Scheffler Immo E. Mitochondria

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MOLECULAR GENETICS OF HUMAN MITOCHONDRIAL DISEASES 371

general population (0.1% to 10%, probably variable in different ethnic groups)

(102) . Additional data on population genetics and history will be required to

establish whether other rare mutations are mildly pathogenic, or secondary,

or of no consequence at all. It will be important to resolve whether LHON is

caused exclusively by mutations affecting complex I.

While there is agreement that the primary mutations in the ND1, ND4, and

ND6 genes represent the major risk factor in LHON, there is more controversy

about the actual effect of these mutations on the rate of electron transport

and oxidative phosphorylation. It would seem straightforward to look at the

speciﬁ c activity of complex I (the NADH - CoQ oxidoreductase) in mitochon-

dria from LHON patients and normal controls. Nevertheless, results in differ-

ent laboratories have been contradictory, depending on what was measured

(summarized by reference 100 ). One type of measurement involves oxygen

consumption with NADH - linked substrates, and respiration with succinate is

used as a control and for normalizations. Care must be taken to measure

complex I activity by demonstrating the sensitivity to rotenone. Experiments

can be performed with white blood cells and platelets from patients, or after

construction of cybrid lines containing mitochondria from LHON patients and

nuclei from ρ

lines. Another approach is to measure the pyruvate : lactate ratio

as an indicator of the NADH/NAD balance in ﬁ broblasts. The method has

pitfalls discussed by Howell (100) .

Why does a reduced activity of complex I in homoplasmic patients cause

LHON, while a reduction in electron transport and OXPHOS capacity in a

patient with heteroplasmy and mtDNA deletions cause KSS? In homoplasmic

LHON patients there is no mechanism for a further shift toward a worse

condition in individual cells or tissues. In contrast, in KSS patients, sampling

of available tissue may indicate 50% heteroplasmy, for example, but this is an

average, and individual cells in the brain or myotubes in muscle may be more

severely compromised, causing clinical symptoms. It is clear that much remains

to be learned about the distinction between localized defects in individual

complexes of the electron transport chain and an overall reduction in several

or all complexes due to a mtDNA deletion. The metabolic consequences are

almost certainly more complex that a simple reduction in ATP supply.

7.3.6.2.2 Neuropathy, Ataxia, Retinitis Pigmentosa (NARP) If there is a

“ bottleneck ” in oxidative phosphorylation, it can easily be imagined in complex

V, as already indicated by the well - known phenomenon of coupling in mito-

chondria and the dependence of electron transport on the ADP concentration.

A more rigorous statement is that ATP turnover (synthesis and consumption)

has the highest control coefﬁ cient of all the reactions of OXPHOS, and changes

in this rate would be expected to have the largest effect on the overall rate of

oxidative phosphorylation (see Chapter 5 ). One may, therefore, be able to

rationalize that mutations in ATP synthase (complex V) subunits are rare, and

in the one reported case the symptoms are relatively broad. A mutation in

subunit 6 (nucleotide 8993; Leu → Arg) leads to neuropathy, ataxia, and reti-

372 MITOCHONDRIAL MUTATIONS AND DISEASE

nitis pigmentosa (NARP). Since the structure of complex V is beginning to

become resolved at high resolution (see Chapter 5 ) the amino acid substitution

can be related to the observed inhibition in ATP synthesis: An additional posi-

tive charge is positioned in the proton channel. In fact, the inhibition appears

to be severe, and NARP patients are heteroplasmic, with the severity of the

symptoms related to the fraction of mutated genomes. At one end of the

spectrum are mild neurological signs such as migraine headaches and retinitis

pigmentosa, and at the other end one observes necrotizing encephalopathy or

Leigh syndrome (102 – 104) . A pedigree has been described in which the mother

was mildly affected (87% mutant mtDNA), but all four offspring had > 90%

mutant DNA and were severely affected; two daughters died of Leigh syn-

drome at an early age.

