[extropy-chat] Bene Tleilaxu and your mitochondria

[Rafal Smigrodzki]

A few months ago I promised to post an article on mitochondria and
aging which I was writing with Shaharyar Khan, and finally I can keep
my promise. "Mitochondrial microheteroplasmy and a theory of aging and
age-related disease" will be published in Rejuvenation Research in
August. Here is the text (without figures) and I can send the pdf to
anyone interested.

Questions and comments welcome.

Rafal 

Mitochondrial microheteroplasmy and a theory of aging and age-related disease

Rafal M. Smigrodzki, MD PhD*
and
Shaharyar M. Khan, PhD

Address for correspondence and reprints: Gencia Corporation, 706B
Forest St, Charlottesville VA, 22903, Phone 434-295-4800, Fax:
434-295-4951, Email: rafal at genciabiotech.com

Keywords: Microheteroplasmy, aging, AD, PD, diabetes, hypertension

*Corresponding author to whom communications should be addressed. 

Abstract

We implicate a recently described form of mitochondrial mutation,
mitochondrial microheteroplasmy, as a candidate for the principal
component of aging. Microheteroplasmy is the presence of hundreds of
independent mutations in one organism, with each mutation usually
found in 1 - 2% of all mitochondrial genomes. Despite the low
abundance of single mutations, the vast majority of mitochondrial
genomes in all adults are mutated. This mutational burden includes
inherited mutations, de novo germline mutations, as well as somatic
mutations acquired either during early embryonic development or later
in adult life. We postulate that microheteroplasmy is sufficient to
explain the pathomechanism of several age-associated diseases,
especially in conditions with known mitochondrial involvement, such as
diabetes (DM), cardiovascular disease, Parkinson's disease (PD), and
Alzheimer's disease (AD) and cancer.  The genetic properties of
microheteroplasmy reconcile the results of disease models (cybrids,
hypermutable PolG variants and mitochondrial toxins), with the
relatively low levels of maternal inheritance in the aforementioned
diseases, and provide an explanation of their delayed, progressive
course.

Article Outline

1. Introduction
2. Review of mitochondrial biology and genetics
	2.1 Mitochondrial biology and mtDNA
2.2 Changes in mitochondrial genomes during early ontogeny
2.3 Replication Induced Mutations
2.4 Heteroplasmy
2.5 Microheteroplasmy and certain other acquired mitochondrial mutations
3. Mitochondria in age-related disease
3.1 Lines of evidence
3.2 Neurodegeneration
3.3 Diabetes and hypertension
3.4 Cancer
	3.5 Animal models of mitochondrial aging
4. Mitochondrial theory of aging
	4.1 Objections to the mitochondrial hypothesis of aging
	4.2 Microheteroplasmic mitochondrial theory of aging
	4.3 Cellular responses to microheteroplasmy
	4.4 Focal microheteroplasmy
4.5 Microheteroplasmy in stem cells
5. Competing hypotheses
5.1 Relationship to competing hypotheses
	5.2 Unresolved issues
6. Predictions of the microheteroplasmic mitochondrial theory of aging
	6.1 Age-related diseases
	6.2 Normal aging
6.3 Therapeutic opportunity
7. Conclusion
	7.1 All roads lead to Rho (¦Ñ)
8. Acknowledgements.
9. References

1. Introduction

Aging, broadly defined, is the decline and failure of biological
processes to maintain the complexity and contiguity of an organism
over time. Maintaining this complexity and contiguity in the face of
entropic forces requires continuous energy appropriation and
dissipation 1. Another element crucial to maintenance of complexity is
integrity of genetic information, as evidenced by progerias,
conditions where failure of genomic maintenance leads to accelerated
development of aging phenotypes 2. Interestingly, many progerias
primarily affect stem cells and their generation of oxidative stress,
preventing the repopulation of tissues damaged with age 3.  These
diseases provide significant insights into the relevance of
maintaining genomic integrity against the ravages of time.

