

pancreatic islets were quantified by laser densitometry and last cycle hot PCR labeling and revealed that they were 63 ± 5% (Kobayashi et al., 1997). Similarly,

immunohistochemical studies of pancreas from patients with MELAS syndrome

revealed a reduction in the number of pancreatic β- and α-cells and in total islet mass.

The level of heteroplasmy for the A3243G tRNALeu mutation found in this study was

43% (Otabe et al., 1999). Interestingly, none of these studies revealed apoptosis markers in pancreatic islets.

ROS formation in respiratory chain deficient cells

Fibroblasts of patients with Complex I deficiency and different mitochondriopathies

showed a significant increment in ROS formation (Luo et al., 1997; Pitkanen and

Robinson, 1996) along with upregulation of SOD2 (Pitkanen and Robinson, 1996). Correlations between the levels of Complex I deficiency, ROS production, lipid

peroxidation and apoptosis were observed in studies of human xenomitochondrial cybrids harboring a 40% Complex I deficiency. Cell death was in this case

quantitatively associated with ROS production rather than with Complex I deficiency

(Barrientos and Moraes, 1999). Complex II mutations causing hereditary paraganglioma likely result in increased O2

-· formation as a consequence of impaired

52

ubiquinone binding (Yankovskaya et al., 2003). In transmitochondrial cybrids

containing a mitochondrial cytochrome b gene mutation of a patient with MELAS and parkinsonism, high levels of the mutation were associated with Complex III

deficiency and increased intracellular hydrogen peroxide levels (Rana et al., 2000), Fibroblasts of patients with the NARP mutation of the mitochondrial ATP synthase 6

gene show massive superoxide generation along with upregulated SOD activity

(Geromel et al., 2001). Fibroblasts of patients with MELAS or MERFF display increased intracellular H2O2 levels and oxidative damage to lipids and DNA (Wei et

al., 2001b). In whole blood cells of patients with MELAS-related mitochondriopathy, ROS-associated telomere shortening was observed (Oexle and Zwirner, 1997). In a

mouse model of sarcopenia, the age-related decline of muscle mass and function,

mtDNA deletions were found to colocalize with sites of oxidative damage to nucleic acids (Wanagat et al., 2001). Finally, mtDNA depleted (rho-0) cells showed an

induction of SOD2 and other antioxidant defenses conferring enhanced resistance

against oxidative stress-induced cell death (Park et al., 2004). The molecular mechanisms whereby mtDNA mutations induce ROS formation are not well defined.

Mutations of respiratory subunits may block electron transfer within or between the respiratory complexes leading to electron leakage and superoxide formation. The

mutations can also impair complex assembly as demonstrated for complex III (Rana

et al., 2000), IV (D'Aurelio et al., 2001; Hanson et al., 2001), and V (Nijtmans et al., 2001), leading to loss of the respective complex or to a misassembled dysfunctional

complex (Nijtmans et al., 2001), block in electron transfer and/or electron leakage and superoxide formation (Rana et al., 2000). Finally the mutations may inhibit

ubiquinone binding to the complex causing electron leakage (Yankovskaya et al.,

2003). Impaired mtDNA expression, caused by tRNA gene mutations, mtDNA deletions or mtDNA depletion may result in misassembled complexes and ROS

formation. However, there are 2 tendencies that potentially counteract ROS formation in this situation: (i) the lack of mtDNA-encoded subunits could block assembly of

several complexes and only deplete the number of fully active complexes without

causing any block or leak in electron transfer; (ii) the combined deficiency of several respiratory complexes lowers oxygen consumption dramatically.

53

Metabolic alterations and cell death in respiratory chain deficient cells

Mitochondria have a central role in energy metabolism. Firstly they provide the vast

amount of cellular ATP though oxidative phosphorylation. Secondly, glycolysis and mitochondrial fatty acid β-oxidation are linked to respiration (Stryer, 1995). Severe

RC deficiency increases the [NADH]/[NAD+] ratio and slows down the activities of

the TCA cycle and fatty acid β-oxidation that are both primarily regulated by the

[NADH]/[NAD+] ratio (Erecinska and Wilson, 1982). Deficient oxidative

phosphorylation lowers the [ATP]/[ADP] ratio that serves as the primary cytosolic sensor for altered energy metabolism and controls glycolytic rates (Erecinska and

Wilson, 1982). Lowered mitochondrial ATP production is compensated for by up-regulation of glycolysis (Hansson et al., 2004; Wang et al., 2001a) and increased

mitochondrial biogenesis (Hansson et al., 2004; Heddi et al., 1999; Wredenberg et al.,

2002). This is also reflected by the formation of ragged red fibres in muscle (DiMauro and Schon, 2003). The increased mitochondrial biogenesis increases the overall

mitochondrial ATP production rate in respiratory chain-deficient skeletal muscle

(Wredenberg et al., 2002) but not in respiratory chain-deficient heart muscle (Hansson et al., 2004). Respiratory chain-deficient heart muscle displays a switch from fatty

acid metabolism to glycolysis early in the progression of cardiac mitochondrial dysfunction prior to the increase in mitochondrial mass (Hansson et al., 2004). The

observed switch from fatty acid metabolism to glycolysis is unlikely to benefit energy

homeostasis in the respiratory chain-deficient hearts because fatty acid oxidation yields more ATP than glycolysis and is the chief myocardial energy source (Barger

and Kelly, 1999). In most cancer lines with deficient oxidative phosphorylation, the energy for growth is almost exclusively derived from glycolysis. One reason for this

is that glycolysis provides ATP at a low yield but high rate whereas oxidative

phosphorylation produces ATP at a lower rate but higher yield (Pfeiffer et al., 2001). Upregulation of glycolysis results in lactic acidosis due to lowered pyruvate

utilisation by the TCA cycle. Severe RC deficiency is thus expected to result in lowered mitochondrial ATP production, lowered TCA cycle activity, impaired amino

acid and heme biosyntheses, impaired fatty acid degradation, up-regulation of

glycolysis and increased mitochondrial biogenesis. MtDNA depleted (rho-0) cells can grow in glucose medium supplemented with uridine and pyruvate. The growth rate is

lowered but there is no apparent increased cell death rate under these conditions (Dey

54

and Moraes, 2000; Jiang et al., 1999; Wang et al., 2001a). Oxygen consumption of

rho-0 cells is almost abolished but rho-0 cells still preserve the mitochondrial membrane potential to some extent (Jiang et al., 1999). This occurs through the

reverse action of the F1-ATP synthase that acts as a soluble ATP hydrolase in the absence of the mtDNA-encoded subunits of the Fo part. The electrogenic exchange of

mitochondrial ADP3- for cytosolic ATP4- maintains the membrane potential in rho-0

cells and upregulation of glycolysis provides ATP4-. Preservation of the mitochondrial membrane potential is likely required for protein import and mitochondrial biogenesis

in order to maintain some important metabolic functions, for example biosynthetic pathways (Stryer, 1995) or buffering of apoptotic factors. Pharmacological inihibition

of the reverse action of the soluble F1 ATPase in rho-0 cells has detrimental effects