7.3.6.2.3 MELAS, MERRF, and MIMyCa Point mutations responsible

for mitochondrial encephalomyopathy with lactic acidosis and stroke - like epi-

sodes (MELAS), myoclonus epilepsy and ragged red ﬁ bers (MERRF), and

maternally inherited disorder with adult - onset myopathy and cardiomyopathy

(MIMyCa) are found in speciﬁ c tRNA genes. The MERRF mutation (tRNA

Lys

)

is heteroplasmic. Although the symptoms vary from mild (hearing loss, mito-

chondrial myopathy, electrophysiological changes) to severe (uncontrolled

myoclonic jerking, dementia, cardiomyopathy, nephropathy, neurosensory

hearing loss, altered VER and EEG), there is no strict correlation with the

percentage of mutant mtDNAs. In the view of Wallace et al. (105) , age strongly

inﬂ uences the expression of the mutation. This group has become the strongest

advocate of the hypothesis that an accumulation of mitochondrial mutations

in somatic cells with age causes a general decline in OXPHOS capacity with

age, possibly at different rates in different tissues. For each tissue there is a

critical threshold below which the energy needs of the cells can no longer be

met. In an individual starting out with a lower capacity due to a tRNA

Lys

muta-

tion, the drop in capacity with age will reach the threshold sooner, and symp-

toms start to appear. The nervous system and muscle with the highest minimal

requirements are predictably the ﬁ rst tissue to be affected (102) . Based on the

observations in pedigrees, an individual with > 90% of mutant tRNALys could

nevertheless be normal at a young age, but would deteriorate as he/she aged.

This hypothesis has to some extent prompted research and is now supported

by data showing an increase in mtDNA mutations with normal aging in various

tissues (see Section 7.4.2 below).

7.3.6.2.4 Sensorineural Hearing Loss Deafness can arise from many causes,

including overexposure to rock concerts and iPods. An interesting genetic case

are speciﬁ c mutations in the 12S rRNA gene that lead to aminoglycoside -

induced and nonsyndromic deafness (106 – 108) . The investigation of several

large pedigrees from different ethnic origins has clearly established maternal

inheritance and the 12S rRNA alteration as the primary factor, which is,

however, not sufﬁ cient by itself to produce the phenotype. Guan and his

MOLECULAR GENETICS OF HUMAN MITOCHONDRIAL DISEASES 373

colleagues have postulated that modiﬁ er genes in the nucleus must contribute

to the expression of the phenotype. A very exhaustive and thorough genotypic

analysis has focused on a gene (TRMU) encoding a mitochondrial protein

required for tRNA modiﬁ cation (post - transcriptional) (106) . The role of this

gene product had ﬁ rst been elucidated in bacteria. The same group has also

identiﬁ ed a deafness - associated mitochondrial tRNA(Ser(UCN)) T7511C

mutation, which becomes highly penetrant in families in conjunction with

homoplasmic ND1 T3308C and tRNA(Ala) T5655C mutations (109) .

7.3.7 Nuclear Mutations and Mitochondrial Disease

7.3.7.1 Defective Electron Transport Chain The term “ mitochondrial

disease ” was at ﬁ rst associated with pathologies arising from mutations in

mtDNA, and therefore with partial defects in energy metabolism. However, it

is clear that a substantial number of nuclear genes encode subunits of the

complexes of the oxidative phosphorylation system, or proteins that are essen-

tial for the biogenesis of mitochondria and the assembly of such complexes.

Mutations in these genes could cause similar phenotypes/pathologies, and they

are therefore included in the discussion. On the other hand, many “ inborn

errors of metabolism ” result from the compartmentalization of metabolism

and from defective mitochondrial enzymes. Such “ metabolic disorders ” result-

ing from nuclear mutations will not be further discussed here. Adding up all

the nuclear - encoded subunits of complexes I – V would suggest ∼ 70 nuclear

genes contributing to the biogenesis of the ETC and ATP synthase. The number

of assembly factors required is still unknown. One can add to this number (1)

the genes/gene products required for mtDNA replication, maintenance, repair;

(2) gene products required for transcription and processing of the polycis-

tronic transcripts; and (3) gene products for the mitochondrial translation

machinery. Thus, at least 200 genes contribute directly to the biogenesis of the

OXPHOS machinery, and defects in such genes could lead directly to an

“ energy crisis. ” Many more genes are needed for mitochondrial protein import,

metabolite exchange between the cytosol and the matrix, and the biosynthesis

of essential cofactors and prosthetic groups (hemes, Fe – S centers, ubiquinone).

A deﬁ ciency would probably affect energy metabolism, but may cause a more

complex pathology. The term “ mitochondrial disease ” is at risk of becoming

less speciﬁ c since so many functions are associated with mitochondria.