In contrast to considering aging from the perspective of fundamental
physical and systems-theory principles, practical approaches to the
pathophysiology of most aging-related diseases focus on specific
biochemical changes that accompany aging. Thus, mutations in various
nuclear genes have been found in familial cases of such late-onset
conditions as AD  4, 5,  PD  6, 7, 8, 9, 10 and DM (multiple genes,
reviewed in 11). Some nuclear polymorphisms have been found to
correlate with an increased risk of developing AD 12, PD 13, DM 11,
cardiomyopathy 14, or atherosclerosis 15. Involvement of toxins, such
as the complex I inhibitors rotenone and other pesticides is
hypothesized in PD 16. Based on the analysis of familial models of
such diseases, a number of biochemical processes, such as protein
folding, post-translational processing, protein degradation, and
accumulation of toxic products are believed to be involved in the
sporadic, age-related forms of these diseases, although no causative
nuclear gene mutations have been identified in the vast majority of
cases. This observation is significant: absence of mutations should
exclude a gene from consideration as a cause, relegating it and the
relevant processes to a secondary role in pathomechanism. Despite
this, aggregated proteins and lipids in the form of neurofibrillary
tangles, Lewy bodies and other aggregated proteins, or lipofuscin have
been postulated as causative in aging.  Weakening their case, however,
is that none has so far been unequivocally shown to persist throughout
ontogeny. Indeed, the accumulation of at least some of them (NFT,
lipofuscin, A-beta) is reversible 17 and 18, while other misfolded
protein aggregates are known to be protective 19.

A molecular substrate of aging and the permissive factor for
age-related disease would have to persist for decades and accumulate
change over this time. The nuclear and mitochondrial DNA appear to
have the requisite characteristics: long half-life, and the
accumulation of mutations (information loss) which persist even
through replication, a feat not typical of proteins or lipids, whose
turnover erases changes accumulated after synthesis. Thus, while there
may be other molecular mechanisms for long-term accumulation of change
important in some contexts, such as prions, protein glycation and
other chemical reactions, primarily in avascular tissues with slow
turnover of macromolecules, DNA appears to be the most likely
candidate for the substrate of aging.

Based on the above considerations, aging should be conceptualized
primarily as a disease of our somatic cell DNA, where mutations
accumulate with time, and lead to cellular dysfunction. Thus, aging is
an integral of information loss over time. Information loss may take
the form of not only a total loss of genes but also accumulation of
corrupted versions of genes (the importance of this point should
become apparent in our later discussion of the biochemical mechanism
of aging). Other factors related to aging, such as oxidative stress,
nitrosative stress, inflammation, or protein aggregation would then
act through a common DNA-related mechanism or represent secondary
events.

The accumulation of mutations in somatic DNA is not a new concept. 
Originally postulated by Knudson, carcinogenesis is thought to involve
serially accumulated mutations in oncogenes and anti-oncogenes that
lead to uncontrolled proliferation 20, 21.  For other features of
aging, the pre-eminent theory implicates mitochondria. According to
the mitochondrial theory of aging, first proposed by Harman in 1972
22, mitochondrial genomes accumulate mutations as a result of damage
from reactive oxygen species, replication and/or repair errors,
leading to impairment of oxidative phosphorylation, failure of ATP
production, and slowing of cellular maintenance processes. Since
mitochondria have been shown to be crucial for programmed cell death
23, damage to their genomes may lead to apoptosis and loss of tissue
function. Furthermore, as the producers of energy, informational loss
in mitochondrial genomes would predict an exponential decline due to
entropic forces, persistent energy production being the primary
provider of informational contiguity. No other theory so closely
accounts for both the energetic/metabolic and informational decline
that occurs with age.  However, this theory does not explain why
specific age-related diseases appear only in a fraction of the
population, even though the mutations would accumulate in all adults,
and it fails to account for the wide range of ages of onset.

We propose that the recently described form of DNA damage,
mitochondrial microheteroplasmy 24, 25, 26, 27, 28, 29 and the
individual differences in its accumulation confer a proclivity to
develop specific age-related conditions, such as PD, AD, DM,
cardiovascular disease, as well as determine to a great extent the
rate at which we age. The rate of accumulation of mutations in mtDNA
will determine the timing of onset, subject to modulation by nuclear
genetic and environmental factors; thus acting as the principal
component of the molecular clock of aging. Microheteroplasmy has also
important methodological implications ¨C the current practice of direct
sequencing of mtDNA products is very likely to miss most pathogenic
mutations and should be supplemented by clonal sequencing. This
represents an extension and refinement of the mitochondrial theory of
aging.

In the following exposition we first review data on mitochondrial
genetics, then explore the literature on the biochemical connections
between mitochondria and age-related disease, and finally attempt to
outline the case for mitochondrial microheteroplasmy as the causative
factor in aging and age-related diseases. We also provide a
methodological explanation for the failure of many in the field of
mitochondrial genomics and aging to identify low level mutations
present in nearly all mitochondrial genomes.