(Buchet and Godinot, 1998). It is therefore not clear to what extent RC function is indeed required for cell maintenance in vivo. RC deficiency lowers the mitochondrial

membrane potential and may increase ROS formation rendering the cell more

susceptible to induction of PT (Susin et al., 1998). RC deficiency may significantly impair Ca2+ homeostasis by decreasing the mitochondrial Ca2+ buffering capacity, the

activity and expression of ATP dependent endoplasmic and plasma membrane-bound Ca2+ pumps (Arai et al., 1994; Carafoli, 2004; Wang et al., 2001a; Wankerl and

Schwartz, 1995) leading to cytosolic Ca2+ overflow, mitochondrial Ca2+ uptake and

either necrosis or apoptosis. Apoptosis is an energy dependent process, for example the formation of the apoptosome requires ATP (Liu et al., 1996). Furthermore, the

intracellular ATP level determine the cell death fate, either by necrosis or apoptosis (Ankarcrona et al., 1995; Eguchi et al., 1997; Leist et al., 1997). Enhanced glycolytic

ATP production in the absence of oxidative phosphorylation can allow execution of

apoptosis (Leist et al., 1997). Nevertheless, mtDNA depleted cells are more resistant against induction of apoptosis in culture (Dey and Moraes, 2000; Park et al., 2004).

Reports regarding apoptosis/cell death in respiratory chain disease are very scarce. It is very difficult to monitor cell death in vivo and the availability of patient material is

rather limited. Cell death is a well-documented pathological hallmark of Leigh

syndrome and LHON. Cell loss has also been documented in diabetic carriers of the A3243G tRNALeu mutation exhibiting a reduction in the number of pancreatic insulin-

producing β-cells in post-mortem pancreas sections (Kobayashi et al., 1997; Otabe et

al., 1999). Apoptosis has also been observed in COX-negative muscle fibres of

55

patients carrying > 40% mtDNA deletions and > 70% tRNA point mutations

(Mirabella et al., 2000). Cultured fibroblasts of patients with NARP harboring the 8993 mutation of the mitochondrial ATP synthase 6 gene display massive superoxide-

induced apoptosis (Geromel et al., 2001). Thus, it is possible that increased cell death is a general pathogenic mechanism of RC disorders.

Mouse models for respiratory chain disease

It is often assumed that ATP deficiency is the main cause of RC disease but there is evidence that additional processes influence the phenotype (Hansson et al., 2004;

Wredenberg et al., 2002). The limited availability of human tissue makes it necessary

to perform studies of molecular pathogenesis in model organisms. Mice and humans display quite similar gene content, comparable types of internal organs and

physiological processes. The generation of transgenic mice with mutant mtDNA molecules is a significant technical problem because attempts to transfect

mitochondria with whole mtDNA have been unsuccesful. This problem has been

solved by two alternative approaches: (i) cybrid-mediated transfer of mutant mtDNA into ES cells or pro-nucleus stage embryos followed by propagation of the mutation

through the mouse female germline; (ii) inactivation of mtDNA gene expression through disruption of the gene encoding Tfam. In addition to mouse models that

directly affect the expression of subunits of the RC, mouse models have been created

that alter the homeostasis of the mitochondria and thereby indirectly affect RC function, for example gene ablation of SOD2, Gpx1, ANT1.

Cybrid-mediated transfer of mtDNA has been used to create: (i)

Heteroplasmic mice harboring mtDNA haplotypes from two different mouse strains by fusion of enucleated zygotes from one mouse strain to pronucleus-stage embryos

from another mouse strain (Jenuth et al., 1996; Jenuth et al., 1997); (ii) Mice carrying deleted mtDNA (ΔmtDNA) (Inoue et al., 2000). Mouse cell lines heteroplasmic for

mtDNA with a 4969bp-deletion were propagated in a mouse rho-0 cell line. These

cybrids were enucleated and fused to pro-nucleus-stage embros. Heteroplasmic mice

transmitted the ΔmtDNA to several generations, displayed high levels of rearranged

mtDNA in most tissues and RC deficiency in heart, skeletal muscle and kidney leading to myopathy and kidney failure. Surprisingly, a partially duplicated mtDNA

56

molecule was identified in postmitotic tissues despite being undetectable in the

cybrids used to create these mice and it is therefore unclear wether ΔmtDNA was

transmitted intact through the female germline or generated later by intramolecular rearrangements. ΔmtDNA syndromes in humans have in most instances an adult-

onset, are not transmitted, exhibit high levels of ΔmtDNA in postmitotic tissues, such

as brain, heart and skeletal muscle and cause no kidney failure. Thus the limitiation of

this mouse model is that it does not reproduce well the pathology found in adult

patients with ΔmtDNA (Larsson and Rustin, 2001). (iii) Mice carrying a mutation in

the 16S rRNA gene (chloramphenicol resistance, CAPR mtDNA) (Sligh et al., 2000). CAPR mtDNA was propagated in a rho-0 cell line. The enucleated cybrids were fused

to a female ES cell line. The cybrid ES cells were injected into C57/Bl6 blastocysts and high degree chimeras were obtained. Mice heteroplasmic for CAPR mtDNA

transferred the mutant mtDNA through the female germline for several generations

and exhibited a severe phenotype including myopathy and cardiomyopathy but the respiratory defects in these mice have not been demonstrated yet.

Disruption of the Tfam gene causes depletion of mtDNA, mitochondrial

transcripts, loss of mtDNA encoded polypeptides and severe RC deficiency (Larsson et al., 1998; Wang et al., 1999). Tfam KO mice therefore mimic human disease caused

by impaired mtDNA gene expression, for example ΔmtDNA syndromes, mtDNA

mutations of tRNA genes, and mtDNA depletion syndromes. Homozygous Tfam KO mice show severe developmental delay and die between embryonic day 8.5 and 10.5.