Some of these genes are known to be X - linked. A severe deﬁ ciency would

most likely be embryonic lethal in males. In X - linked genes, only missense

mutations encoding partially functional proteins would be expected in affected

males. It is interesting to speculate whether X - inactivation in a heterozygous

female might be biased during development to lead to a functional OXPHOS

system in surviving/selected cells. Random X - inactivation would lead to a

deﬁ ciency at the cellular level, and mosaicism may not be compatible with a

viable tissue. Severe autosomal mutations appear to be rare. In a homozygous

condition, one would most likely ﬁ nd embryonic lethality. Thus, patients, even

374 MITOCHONDRIAL MUTATIONS AND DISEASE

newborns with a short life expectancy, must have some residual OXPHOS

activity. How much is detectable can depend on the tissue in which it is mea-

sured (and which tissue is available for autopsy from a live patient). Dominant

autosomal mutations in any of the above - mentioned genes have not been

reported. Large pedigrees for patients with severe mitochondrial diseases due

to nuclear mutations are rare.

A signiﬁ cant number of patients have now been characterized with an

OXPHOS deﬁ ciency due to nuclear mutations affecting the activity of either

an isolated complex or the entire electron transport chain (see references 59 ,

60 , and 110 – 112 for reviews). Mutations in genes encoding structural proteins

(subunits) generally cause a deﬁ ciency in a speciﬁ c complex, but there are

exceptions. Patients with an isolated complex I deﬁ ciency represent a majority

of such patients (113 – 122) . In most or all of such patients a reduced complex

I activity results from a reduced level of the assembled complex I, rather than

from a reduced speciﬁ c activity of the complex. It is noteworthy that in a large

fraction of such patients the defective gene has not been identiﬁ ed, despite

the determined efforts by several laboratories where all known nuclear and

mitochondrial genes for subunits of complex I were sequenced. A majority of

such patients are male, and the possibility of an as yet unidentiﬁ ed X - linked

gene required for complex I assembly or activity must be considered. The

existence of such an X - linked gene is suggested by experiments in the Schefﬂ er

laboratory, where Chinese hamster mutant cells lacking complex I could be

complemented with a wild type mammalian X chromosome (36) . These

mutants constitute a third complementation group distinct from the mutants

with defective NDUFA1 (MWFE) or NDUFB11 (ESSS) genes.

Patients with a partial complex II activity have also been described by

several laboratories (123 – 125) . While some of these have the expected symp-

toms from an OXPHOS deﬁ ciency, mutations in the SDHB, SDHC , and SDHD

genes can cause the formation of an unusual class of tumors called paragan-

gliomas, a tumor of the carotid body (the main arterial chemoreceptor that

senses oxygen levels in the blood) (126 – 130) . Isolated complex III deﬁ ciencies

are very rare. There is one report of a deletion in the nuclear gene UQCRB

encoding the human ubiquinone - binding protein of complex III (QP - C subunit

or subunit VII) in a consanguineous family (131) . Among the patients with an

isolated complex IV deﬁ ciency, several defective nuclear genes have been

characterized that do not encode subunits of the mature complex. The observed

low activity of cytochrome oxidase was proved to be due to missing assembly

factors. As described above, many genes/proteins have been shown to be

required for complex IV assembly. In human patients with Leigh syndrome,

severe mutations have been described in the SURF1 gene (132 – 135) , and other

patients have been identiﬁ ed with mutated SCO1 or SCO2 genes (59, 136) .

The products of these genes have similar but not redundant functions in the

delivery of copper to the complex. In contrast to patients with a complex I

deﬁ ciency, the relevant complex IV - related genes are found to have homo-

logues in yeast, and thus any suspected mutation or deﬁ ciency can be tested

MOLECULAR GENETICS OF HUMAN MITOCHONDRIAL DISEASES 375

in this model system to verify its effect on complex IV assembly/activity.

Nuclear mutations responsible for complex V deﬁ ciencies have not been

detected in humans so far.