2. Review of mitochondrial biology and genetics

2.1 Mitochondrial biology and mtDNA

Mitochondria are intracellular organelles found in almost all
eukaryotes, derived from ¦Á-proteobacteria and possessing their own
genome 30, 31. They are involved in many biochemical processes, most
notably oxidative phosphorylation, and in apoptosis, or programmed
cell death.  In humans, the mitochondrial genome (mtDNA, at the time
of its discovery referred to as ¦Ñ DNA) is a 16.5 kb circular dsDNA
molecule which codes for 13 protein subunits of the electron transport
chain (ETC), as well as the tRNA and rRNA genes necessary for the
translation of the protein genes. In each cell there are usually
between 1,000 and 100,000 copies of mtDNA, on average 4,900 mtDNA
genomes per nuclear genome 32. Since each copy may be independently
replicated, mutations can accumulate in various proportions of the
genomes. The presence of a mutation in 100% of the genomes is termed
homoplasmy, while heteroplasmy is a mixture of mutated and wild-type
sequences for a given locus.

MtDNA is almost exclusively inherited through the maternal line 33
which allows the detection of mitochondrial contributions to phenotype
by observing the degree of matrilineal inheritance of a trait 34. A
number of strictly maternally inherited conditions have thus been
linked to mtDNA: MELAS, MERRF, LHON, CPEO, MILS, and others 35. In all
of them the causative mutation turns out to be either a homoplasmic
mutation or a heteroplasmic mutation present in a high percentage of
genomes, usually higher than 50%, though 20% heteroplasmy can also
produce phenotypes. The phenotypic manifestation of these mutations
tend to develop in infancy, childhood, or early adulthood, and
frequently include encephalopathy, myopathy, and diabetes, in addition
to other specific symptoms which serve to define the above-mentioned
syndromes.

2.2 Changes in mitochondrial genomes during early ontogeny

Cells in the maternal germ line spend many decades in a largely
quiescent, amitotic state, arrested in the metaphase of the second
meiotic division 36 ¨C despite this, their mitochondrial genomes
undergo continuous turnover. In all tissues, whether consisting of
mitotic or postmitotic cells, mtDNA undergoes continuous replication
and replacement, with a half-life of a few weeks 37. Accumulation of
mutations thus occurs constantly throughout ontogeny 38, 39, 40(also
reviewed in 41), and is due to both polymerase errors and errors in
the repair of DNA damaged by environmental influences.

Since uncorrected accumulation of mutations would within a very small
number of generations become incompatible with survival, there are
mechanisms for selection against mtDNA mutations. Currently it is
believed that the majority of female germ line cells undergo apoptosis
42 in the final stages of maturation. Accumulation of mtDNA mutations
above a certain threshold was postulated as the causative mechanism
43, although the precise levels and types of mtDNA mutations necessary
to trigger apoptosis have yet to be defined. An element of the sensing
mechanism which detects the accumulation of mutations is the
production of ROS (reactive oxygen species) 44, as evidenced by
susceptibility of oocytes to pulses of ROS. ROS are byproducts of
oxidative phosphorylation, generated mainly by electron leakage from
complexes I and III of the ETC 45. They are capable of damaging
proteins, lipids and nucleic acids, and are believed to be the
principal agent directly causing mutations in human DNA. During the
maturation of germline cells, the oocyte switches from the anaerobic
metabolism it used for 20 to 40 years (time from formation of ovaries
in the fetus to initiation of ovulatory cycle in the woman) 46 to
metabolism based on oxidative phosphorylation and increasing amounts
of ROS are generated. In germline cells and early embryos with
severely damaged mtDNA, and correspondingly high ROS output, apoptosis
is initiated, thus favoring the selective production of ova and
embryos with largely intact mtDNA 47 (Fig 1).

However, as noted above, there are many conditions where this
cleansing mechanism fails. Some deleterious mtDNA mutations, such as
the high-level heteroplasmic A3243 mutation responsible for many cases
of MELAS 48, are compatible with the survival of the ovum and fetus.
Lower levels (<5%,) 49 of this mutation may be present in the maternal
line for generations, with few phenotypic manifestations but if their
concentration rises to the range >20% (see also  50), increasingly
early and/or severe phenotypes develop.

2.3 Replication Induced Mutations

The mitochondrial DNA polymerase, PolG, is a family A polymerase
related to phage polymerases that contains two subunits, a catalytic
domain possessing exonuclease capability and a processivity subunit. 
Like all family A polymerases, PolG introduces mistakes during
polymerization but that are quickly repaired by the exonuclease
domain.  Several investigators have taken advantage of this fact and
introduced mutations in the exonuclease domain to create PolG mutants
that accumulate mutations in mtDNA 51, 52.  These models have provided
revealing insights into mutational accumulation in mtDNA throughout
ontogeny.  Critics, however, have continued to point to the excellent
in vitro exonuclease capability of PolG as evidence that these models
have little to do with mammalian aging.  As such, this criticism has
shifted focus from mtDNA polymerization to mtDNA repair as a major
bottleneck in mtDNA accumulation.