They exhibit complete absence of Tfam protein, complete absence of mtDNA and

abolished oxidative phophorylation. Heterozygous Tfam KO mice have a 50% reduction in Tfam transcripts and proteins, a 34% reduction in mtDNA copy number,

a 22% reduction in mitochondrial transcripts and a mild respiratory chain deficiency in heart (Larsson et al., 1998). Tissue-specific inactivation of Tfam in heart results in

postnatal death due to dilated cardiomyopathy. These animals develop conduction

defects with prolongation of the PQ interval and intermittent atrioventricular blocks under anesthesia. Heart-specific Tfam KO mice exhibit a reduction in Tfam protein,

mtDNA levels and mtRNA levels in heart and muscle, and reduced activities of Complex I and IV. Histochemical analyses of the Tfam KO hearts revealed a mosaic-

staining pattern with COX-negative and SDH-hypereactive cardiomyocytes. The

57

heart–specific Tfam KO mice thus reproduce typical morphological and physiological

features of human mitochondrial cardiomyopathy (Wang et al., 1999). ANT1-/- mice develop a myopathy with ragged red fibres and a hypertrophic

cardiomyopathy associated with a marked proliferation of mitochondria (Graham et al., 1997). The inhibition of ADP/ATP exchange deprives the ATP synthase of

substrate leading to hyperpolarization of the mitochondrial membrane potential,

inhibition of electron transport and increased superoxide production. Consistently, ANT1-/- mice exhibit six- to eight fold increased H2O2 production in skeletal muscle

and heart mitochondria and upregulated SOD2 and GPx1 enzyme activities (Esposito et al., 1999). MtDNA rearrangements were observed in hearts and skeletal muscle of

aged ANT1 -/- mice and may be explained by the enhanced ROS formation acting as a

mutagen or alternatively by alterations of the mitochondrial nucleotide pool impairing mtDNA replication (Esposito et al., 1999).

SOD2 and GPx1 KO mice were created to assess the consequences of

enhanced superoxide and hydrogen peroxide formation, respectively, on mitochondrial function. On the CD1 background, SOD2-/- mice die of dilated

cardiomyopathy at the age of 8 days (Li et al., 1995). On the B6 background, SOD2-/- mice die at the age of 18 days due to neurodegeneration (Lebovitz et al., 1996). While

inactivation of SOD2 proves to be lethal early in life, the inactivation of either the

cytosolic Cu/Zn-SOD (SOD1) (Reaume et al., 1996) or the extracellular SOD (SOD3) (Carlsson et al., 1995) have little effect on the viability of the mice. This suggests that

mitochondrial production and toxicity of O2-· is far more deleterious than the cytosolic

and extracellular formation of O2-·. SOD2-/- mice develop massive lipid droplets in the

liver, deficient activities of Complex I and II, citrate synthase and aconitase in heart.

Increased O2-· concentrations thus inactivate enzymes containing mitochondrial iron-

sulphur centers blocking the TCA cycle and electron transport (Melov et al., 1999).

Respiratory measurements demonstrated signs indicative of increased mitochondrial basal proton leak. Mitochondria from SOD2-/- mice are more susceptible to activation

of the PTP indicating that O2-· sensitizes the PTP (Kokoszka et al., 2001). Similar

effects on respiration and accumulation of ROS-induced protein and lipid damage are observed in middle-aged SOD2+/- mice and in wild-type mice at a later age.

Furthermore SOD2+/- mice display a three-fourfold increased apoptosis-rate in liver (Kokoszka et al., 2001).

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Prior to generating GPx1 KO mice, a reporter cassette (β-galactosidase) was

inserted into the GPx1 mouse gene and revealed that the GPx1 gene locus is mainly

active in liver, brain, kidney and weakly in heart and skeletal muscle. GPx1 protein is found in the cytosol and the mitochondria of liver and kidney. GPx1-/- mice are viable

but display a 20% weight reduction. H2O2 levels are increased fourfold in liver mitochondria. Respiratory measurements indicated an increased mitochondrial

uncoupling in liver. However augmented mitochondrial H2O2 production in liver,

brain and kidney appears to cause a rather mild phenotype in the living animal compared to inactivation of SOD2 (Esposito et al., 2000).

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Aims of this study

RC deficiency causes specific mitochondrial disorders. In addition, various types of

evidence links RC deficiency with age-associated common disorders and ageing. RC deficiency is expected to lower mitochondrial ATP generation. However, there are

potentially other factors contributing to the pathophysisology of RC dysfunction. The specific aims of this proposal are to investigate whether:

1) RC deficiency in pancreatic β-cells impairs glucose-stimulated insulin release and

β-cell survival.

2) RC deficiency affects cell survival. 3) RC deficiency causes ROS formation.

4) Mitochondrial superoxide regulates uncoupling protein activity, RC function and

energy expenditure.

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Comments on methods

All methods used in this study have been described in the original publications that

are included in the end of the text. Central methods that have not been described or are only referred to in the original publications will be described in this section.

Tfam knockout strategy

The mouse Tfam gene consists of 7 exons. LoxP sites were introduced into the mouse

Tfam gene by homologous recombination of ES cells (Larsson et al., 1998). The loxP sites flank exon 6 and 7 of the mouse Tfam gene including the polyadenylation signal.

Mice homozygous for the floxed Tfam allele (TfamloxP/TfamloxP) were mated to

different transgenic strains expressing cre-recombinase from cell-type specific promoters: (i) mice expressing a β-actin-cre transgene (Lewandoski et al., 1997)

ubiquitously in the preimplantation embryo to generate germline Tfam KO mice

(paper II); (ii) mice expressing cre-recombinase from the rat insulin 2 promoter (RIP2-cre) (Postic and Magnuson, 1999) to generate pancreatic β-cell specific Tfam

KO mice (paper I); (iii) mice expressing cre-recombinase from the muscle creatine

kinase promoter (Ckmm-cre) (Bruning et al., 1998) to disrupt Tfam in cardiomyocytes

(paper II); (iv) mice expressing cre-recombinase from the calcium dependent calmodulin kinase II promoter (CaMKII-cre) (Xu et al., 2000) to disrupt Tfam

specifically in forebrain neurons (Paper III). Cre-mediated excision of exon 6 and 7 of

the Tfam gene results in loss of the polyadenylation signal and Tfam transcript instability.

Enzyme histochemistry

Cryostat sections were prepared and stored at -20o C. To detect COX enzyme

activities, cryostat sections were incubated 30-60 min at room temperature with a reaction mix containing 1mg/ml cytochrome c (Sigma-Aldrich Sweden AB), 2mg/ml

catalase (Sigma-Aldrich Sweden AB), 75mg/ml sucrose (BDH AnalaR, England),

0.5mg/ml 3,3’- diaminobenzidine tetrahydrochloride dihydrate (DAB, Fluka Chemicals, Switzerland) in 0.05M phosphate buffer (0.01M NaH2PO4, 0.04M

Na2HPO4, pH 7.4). To stain for succinate dehydrogenase (SDH) enzyme activity,

61

cryostat sections were incubated 30-90 min at 37 o C with a reaction mix containing

54mg/ml succinate acid Na-salt (Sigma-Aldrich Sweden AB), and 1mg/ml 4-nitro blue tetrazolium chloride (NBT) (Boehringer Mannheim Scandinavia, Sweden) in

0.2M phosphate buffer (0.16M Na2HPO4, pH 7.2-7.6). COX/SDH double staining was performed by first staining for COX and then for SDH.