7.3.7.2 MtDNA Maintenance and Replication Although mtDNA replica-

tion is relaxed (not tightly coupled to nuclear DNA replication), there must be

controls that maintain the copy number in a range appropriate for a particular

tissue or organ. A number of patients have now been characterized with muta-

tions affecting this pool of mtDNAs. Symptoms include progressive external

ophthalmoplegia (PEO), exercise intolerance, ataxia, cardiomyopathy, gastro-

intestinal symptoms, and more (see references 59 and 137 – 139 ). The symptoms

appear between 18 and 40 years of life and are associated with extensive and

increasingly large proportions of mtDNA deletions. Such deletions, detectable

by Southern blotting, are not found in all tissues, and it appears that they arise

in nondividing tissues such as muscle, in contrast to leukocytes. The deletions

are the result of defective DNA replication, which can be caused by mutated

enzymes of the replication machinery itself (DNA polymerase γ , or the heli-

case/primase TWINKLE) or by perturbations in the mitochondrial nucleotide

pool(s) involving enzymes such as the thymidine phosphorylase (140) or trans-

porters such as the adenine nucleotide translocator (141) .

There are also syndromes resulting from mtDNA depletion. Children

healthy at birth may develop severe hepatopathy, myopathy, nephropathy, or

encephalopathy from a tissue - speciﬁ c loss of mtDNA down to levels of ∼ 10%

of normal. Mitochondria from such a patient could be introduced into ρ

cells

and repopulate such cells with mtDNA (142) . Thus, the unaffected parents

were believed to be heterozygous for a recessive mutant allele, a conclusion

strengthened by the ﬁ nding of two affected sons and an affected daughter in

a consanguinous marriage (143) . The restriction of the loss of mtDNA to spe-

ciﬁ c tissues is still much of a puzzle, especially since variable tissue involve-

ment has been reported in a single family (137) .

Because affected families are rare and usually small, linkage studies are

difﬁ cult. One approach has been to select candidate genes and carry out hap-

lotype analysis with closely linked satellite markers. Among the candidates,

the genes for the mitochondrial transcripion factor (mtTFA), the nuclear

respiratory factor 1 (NRF - 1), the mitochondrial single - stranded DNA binding

protein (mtSSBP), and endonuclease G were examined, but mtTFA remained

the only candidate, if inheritance is indeed recessive (143) . A limited or bio-

chemical analysis has again implicated perturbed mitochondrial dNTP pools.

In a small number of families, defects were identiﬁ ed either in the deoxy-

guanosine kinase (144) or in the mitochondrial thymidine kinase (TK2) (138,

145) . The puzzling aspect of these ﬁ ndings is that certain tissues (skin, blood)

are completely spared. It may be noted in this context that HIV patients being

treated with drugs such as azidovine (nucleoside analogues) may also suffer

from mtDNA depletion in various tissues after prolonged treatment. The

mtDNA populations recover after withdrawal of the drug.

376 MITOCHONDRIAL MUTATIONS AND DISEASE

7.3.7.3 Friedreich’s Ataxia The discussion of mitochondrial diseases

cannot be exhaustive in this volume, but two more syndromes caused by very

speciﬁ c mitochondrial protein defects should be mentioned here. Friedreich ’ s

ataxia is the most common hereditary ataxia, and it is considered a nuclear -

encoded mitochondrial disease because it is clearly caused by a deﬁ cit in the

mitochondrial protein frataxin (146 – 149) . The deﬁ cit results from a reduced

level of frataxin mRNA in the cytosol. The frataxin gene on chromosome 9q13

has an unstable GAA trinucleotide repeat in the ﬁ rst intron; an expanded

repeat leads to inefﬁ cient splicing. The severity of the phenotype is correlated

with the size of the expanded repeat. Although the corresponding yeast gene

had been identiﬁ ed and used to reproduce the phenotype in this model organ-

ism, it has nevertheless been a challenge to characterize the precise role of

frataxin in mitochondria. It is generally agreed that frataxin plays a role in

mitochondrial iron homeostasis and may be the donor of iron in the synthesis

of heme and of iron – sulfur clusters. A deﬁ ciency reduces the supply of these

important constituents of the electron transport complexes and hence reduces

ATP production by oxidative phosphorylation. However, further complica-

tions arise. Frataxin may bind surplus iron (it binds six Fe

per monomer);

and, when depleted, iron accumulates in mitochondria. This surplus/free iron

can facilitate the interconversion of ROS into the highly reactive hydroxyl

radical by the Fenton reaction and thus accentuate oxidative stress. In other

words, frataxin contributes to the antioxidant defense, in addition to its iron

chaperone function in heme and ISC synthesis. There is also a contrary point

of view suggesting that increased superoxide production could not explain by

itself the FRDA cardiac pathophysiology (150) .