A recent study has revealed that in vitro exonuclease capacity of PolG
can be significantly impaired by imbalances in nucleotide
concentrations 53.  The study finds that mitochondrial dNTP pools are
highly asymmetric in vivo with an overabundant representation of dGTP.
 This overrepresentation is sufficient to drive mutagenesis with a 4
fold increase in single base pair error over that occurring at
equimolar dNTP reactions.  Such a high error rate would predict high
mtDNA mutation accumulation rates and may provide an explanation for
the mechanism of mtDNA mutation accumulation even in the absence of
additional processes, such as ROS-induced damage.

2.4 Heteroplasmy

Heteroplasmy, the presence of varying fractions of mutated mtDNA in
tissues, has been extensively studied in many paradigms. Usually,
mitochondrial DNA is used as a template for PCR, and the amplified DNA
is directly sequenced. This method allows only the reliable detection
of mutations present in no less than 30% of mitochondrial genomes.
Since the PCR product is a mixture of DNA species, in heteroplasmic
loci there are superimposed peaks on the sequencing chromatogram ¨C the
lower peak corresponding to the minor component cannot be easily and
reliably distinguished from instrument noise. With additional
refinements it is possible to increase sensitivity to about 10%
heteroplasmy 38 but as alluded to before, this is still well below the
sensitivity needed to detect the class of mutations which are the
focus of this article. Another technique used to analyze mutations is
denaturing HPLC (DHPLC) 54 but even there the sensitivity is usually
not better than 3% and confirmatory sequencing is still necessary to
localize the mutation. The state of the field is much akin to being
forced to study protein structure using a conventional light
microscope - simply put, we would be forced to postulate that proteins
do not exist

There is an enormous body of experimental data describing high-level
heteroplasmic and homoplasmic mutations. Homoplasmic mutations have
been found to be causative in the classical mitochondrial diseases, in
diabetes 55, deafness 56, the metabolic syndrome 49, and others. There
are homoplasmic mutations which correlate with sporadic PD 57,
cardiomyopathy 58, bipolar disorder 59, and other common conditions.
All of these, however, are found only in a small percentage of cases.

2.5 Microheteroplasmy and certain other acquired mitochondrial mutations

The other form of mtDNA mutational load, low-level heteroplasmic
mutations (microheteroplasmy), is more difficult to detect. To detect
an unknown mtDNA mutation present in 1 to 2 % of genomes it is
currently necessary to clone the amplified mtDNA PCR fragment and
sequence hundreds of clones. With cloning, the signal from minor
mutated species can be observed separately and the percentage of
mutation can be calculated from the ratio of wild-type vs. mutated
clones. This procedure is more than a hundred times more expensive
than direct PCR sequencing. Consequently, there is much less data on
microheteroplasmy. Available studies 24, 25, 26, 27, 28, 29, indicate
that microheteroplasmy is present in all tissues, at all ages
examined, and in all subjects. There is progressive accumulation of
mutations over many decades in most tissues, with the notable
exception of the substantia nigra, the brainstem nucleus affected in
PD, where a high level of microheteroplasmy is present even at birth.
Thus, in contrast to homoplasmic mutations and most high-level
heteroplasmic mutations, microheteroplasmy is largely an acquired form
of mutational burden. Each of the mutations detected is usually
present at a 1-2 % level or less, although some of them may be present
in up to 10% in homogenates and in 10-20 % level in single cells 27.
With hundreds of different mutations in each patient their total
burden adds up to a level comparable with the mutation loads present
in classical mitochondrial disorders. There appears to be a roughly
smooth distribution of mutations along the genome, except for the
mitochondrial control region, or the D-loop, where the mutation
frequency is approximately ten times higher than in the coding parts
of the genome. The estimated percentage of mitochondrial genomes with
at least one amino acid-changing mutation, a mutation  in a tRNA  rRNA
gene, or in a control region, is > 90%, based on the mutation level of
59.3 mutations per million basepairs in complex I and 640 mutations in
non-coding regions 24. If the higher mutation level estimates from 27,
28 and 26 are used, the average mitochondrial genome from an aged
adult is calculated to have about 3 point mutations, with the majority
of them present in protein-coding regions. This means that at an
advanced age there are virtually no wild-type mitochondria left.