62

Results and Discussion

Pathogenesis studies in a mouse model of mitochondrial diabetes (Paper I) Mutations of mtDNA cause approximately 0.5-1% of all types of diabetes mellitus

and studies of mtDNA depleted pancreatic β-cell lines suggest that RC function is

critical for insulin release. To study the pathogenesis of mitochondrial diabetes, we inactivated the Tfam gene specifically in pancreatic β-cells by cre-mediated excision

of a loxP-flanked Tfam allele. To this end, transgenic mice expressing cre-

recombinase from the rat insulin-2 promoter (RIP2-cre) (Postic and Magnuson, 1999)

were bred to knockin mice harboring a loxP flanked Tfam allele (Larsson et al., 1998). The RIP2-cre strain expresses cre-recombinase specifically in pancreatic β-cells and

in certain hypothalamic regions as evidenced by crosses between the RIP2-cre strain

and a mouse strain harboring a conditional lacZ gene (Soriano, 1999) (Gannon et al., 2000) and immunohistological analyses of cre-recombinase expression (Gannon et

al., 2000). As expected, PCR analyses demonstrated the presence of the Tfam KO

allele only in pancreas and brain of young (7-week old) homozygous Tfam KO mice (RIP2-cre/+, TfamloxP/TfamloxP).

At the age of 7 weeks there was a highly efficient inactivation of mtDNA gene

expression in homozygous Tfam KO mice as revealed by: (i) in situ hybridization studies of pancreas sections showing severe mtDNA depletion in islets of mutant

mice; (ii) enzyme histochemical double staining for cytochrome c oxidase (COX) and succinate dehydrogenase (SDH) activities showing a severe COX deficiency and

normal SDH activities in islets of mutant mice. This result was expected because

COX contains mtDNA encoded catalytic subunits whereas SDH is completely nucleus-encoded; (iii) EM pictures of insulin producing β-cells demonstrating

abundant, abnormally appearing mitochondria in mutant islets.

The homozygous Tfam KO mice developed diabetes with decreased blood insulin concentrations from the age of about 5 weeks onwards. Insulin tolerance tests

showed normal insulin sensitivity in younger (12-week old) mutant mice and

enhanced insulin sensitivity in older (27-33 week) mutant mice. Histochemical analyses of islet cell composition showed a normal cell-type composition and point

counting morphometry demonstrated normal β-cell mass in young (7-week old)

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mutant mice. Older (33-39 week old) mutant mice showed a marked change in the

cell-type composition of the islets and a reduction in β-cell mass. There was a patchy

loss of β-cells and a relative increase in glucagon- and somatostatin-producing α- and

δ-cells, respectively. These data conclusively demonstrate that mutant β-cells had

been lost over time. However, we found no evidence for increased apoptosis (<1% of TUNEL reactive islet cells) or islet inflammation as a consequence of necrosis in

young 7-week old mutant mice.

Next, we analyzed the β-cell stimulus-secretion coupling at the age of 7

weeks, when β-cell mass was still normal in mutant mice. The β-cell stimulus-

secretion coupling is the signal transduction pathway whereby glucose stimulation of the β-cell results in insulin secretion. We found a decreased hyperpolarization of the

mitochondrial membrane potential in isolated mutant islets in response to glucose

stimulation consistent with a RC deficiency. We then monitored intracellular Ca2+

after glucose stimulation of isolated islets. We found a decreased rise in intracellular Ca2+ and an almost complete absence of the fast Ca2+ oscillations in mutant islets.

When both, mutant and control islets, were exposed to extracellular K+ which depolarizes the plasma membrane potential, both had normal intracellular Ca2+ rise

indicating that the stimulus-secretion pathway downstream of ATP-dependent K+

channels worked normally in mutant islets but also demonstrating that the lack of a Ca2+ rise in response to glucose stimulation was not due to cytosolic Ca2+ overflow.

Next, we studied the kinetics of glucose-induced insulin release by perfusing islets isolated at the age of 7 weeks. Insulin release was significantly reduced in mutant

islets. We also determined insulin content and found no significant differences

between mutant and control islets. The homozygous Tfam KO mice thus displayed impaired stimulus-secretion coupling at the age of 7 weeks.

Blood glucose levels dropped in older mutant mice but remained elevated. Older (14- and 27-39-week old) mutant mice showed presence of the Tfam allele only

in brain but not in pancreas. Consistently, islets of older mutant mice showed normal

COX activity and normally appearing mitochondria on EM pictures. Immunofluorescence analyses showed that cre-recombinase was abundantly

expressed in islets of 7-week old mutant mice while cre-recombinase was not

expressed at all or only in very few islet cells in older (22-25 week old) mutant mice. Thus, the recombination event inactivating the Tfam allele and the RC deficiency

64

ceased when the mutant mice became older. The diabetic phenotype in older mutant

mice is thus caused by the loss in β-cell mass and the improvement in glucose

homeostasis observed in the older mutant mice is likely attributable to expansion of β-

cell mass over time and enhanced insulin sensitivity. This study provides the first genetic in vivo evidence that loss of mitochondrial

DNA gene expression in pancreatic β-cells causes diabetes. This study identified two

phases in the progression of mitochondrial diabetes: mutant β-cells first display

impaired stimulus-secretion coupling. This is later followed by β-cell loss. This

mouse model reproduces the clinical features of patients with mitochondrial diabetes,

i.e. impaired pancreatic insulin secretion and β-cell loss.

Apoptosis studies of cells and tissues lacking mtDNA gene expression (Paper II) Apoptosis may contribute to the pathology of RC disease. Mitochondria are key

regulators of apoptosis and a RC deficiency may directly activate cytochrome c

release because of lowered mitochondrial membrane potential and ROS formation (Susin et al., 1998). MtDNA depleted (rho-0) cells do not exhibit increased cell death

in culture and are reportedly more resistant against apoptosis induction suggesting that intact RC function is required for apoptosis execution (Dey and Moraes, 2000).

In order to clarify wether respiratory chain-deficient cells are able to undergo

apoptosis and are more apoptosis-prone in vivo we investigated heart-specific Tfam KO mice, Tfam KO embryos and an mtDNA depleted osteosarcoma rho-0 cell line.