Oxidative stress caused by frataxin deﬁ ciency leads to the selective loss of

dorsal root ganglia neurons, cardiomyocytes, and pancreatic beta cells. Why

these nondividing cells are particularly sensitive to frataxin loss is still a puzzle,

since frataxin is widely expressed in all cells and tissues. From this selective

cell loss, one can rationalize the cardiomyopathy and diabetes that form part

of the clinical manifestations of Friedreich ’ s ataxia. The disease is relatively

common because the trinucleotide GAA expansion is an event occurring at a

higher frequency compared to other mutations in nuclear genes.

7.3.7.4 Deafness and Dystonia Syndrome (Mohr –Tranebjaerg Syndrome)

DDS is an X - linked recessive disorder (151) . Sensorineural hearing loss, dys-

tonia, mental deterioration, paranoid psychotic features, and optic atrophy are

indicative of progressive neurodegeneration. The corresponding human gene

was identiﬁ ed and cloned by molecular – genetic techniques after careful

mapping, and when the amino acid sequence of the human DDP1 protein was

“ blasted ” against other proteins in the whole protein database, it was found

to be homologous to the Tim8p of yeast (152 – 154) . DPP1 (alias TIMM8a)

assembles with TIMM13 in a 70 - kDa complex in the intermembrane space

and assists in the integration of the hTim23 protein into the inner membrane

(see Chapter 4 ). The elucidation of the mechanism was greatly helped by

molecular genetic manipulations of the homologous genes in yeast ( TIM8,

TIM13 ). The DPP1 protein was not present in ﬁ broblasts from a patient with

this syndrome, presumably because a missense mutation, Cys66Trp, abolished

a conserved CX (3) C ’ motif, preventing the protein from assembly with

TIMM13 (as predicted from similar studies in yeast). Thus, a satisfactory

molecular explanation can be provided for the observed symptoms. On the

other hand, one might have predicted that the loss of hTim8 and hence the

effect on the import of Tim23 would have an effect on a much wider range of

tissues.

7.3.8 Conclusion

A migraine headache or muscle fatigue should not cause an immediate panic

and provoke thoughts about mutations affecting mitochondrial functions, but

a chronic condition does deserve attention. A little over two decades ago,

physicians may have had a distant memory of mitochondria from the basic

biochemistry course in their ﬁ rst year in Medical School. Today, histochemical,

biochemical, and molecular tests of mitochondrial dysfunction have become

almost routine in the analysis of a broad spectrum of neuropathies and myopa-

thies. Both nuclear and mitochondrial mutations can contribute to defective

mitochondrial energy metabolism. Severe nuclear mutations in homozygous

individuals would almost certainly be lethal. If heterozygotes have no increased

ﬁ tness or selective advantage (as may be the case in sickle cell anemia and

possibly in the case of cystic ﬁ brosis) and homozygotes are not viable, the

frequency of speciﬁ c alleles in the population may remain small. However, the

large number of genes required for an active oxidative phosphorylation system

represents a large target for mutational damage, and hence the overall fre-

quency of individuals with mitochondrial diseases is potentially very large. The

number may also depend on where one draws the line between a headache, a

fatigue, and exercise intolerance, and a disease. Ask your doctor, as the TV ads

advise.

7.4 MITOCHONDRIAL DNA AND AGING

The most important inventions in evolution are sex and death.

— F. Jacob

7.4.1 Introduction

The achievements of the last decade have revitalized research on mitochon-

dria, not only because of new insights gained into the genetic basis of mito-

chondrial function and dysfunction, but also because of the realization of their

vital role in maintaining a healthy individual. It is, with hindsight, perhaps not

too surprising that mitochondria as the “ powerhouse ” of the cell would play

MITOCHONDRIAL DNA AND AGING 377

378 MITOCHONDRIAL MUTATIONS AND DISEASE

a critical role in the health (function and survival) of cells. Turnover of all

components is a constant activity, consuming as much as 50% of the total

energy supply, and in the broad sense turnover is clearly related to “ mainte-

nance and repair. ” When the energy supply becomes limiting, the infrastruc-

ture slowly deteriorates, and more severe consequences may be triggered by

catastrophic failures at localized sites. This view, particularly as related to the