Mice with heart-specific inactivation of the Tfam gene exhibit postnatal onset of

severe mitochondrial cardiomyopathy (Wang et al., 1999). We found increased apoptosis in homozygous heart-specific Tfam KO hearts as reflected by an increase in

TUNEL reactive cells, activation of caspase 3 and caspase 7, and apoptotic DNA fragmentation. Trichrome staining of heart sections did not reveal fibrosis in mutant

hearts as a consequence of necrosis. Northern blot analysis revealed increased

expression of genes regulating apoptosis, namely BAX and BCL-xL, and massive up-regulation of glycerladehyde-3-phosphate dehydrogenase (Gapdh) indicative of

increased glycolysis. We next analyzed homozygous Tfam KO embryos. Homozygous Tfam KO embryos die between E8.5 and 10.5 and have abolished

oxidative phsophorylation (Larsson et al., 1998). We found massive induction of

65

apoptosis in homozygous Tfam KO embryos at E9.5 as reflected by massive TUNEL

reactive cells and activation of caspase 3, followed by resorption of the embryo at E10.5. We next reexamined wether mtDNA depleted (rho-0) human osteosarcoma

cells were able to undergo apoptosis. We stimulated apoptosis using Staurosporine (STP), TNFα and anti-Fas antibodies (anti-Fas) that stimulate Fas, the receptor of Fas

ligand. STP stimulates mitochondrial cytochrome c release while TNFα and anti-Fas

antibodies (Anti-Fas) activate caspase 8 which activates the effector caspase 3 or the pro-apoptotic BID protein (tBID) that in turn induces mitochondrial cytochrome c

release. Human osteosarcoma cells were scored for apoptosis by simultaneous labeling of the cells with Annexin V and Propidium iodide followed by flow

cytometry to determine the percentage of apoptotic cells (Annexin positive and

Propidium iodide negative). Rho-0 cells were able to undergo apoptosis in response to all apoptotic stimuli and they were less sensitive to induction of apoptosis by STP

and more sensitive to induction of apoptosis by TNFα and Anti-Fas. Apoptosis was

further confirmed by detection of DNA fragmentation. Thus, the human rho-0 osteosarcoma cells exhibited an increased threshold for

induction of the cytochrome c release pathway as previously reported (Dey and

Moraes, 2000) and were more sensitive to induction of the death receptor pathway as previously observed in cytochrome c KO cells (Li et al., 2000). The significance of

these differences however remainns unclear because cancer-cell lines carry

chromosomal rearrangements, and thus likely consist of a mixed genetic background. We explored whether ROS formation was associated with the observed in vivo

apoptosis of respiratory chain-deficient cardiomyocytes. We found significantly increased transcript levels of SOD2 and GPx. Next we determined total SOD and GPx

activities and found a significant increment in GPx activity, only. Furthermore, the

activities of Complex II and of aconitase that are readily impaired by ROS due to inactivation of iron-sulphur clusters proved normal.

This study shows that RC deficient cells can undergo apoptosis and that RC deficient cardiomyocytes and embryos are more prone to undergo apoptosis in vivo.

Apoptosis may thus contribute to RC disease. There is evidence for increased ROS

formation in RC deficient hearts but the normal activities of enzymes readily impaired by ROS suggest that the enhanced ROS formation is compensated for by upregulation

of antioxidant defenses.

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Analysis of the relationship between prolonged respiratory chain deficiency, ROS formation and cell death in late-onset neurodegeneration mice (Paper III) Correlative data suggest a connection between deficient RC function, ROS production

and apoptosis induction in age-associated neurodegeneration (Wallace, 1992). We performed a genetic experiment to test whether these processes are linked. To this

end, we inactivated the Tfam gene in neurons of the frontal neocortex and the

hippocampus. Mice expressing cre-recombinase from the calcium dependent calmodulin kinase II promoter were crossed to mice with a loxP flanked Tfam allele to

obtain homozygous tissue-specific Tfam KO mice (TfamloxP/TfamloxP, +/CaMKII-cre) called MILON mice (Mitochondrial late onset neurodegeneration mice). In the

CaMKII-cre transgenic line, cre-recombinase is expressed from postnatal day 14

(P14) and maximal recombination of loxP flanked alleles is observed at P29 causing recombination in about 50% of the neurons in neocortex and hippocampus (Xu et al.,

2000). Consistent with these earlier observations we found identical levels of the mutant Tfam allele at 1 month (relative level, 24 ± 3%) and 5 months of age (relative

level, 25 ± 2%) in pre-symptomatic MILON mice confirming that maximal

recombination was obtained by 1month of age. Tfam protein levels in neocortex of MILON mice were reduced from the age of 2 months onwards. MtDNA copy number

and mtRNA levels were reduced by 40% from the age of 2 and 4 months, respectively. In situ hybridization showed a dramatic reduction of mtRNA in necortex

and hippocampus. Biochemical measurements demonstrated reduced activities of

Complex I and IV in 4- and 5-month old MILON mice. Enzyme histochemical stainings revealed severe COX deficiency in individual neurons of hippocampus and

neocortex of MILON mice at the age of 4 months. The reduction in RC function in tissue-extracts of neocortex is thus the result of profound COX deficiency of

individual Tfam KO neurons.

MILON mice displayed onset of rapidly progressing, severe neuro-degeneration in hippocampus and neocortex at 5-5.5 months and died at the age of

5.5-6 months. Cresyl violet staining showed massive loss of neocortical neurons and of hippocampal neurons of the CA1 and to a lesser extent of the CA3 region of the

hippocampus accompanied by a cellular infiltration with macrophages and gitter cells.

The S-phase marker PCNA demonstrated numerous dividing glia or inflammatory

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response cells in neocortex and hippocampus as judged from their morphological

appearance. Immunostainings for neurofilament (NF-10) showed axonal degeneration and immunostainings for GFAP to detect astrocytes showed reactive astrocytosis

further confirming the presence of neurodegeneration and an inflammatory response in end-stage MILON mice. End-stage MILON mice, displayed massive induction of

TUNEL positive cells in hippocampus and neocortex. In the hippocampus, the first

areas displaying TUNEL positive cells were the CA1 and to some extent the CA3 areas, followed by appearance of TUNEL positive cells in the dentate gyrus at a later

stage. In order to distinguish between apoptosis and necrosis, we performed additional analyses. Gel electrophoresis of DNA from neocortex and hippocampus revealed no

apoptotic DNA fragmentation pattern. However, a more sensitive PCR assay detected

DNA fragmentation in endstage MILON mice. Immunostainings did not show presence of activated caspase 3 or 7 in brain sections of end-stage MILON mice.

Northern blot analysis revealed no changes in the transcript levels of the proapoptotic

BAX and anti-apoptotic BCL-xL. Thus apoptosis markers were mainly negative despite massive neuronal cell death suggesting that a major fraction of dying neurons

underwent necrosis rather than apoptosis. Northern blot analyses showed normal levels of the transcripts for glycerladehyde-3-phosphate dehydrogenase (Gapdh)

suggesting that glycolysis is not upregulated in MILON mice.

Transcript levels and enzyme activities of SOD2 and GPx were determined at 2-, 4- and 5-months of age and revealed only an increase in GPx transcripts in 5-

month old endstage MILON mice. Western Blot analyses and immunohistochemical stainings showed no enhanced protein nitrosylation, another marker for oxidative

stress induced by the conversion of superoxide to peroxynitrite, in 4-6 month old

mutant animals. The activity of the nucleus encoded Complex II whose activity is readily impaired by ROS was not changed in mutant mice determined at 2-, 4- and 5-

months of age. These results suggest unexpectedly low levels of ROS production in MILON mice at all investigated ages, i.e. at all stages of development of severe RC

deficiency. Next we explored whether RC deficient neurons are more susceptible to

an excitotoxic challenge with Kainic acid (KA). Excitotoxic insults result in apoptosis or necrosis dependent on the level of ATP present in a neuron (Ankarcrona et al.,

1995). We challenged MILON mice with KA at the age of 4 months when MILON mice displayed severe RC deficiency but no signs of neurodegeneration. MILON

68

mice with level 3-5 seizures (level 5 is the most severe grade) exhibited a much larger

number of TUNEL positive cells in the hippocampus (CA3 region) 24 hours after injection of KA. Thus, RC deficient neurons are more susceptible to an excitotoxic

challenge. We conclude that neurons with severe RC deficiency are viable for a

prolonged period before they undergo cell death. Surprisingly, there is no apparent

upregulation of glycolysis suggesting that glial cells may sustain neuronal energy metabolism. RC deficient neurons likely die by necrosis rather than by apoptosis and

are more sensitive to excitotoxicity. Most surprisingly, RC deficient neurons did not exhibit significant induction of ROS defenses or signs of oxidative protein damage

during the development of severe RC deficiency.

Regulation of uncoupling protein activity and mitochondrial bioenergetics by superoxide (Paper IV) Mitochondrial generation of superoxide (O2

-·) and other ROS species has been

implicated in ageing. However, it is at present not clear whether mitochondrial O2-·

also regulates RC function and energetic efficiency. A recent study reported

superoxide-mediated activation of mitochondrial uncoupling proteins (UCP) (Echtay et al., 2002b). Activation of UCPs by O2

-· is expected to increase the proton leak

across the mitochondrial inner membrane, to increase respiration and reduce the

generation of O2-· (Korshunov et al., 1997). The mechanism of superoxide-mediated

activation of UCPs would thus provide a feedback control to lower further O2-·

production by the RC (Echtay et al., 2002b). The activation of UCP occurs from the mitochondrial matrix side (Echtay et al., 2002a). Superoxide-mediated activation of

UCPs is expected to have a significant regulatory effect on whole body metabolism

due to the widespread tissue expression of UCPs. For example in BAT, O2-· may

enhance UCP1 function and facilitate nonshivering thermogenesis. In pancreatic

islets, O2-· may activate UCP2 function and lower insulin release. In muscle, O2

-· may

activate UCP3 and influence energetic efficiency. The hypothesis that O2-· activates

UCPs is not universally accepted (Couplan et al., 2002). The study reporting

superoxide-mediated activation of UCPs was performed using isolated mitochondria exposed to artificial systems generating very high concentration of exogenous O2

-·

69

(Echtay et al., 2002b) and there is only one study using adenovirus-mediated SOD2

overexpression in islet cells indicating that superoxide-stimulated UCP-mediated uncoupling may also occur in vivo (Krauss et al., 2003).

The aim of this study was to perform a genetic experiment to investigate wether superoxide-activation of UCPs is a regulatory mechanism in vivo. To this end,

we generated a genetically modified mouse strain with a regulated increase of SOD2

enzyme activity. We used P1 artificial chromosomes (PAC) harboring the entire human SOD2 gene to create transgenic mice overexpressing SOD2. PAC mediated

overexpression confers physiological regulation of gene expression due to: (i) the presence of sequence elements needed for physiological regulation of gene expression

within the large genomic fragment; (ii) minimization of positional effects due to the

presence of large amount of flanking sequences (Heintz, 2000); (iii) moderate overexpression of the introduced transgene within a physiologically relevant interval

(Ekstrand et al., 2004). The use of the human SOD2 protein will make it easy to

establish that the transgene is present and expressed in the transgenic mice by using species-specific DNA and antibody probes for human SOD2 (Ekstrand et al., 2004).

PAC clones from a human genomic library were screened for the presence of the SOD2 gene and ordered in a contig by using Southern blotting with end probes

(generated by vectorette PCR) and by size determination with pulse field gel

electrophoresis as previously described (Ekstrand et al., 2004). Three PAC clones with overlapping 5’ and 3’ sequences named PAC662, PAC817 and PAC737 were

used to generate transgenic lines by pronuclear injections of embryos of the inbred FVB/N mouse strain. Southern blots of tail DNA using a human SOD2 cDNA probe

showed integration of the human SOD2 gene into the mouse genome without signs of

rearrangements in all founders. We obtained germline transmission from most founder mice. Two independent lines, PAC662D1 (from the founder D1 of clone

PAC662) and PAC737D2 (from the founder D2 of clone PAC737), were characterized further. The transgenes of these two lines were constantly transmitted at

less than the expected Mendelian ratio of 50% (PAC662D1 34% and PAC737D2

38%; p<0.001 obtained by chi-square test for uneven genotype distribution). Increased expression of SOD2 thus had some effects on fertility, consistent with a

previous report (Raineri et al., 2001). Western blot analyses showed expression of the human SOD2 protein in muscle and kidney of the PAC737D2 line and in most tissues

70

of the PAC662D1 line. SOD enzyme activities were determined in isolated

mitochondria of liver, skeletal muscle, brain and BAT and were increased in skeletal muscle of the PAC737D2 line and in all investigated tissues in the PAC662D1 line.

The observed SOD enzyme activity pattern was thus consistent with the observed human SOD2 protein expression pattern.

SOD2 overexpression did not affect mitochondrial morphology and

mitochondrial biogenesis. EM showed normally appearing mitochondria in heart, skeletal muscle and liver. Mitochondrial mass markers, i.e. mitochondrial volume

density by transmission electron microscopy, mtDNA copy number, mtDNA transcript levels and Tfam, cytochrome c and COXII protein levels, revealed no

differences in mitochondrial mass between PAC662D1 and wild-type mice in skeletal

muscle, BAT and liver. In BAT, following cold acclimation, UCP1 protein and PGC-1α protein levels increased 2-3 fold in both wild-type and PAC662D1mice, but again

no differences were found between the wild-type and the SOD2-overexpressing mice.

Thus, while markers of recruitment and mitochondrial biogenesis in BAT responded

as expected to cold stimulus, the overexpression of SOD2 was without influence on the process.

Oxidative stress damages cellular membranes (Davies, 1995) and renders mitochondria more prone to induction of mitochondrial permeability transition

(Kokoszka et al., 2001). We examined induction of mitochondrial permeability by

Ca2+ and oleate in liver and muscle mitochondria and found as expected that PAC662D1 mitochondria were more resistant to induction of high amplitude swelling

than wild-type mitochondria. The response to alamethicin, a pore forming peptide inducing maximal mitochondrial swelling, was greater in PAC662D1 mitochondria

showing that mutant mitochondria were significantly less swollen than wild-type

mitochondria prior to the addition of alamethicin. SOD2 overexpression thus conferred resistance to induction of mitochondrial permeability.