process of aging, is undoubtely very simplistic. Nevertheless, the concept that

aging and mitochondrial dysfunction may be related has had a powerful appeal

to a number of investigators in the past two decades (62, 155 – 167) . The cita-

tions listed here include some inﬂ uential “ historical ” papers and a sampling

of the most recent reviews. One of the most prominent journals in the biologi-

cal sciences has recently acknowledged that “ aging research come of age ” and

devoted a whole issue to this subject (168) . The present chapter will attempt

to review some of the hard facts and follow up on some of the more promising

speculations. A discussion of the many aspects of senescence is clearly beyond

the scope of this treatise. Familiar characteristics associated with aging are a

decline of muscle power and often, but not always, diminished mental capaci-

ties. From the well - established cause - and - effect relationship between clinically

signiﬁ cant encephalomyopathies and mitochondrial DNA mutations, it is

tempting to look for a similar relationship between normal aging and a reduced

capacity for respiration and oxidative phosphorylation.

Some clear distinctions should be made at the outset. Attempting to deﬁ ne

a single cause for normal aging may be presumptious. However, it is probably

not too far - fetched to claim that a diminution of mitochondrial functions may

either be central to the process or be a strong contributor inﬂ uencing the

timing and the severity of the overall deterioration. The challenge is to set up

a plausible chain of events and to establish a cause - and - effect relationship. A

credible scenario gaining acceptance is based on the postulate of a slow and

stochastic buildup of damage caused by reactive oxygen species (ROS) that

are a normal byproduct of respiration. Superoxide, hydrogen peroxide, and

hydroxyl radicals are constantly being formed. Estimates are that up to 4% of

the oxygen consumed in mitochondria is converted to reactive oxygen species.

This number has recently been challenged and reduced by an order of mag-

nitude, but it is still recognized to be a signiﬁ cant risk of damage to macro-

molecules (163) . Their potential harmfulness can be judged from the

co - evolution of a set of enzymatic defence mechanisms such as superoxide

dismutase, catalase, and peroxidases, enzymes which destroy these reactive

species before damage can be done. A number of experiments with transgenic

ﬂ ies and mice have demonstrated quite convincingly that knocking down these

enzymes can reduce the life span, while increased doses of such enzymes can

prolong the life expectancy (169, 170) . A word of caution: The life extension

of fruit ﬂ ies by this means was successful with short - lived strains of Drosophila

melanogaster , but failed with naturally long - lived strains. Additional substances

(e.g., vitamin E) can act as free radical scavengers, and they have been pro-

posed for megavitamin therapies. Although the largest proportion of oxygen

utilized by a cell is by the mitochondrial electron transport chain, cytochrome

P - 450, cytosolic oxidases, and the oxidations in the peroxisome generate reac-

tive oxygen species outside of the mitochondria, and the protective functions

are found in multiple compartments.

The reactive oxygen species can damage DNA, proteins, or lipids. Thus, it

is at least conceivable that the primary cumulative damage is to mitochondrial

lipids (e.g., cardiolipin), altering membrane ﬂ uidity and ultimately causing

defects in electron transport and respiration; as a result, the generation of

reactive oxygen species may be accelerated. Eventually, defense mechanisms

and repair systems are overwhelmed and damage to mtDNA becomes perma-

nent. Alternatively, one could consider mtDNA to be the primary target of

superoxide or hydroxyl radicals; and as more and more mtDNAs sustain muta-

tions in critical coding regions, complexes in the electron transport chain

become less efﬁ cient or inactive, leading to a decline in mitochondrial function.

H. T. Jacobs reviewed the experimental support for this hypothesis (171) ,

concluding that a rigorous test of the hypothesis remains to be undertaken,

but would require a direct manipulation of the rate of mtDNA mutagenesis,

to test whether this could alter the kinetics of aging. In evaluating these alter-

natives (among still others), attention must be paid to the relative rates of

turnover of the major mitochondrial constituents. Generalizations applying to

all differentiated cells and tissues are likely to be inappropriate.