We performed respiration measurements of isolated mitochondria to assess UCP activity. If matrix O2

-· is a significant activator of UCPs, mitochondria

overexpressing SOD2 would be expected to show a markedly lower basal respiration

because of reduced proton leak. Basal respiration (i.e. state 4, after oligomycin addition) was not changed in liver mitochondria that lack all three UCPs, in brain

mitochondria, that have been reported to express UCP2, and in skeletal muscle

71

mitochondria, that express UCP3 (see Table I of Paper IV). In brown fat

mitochondria, which express UCP1, the equivalent respiration (after GDP addition) was also unchanged (see Table II of Paper IV). Thus in the basal state, mitochondrial

superoxide did not have an observable activating effect on proton conductance mediated by UCPs. Similarly, ADP-stimulated state 3 respiration in liver, brain and

muscle was unaffected by the SOD2 overexpression thus indicating no change in

proton leak (see Table I of the manuscript). However, unexpectedly, in all tissues, SOD2 overexpression increased maximal mitochondrial oxygen consumption

(oxidative) capacity upon addition of a chemical unoupler, FCCP (see Tables I and II of Paper IV). The increase in mitochondrial oxidative capacity upon addition of FCCP

correlated with the increase in SOD enzyme activity. These observations thus indicate

a hitherto unrecognized regulatory effect of endogenous superoxide on mitochondrial oxidative capacity.

It is well-accepted that respiration inhibition by GDP in BAT mitochondria is

mediated by effects on UCP1. SOD2 overexpression did not affect the sensitivity for GDP-mediated inhibition of UCP1 in PAC662D1 mitochondria compared to wild-

type mitochondria (Km of wild-type = 162 ± 25 µM, n = 3 and of PAC662D1 = 168 ±

28 µM, n = 3). Superoxide-mediated activation of UCPs is fatty-acid dependent

(Echtay et al., 2002). SOD2 overexpression did not influence the sensitivity of UCP1 to fatty acids (Km of wild-type = 24 ± 5 nM, n = 5 and of PAC662D1 = 26 ± 8 nM, n

= 5). We repeated these measurements with glycerol-3-phosphate which gives rise to

reverse electron flow and increased superoxide generation at complex I (Turrens,

2003). We found similar oxygen consumption rates in SOD2-overexpressing and control mitochondria when using glycerol-3-phosphate as a substrate, and GDP titration experiments showed no significant difference between wild-type

(Km=169±30, n=5) and PAC662D1 (Km= 146±28, n=5) BAT mitochondria.

Next, we explored UCP3 activities. Purified UCP3 incorporated into liposomes displays a proton translocation capacity that can be stimulated by fatty

acids and inhibited by GDP (Jaburek and Garlid, 2003; Jaburek et al., 1999). As UCP3 is found in skeletal muscle mitochondria, we used fatty acid activation and GDP inhibition to estimate UCP3 activity in isolated skeletal muscle mitochondria

assuming that fatty acid effects in intact skeletal muscle mitochondria are mediated by

UCP3. When respiration was stimulated with pyruvate plus malate, skeletal muscle

72

mitochondria did not exhibit innate GDP-responsive uncoupling, in accordance with a

previous report (Cadenas et al., 2002). However, GDP can inhibit basal proton leak upon induction of reverse electron flow generating high levels of O2

-· (Talbot et al.,

2004). We induced reverse electron flow by using succinate in the absence of rotenone and detected a very small GDP-mediated inhibitory effect on basal

respiration. However, the degree of inhibition was the same in wild-type and

PAC662D1 skeletal muscle mitochondria. Furthermore, we found no significant difference in the sensitivity for fatty-acid mediated activation of UCP3 between wild-

type (Km = 2.6 ± 0.6 µM, n=4) and PAC662D1 skeletal muscle mitochondria (Km = 2.5 ± 0.8 µM, n=4). Also, the maximal response to fatty acid was decreased to the

same extent by GDP treatment of wild-type and PAC662D1 mitochondria. Thus

SOD2 overexpression did not influence the effects of fatty acids or GDP on skeletal muscle mitochondria and, provided that these effects are UCP3-mediated, this

demonstrates that UCP3 activity is not influenced by SOD2 overexpression. It has

previously been indicated that loss of UCP3 activity through gene ablation enhances ATP production of the skeletal muscle (Cline et al., 2001). We determined

mitochondrial ATP production rates (MAPR) in skeletal muscle and found no difference between wild-type and PAC662D1 mice further supporting the conclusion

that SOD2 overexpression did not inhibit UCP3 activities.

We also investigated whether signs of a regulatory role for O2-· could be

observed in the intact animal since the above experiments were performed on isolated

systems and potentially important regulatory factors may have been absent. No difference in body weight was found when wild-type and PAC662D1 mice of both

sexes were compared. The resting metabolic rates determined in mice acclimated to

25°C or 4°C for 4 weeks showed no differences between wild-type and PAC662D1

mice. Thus, SOD2 overexpression does not affect energy expenditure, i.e. the innate UCP activation was apparently not altered. To estimate the importance of superoxide

for UCP1 activity in vivo, we induced nonshivering thermogenesis by norepinephrine (NE) injection of mice acclimated to 25°C or 4°C for 4 weeks and measured oxygen

consumption. The respiratory response to NE was similar in wild-type and PAC662D1 mice. Thus, SOD2 overexpression did not affect UCP1 activity in vivo.

Lowered activation of UCP1 by superoxide could be compensated for by increased BAT organ mass, mitochondrial mass per BAT cell or UCP1 protein levels

73

per cell. We excluded these possibilities by measuring the wet weight and protein

concentration of the left lobe of the interscapular BAT, transcript and protein levels of mitochondrial biogenesis markers in BAT (see above) and Western Blot analysis of

UCP1 expression in BAT following cold exposure of the mice to 4o C for > 3 weeks. Thus there was no indication that a putative decrease in UCP1 activity was

compensated for by augmented recruitment of BAT.

We conclude that SOD2 overexpression did not affect the regulation of UCP1 and 3 activities despite clear effects on other mitochondrial parameters, i.e. oxidative

capacity and resistance to inducers of mitochondrial permeability. Also, in a global measure, that of basal metabolic rate in the intact animal, we found no evidence for

effects of SOD2 overexpression, i.e. no significant part of basal metabolism appears

to derive from superoxide-regulated UCP activity. The absence of a regulatory effect of endogenously generated superoxide on UCP1 and UCP3 activities thus indicates a

basic difference between effects of exogenously generated and endogenously

occurring superoxide.

74

Concluding remarks

We have generated a mouse model for mitochondrial diabetes by inactivating the Tfam gene in pancreatic β-cells. We found that severe RC deficiency of pancreatic β-

cells causes diabetes. Two phases were identified in the progression of mitochondrial

diabetes: first pancreatic β-cells display impaired stimulus-secretion coupling; this is

later followed by β-cell loss.