Very elegant, informative, and provocative studies were published by Tri-

funovich and colleages in the past few years (172 – 174) . Mouse strains were

constructed with homozygous mutations in the mitochondrial DNA poly-

merase γ gene. The mutations had no effect on the polymerase activity, but

eliminated the exonuclease (proofreading) activity of the enzyme. The result

was a three - to ﬁ vefold increase in point mutations in mtDNA, in addition to

increased deletions which was correlated with a signiﬁ cantly reduced life span

(median life span of 48 weeks compared to 2 years of controls). Death was

preceded by many of the typical, age - related phenomena such as weight loss,

hair loss, osteoporosis, curvature of the spine, anemia, heart disease, and

reduced fertility. On the one hand, this study appeared to establish quite con-

clusively a strong connection between the accumulation of mutations in

mtDNA and aging. On the other hand, a follow - up study by the same labora-

tory claimed to refute the favorite hypothesis that mtDNA mutations induced

by ROS were the cause of aging. The mtDNA mutator mice developed severe

respiratory chain dysfunction, but there was no increased production of ROS

and no evidence for increased oxidative stress (measured by protein carbon-

ylation levels), aconitase activity (sensitivity of Fe – S to ROS), or levels (induc-

tion) of scavenger enzymes for ROS. To quote these authors: “ The premature

aging phenotypes in mtDNA mutator mice are thus not generated by a vicious

cycle of massively increased oxidative stress accompanied by exponential

accumulation of mtDNA mutations. We propose . . . that respiratory chain dys-

function per se is the primary inducer of premature aging in mtDNA mutator

mice. ” The emphasis here might be placed on the idea of a vicious cycle, also

MITOCHONDRIAL DNA AND AGING 379

380 MITOCHONDRIAL MUTATIONS AND DISEASE

referred to as the “ error catastrophy, ” originally proposed by L. E. Orgel to

explain cellular senescence in tissue culture and aging (the Hayﬂ ick phenom-

enon) with a focus on nuclear genes. This hypothesis poses that initial muta-

tions in polymerases cause an increase in mutations in other genes on mtDNA,

leading to additional faulty proteins which continue to aggravate the damage

to the point of the ﬁ nal catastrophy.

In the above example the mutator polymerase probably swamps any muta-

tional damage by ROS occurring over a normal life span. Nevertheless, it

should be noted that mutations diminishing OXPHOS activity do not neces-

sarily and inevitably lead to increased ROS production, as is frequently

assumed in many publications. Very similar conclusions were reached by

Kujoth et al. (175) with a very similar but independently constructed mutator

mouse model.

7.4.2 Accumulation of mtDNA Damage and Normal Aging

The idea that the accumulation of mtDNA damage might be part of, or even

responsible for, normal senescence is a relatively novel concept. If it was

expressed before, it would have been pure speculation, since the technical

means for testing it were not available until the late 1980s. The ﬁ rst prerequi-

site was the determination of the human mtDNA sequence. The second chal-

lenge was to ﬁ nd, characterize, and quantitate abnormal mtDNA molecules

present at low abundance in a population of 1000 – 3000 molecules per cell.

Restriction mapping could detect only large rearrangements or deletions in

heteroplasmic populations when the abnormal molecules were a homoge-

neous subpopulation and present as a signiﬁ cant fraction (10%?). A hetero-

geneous mutant population with many different point mutations, each present

at the < 1% level, is not so easily detectable.

As in so many other areas, the polymerase chain reaction has had an enor-

mous impact on the analysis of mitochondrial genomes. Taking a cue from the

observations in patients with myopathies, it became possible to focus the initial

attention on relatively large deletions, and speciﬁ cally the ∼ 5 - kb “ common

deletion ” between the repeats at 8470 and 13447 frequently found in Kearns –

Sayre patients (see Section 7.3.6.1 ). By the appropriate choice of oligonucle-

otide primers, such deletions can be detected when present in just a few

mtDNA molecules (0.1% to 1%). Following the initial observations in the

laboratory of Linnane (176) (and see references 157 and 158 for reviews),

several laboratories have investigated the incidence of age - related deletions

in a variety of human tissues including brain, heart, kidney, liver, assorted

muscle and blood (157, 158, 177 – 182) .

Several generalizations can be made. (1) As suspected, there is an age -

related increase in the frequency of mtDNA deletions. (2) Deletions are

detected readily in nondividing tissue such as brain and skeletal muscle, while

in cells with a relatively short half - life (e.g., blood cells), few, if any, mtDNA

deletions have been detected. There must be a selection against an accumula-