We next explored whether respiratory chain-deficient cells are able to undergo apoptosis and are more apoptosis-prone in vivo. We used the previously characterized

germline Tfam KO embryos and heart-specific Tfam KO mice and also reexamined the capacity of mtDNA depleted human osteosarcoma cells to undergo apoptosis.

Germline homozygous Tfam KO embryos displayed massive induction of apoptosis at

E9.5 while homozygous heart-specific Tfam KO mice showed a moderate induction of apoptosis. MtDNA depleted (rho-0) human osteosarcoma cells were able to

undergo apoptosis in response to various stimuli. Respiratory chain-deficient cells are thus able to undergo apoptosis and are more apoptosis-prone in vivo suggesting that

apoptosis may contribute to RC disease. We also explored whether apoptosis was

associated with increased ROS generation. Respiratory chain-deficient cardiomyocytes showed slight induction of antioxidant defenses but normal activities

of iron-sulphur containing enzymes that are readily impaired by ROS. These findings indicate that increased ROS generation is not a major disease mechanism in mtDNA-

depleted tissues.

The relationship between RC deficiency, ROS generation and cell death was studied

in more detail in a mouse model of mitochondrial neurodegeneration (MILON mice).

MILON mice were obtained by inactivation of the Tfam gene in neurons of the neocortex and hippocampus. MILON mice developed late-onset neurodegeneration of

neocortex and hippocampus at the age of 5-5.5 months. There was a long lag time between onset of severe RC deficiency at the age of 4 months and onset of

neurodegeneration approximately one month later. We observed massive induction of

neuronal cell death at the age of 5-5.5 months. Respiratory chain-deficient neurons died rather by necrosis than apoptosis as concluded from absence of apoptosis

75

markers and presence of an inflammatory reaction. Most surprisingly, MILON mice

displayed almost no upregulation of antioxidant defenses and no signs of oxidative protein damage at different ages, i.e. during the development of severe RC deficiency.

Interestingly, RC deficient neurons were more susceptible to apoptosis induction by an excitotoxic challenge with kainic acid.

It can be concluded that cell death is a common feature of respiratory chain-deficient tissues. Embryonal cells, cardiomyocytes, and neurons likely utilize different cell

death pathways as indicated by the lag time between onset of severe RC deficiency and cell death, presence of apoptosis markers and inflammatory changes. Glycolysis

was significantly upregulated in cardiomyocytes but not in neurons. Upregulation of

glycolysis may allow execution of apoptosis (Leist et al., 1997) in cardiomyocytes and its absence may explain why respiratory chain-deficient neurons rather undergo

necrosis. ROS formation is not a prominent feature of cardiomyocytes and neurons

with a severe RC deficiency induced by mtDNA depletion.

We adressed whether mitochondrial O2-· regulates UCP activity as indicated by

studies of isolated mitochondria that used artificial systems to generate high levels of

superoxide. We created PAC transgenic mice with a regulated increase in SOD2

enzyme activity to investigate whether superoxide-mediated activation of UCP is a regulatory mechanism in vivo. SOD2 overexpression improved mitochondrial

parameters, i.e. oxidative capacity and resistance against induction of mitochondrial permeability, but did not alter UCP1 or UCP3 activities. The resting metabolic rate

and the respiratory response to induction of nonshivering thermogenesis were normal

in SOD2 overexpressing mice further confirming that SOD2 overexpression did not affect UCP activities. BAT organ weight, mitochondrial mass and UCP1 protein

levels were not upregulated excluding the possibility that the failure to observe alterations in UCP activities were compensated for by increased recruitment of the

tissue. We conclude that superoxide does not regulate UCP activities under in vivo

physiological conditions, however we observed a hitherto unrecognized effect of superoxide on mitochondrial oxidative capacity. The absence of a regulatory effect of

endogenously generated superoxide on UCP1 and UCP3 activities thus indicates a

76

basic difference between effects of exogenously generated and endogenously

occurring superoxide.

77

Acknowledgements

I wish to express my gratitude to all those who have made this thesis possible,

especially:

Nils-Göran Larsson, my supervisor. It is a very high privilege to be the student of such a distinguished scientist. You taught me to see the critical issues in science.

Thank you for sharing your experience and knowledge with me, for your trust,

generosity and support throughout my entire PhD period, for your friendship and for every advice I got from you to take important decisions.

Claes Gustafsson for your enthusiasm and optimism, for sharing your wealth of ideas

with me, for your help and for all valuable advice I got from you.

I would like to thank Jan-Åke Gustafsson for providing excellent scientific resources

at the Department of Medical Nutrition.

I would like to thank all the collaborators who have contributed to these studies: Per-Olof Berggren, Martin Köhler, Pierre Rustin, Lars Olson, Eva Lindqvist, Anders Oldfors, Barbara Cannon, Jan Nedergaard, Irina Shabalina, Natasa Petrovic, Emma Backlund, Rolf Wibom and Kjell Hultenby. You have all provided

important expertise in your fields!

I would like to thank present and former members of the Nils-Göran Larsson’s Group: Caroline Graff and Jianming Wang for fruitful and pleasant collaborations, Eric Dufour for showing me the real altruism, for your constant interest and willingness to

help, for valuable inputs and fruitful collaboration, Mats Ekstrand for introducing me into new protocols of Molecular Biology and for support whenever I requested it,

Aleksandra Trifunovic for carefully managing the laboratory and providing a platform to perform scientific work, Anna Hansson, Nicole Hance, Mina Pellegrini, Muegen Terzioglu for pleasant company in the laboratory, Anna Wredenberg for

interesting discussions extending beyond the world of science, Christoph Freyer for helping to organize laboratory issues and for organizing the journal club, Martina Gaspari for cheerful company, Chan Bae Park for support, wisdom and fine sense

78

of humour, Ivan Khvorostov for cheerful company and Russian annecdotes at late

and lonely laboratory hours, Zekye Cansu for support in practical laboratory matters, Lene Sörensen for sharing with me her IT expertise, Anja Rantanen and Hans Wilhelmsson for teaching me the Finish and Swedish Culture.

Furthermore I would like to thank Maria Falkenberg and all her group members for

sharing our scientific activities and for having a great time outside the laboratory.

I wish to acknowledge colleagues and friends in/outside the institute, Maria Eriksson for your friendship and your scientific inputs, Annika Wallberg for extremely

valuable advice regarding post-doctoral studies, Margaret Warner and Magnus Nord for critical inputs into my thesis, Gil-Shin Shim for providing me with protocols and technical help and Charlotta Lindvall for your company in my early

years in Sweden.

Finally I would like to thank my mother and my father, to whom I dedicate my

thesis, for your eternal and unconditional support.

79

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