Deletions are another form of acquired mitochondrial mutational burden
(reviewed in 60). Progressive accumulation of mitochondrial deletions
was observed in the rat and rhesus monkey muscle, with unique
deletions appearing in segments within single muscle fibers at levels
up to 10 ¨C 15% 61. The presence of such unique deletions correlates
with ETC abnormalities, such as COX negativity and leads to sarcopenia
60. Approximately 60% of muscle fibers exhibit such genetic and
phenotypic abnormalities in some muscles in the aged rhesus monkey 62.
Deletions accumulating to approximately 20% lead to a profound
sensitization of cells to pro-apoptotic stimuli 63.

Thus, in stark contrast to the frequently quoted estimate of 0.1%
mutated mitochondrial genomes 64, 65, the true mutational load is
orders of magnitude higher.

As alluded to in the introduction, this finding has important
methodological implications ¨C the, literally, thousands of studies
looking at mtDNA with direct sequencing of PCR products were for
technical reasons alone incapable of detecting the majority of
mutations.

Interestingly, there is no correlation between the total number of
microheteroplasmic mutations and the two conditions where such
correlations have been sought so far, PD and AD. However, in PD there
is a correlation between the phenotype and the presence of mutations
in certain very narrow regions of the mitochondrial genome 25. In the
mitochondrial gene ND5 the presence of aminoacid-changing mutations
between codons 127 and 146 correlates very strongly with PD, and using
a genetic algorithm method it is possible to correctly classify 12 out
of 12 samples as PD or control (p= 0.00024). A prospective study
looking specifically at this region 29 correctly identified 15 out of
16 samples. In contrast to e.g. nuclear gene mutations such as parkin,
or ¦Á-synuclein, these mutations were found in almost all cases of
sporadic PD examined to date. In combination with the other data on
mitochondrial dysfunction in sporadic PD (summarized in the next
section), including cybrid data, this result may be interpreted as
evidence for substantial causal involvement of mtDNA in this disease.
No equivalent studies have been done for AD, however, a preliminary
study of D-loop mutations 66 showed a correlation to AD. We propose to
refer to the presence of small regions where microheteroplasmy
correlates with phenotypes as "focal microheteroplasmy".

To summarize the data on mitochondrial genome: It has the
characteristics necessary for the molecular clock of aging,
accumulates mutations both through inheritance and during aging in all
humans, at levels sufficient to explain phenotypic change, and in some
cases the acquired mutations already have been shown to correlate
with, or explain age-related disease.

3. Mitochondria in age-related disease

3.1 Lines of evidence

There are multiple independent lines of evidence for mitochondrial
involvement in various age-related conditions. Broadly, they can be
categorized as follows:

-	matrilineal inheritance
-	correlation of homoplasmic mtDNA mutations and familial forms of the
conditions
-	detectable enzymatic defects in complexes coded by mtDNA
-	replication of disease phenotype in vitro and in vivo by toxins
acting directly on mitochondria
-	replication of pathological biochemistry in cells receiving mtDNA
from patient samples with sporadic forms of the disease
-	detection of elevated oxidative stress, especially preceding the
development of the phenotype
-	mitochondrial localization/interaction of the products of nuclear
genes known to cause early-onset familial forms of disease
-	replication of aging phenotypes in animals after specific induction
of mtDNA damage
-	correlations between disease susceptibility and mtDNA haplotypes

Below we summarize some of the available evidence in the
neurodegenerative conditions PD, AD, as well as in DM, hypertension
and cancer.

3.2 Neurodegeneration

In Parkinson's disease there is a marked deficiency of complex I of
the electron transport chain. First detected by Parker et al. 67, and
subsequently confirmed in multiple studies, the complex I deficit is
present in all tissues examined, and at all stages of the disease
(reviewed in 24). The cells most affected in PD, the pigmented giant
neurons of the substantia nigra, appear to be very susceptible to
complex I dysfunction, as evidenced by the actions of pharmacological
inhibitors of complex I, such as MPP+ or rotenone. Intake of MPP+ is
sufficient to cause an acute form of Parkinson's disease in humans 68
and experimental animals, while chronic administration of rotenone has
been shown to induce histopathological changes and a phenotype
consistent with PD in rodents 16. It must be noted, however, that in
the condition caused by MPP+ there is symmetric nigral degeneration,
while in PD there is usually pronounced asymmetry in rate of
degeneration between the two sides, which implies that the actual
cause of sporadic PD differs from a simple exposure to complex I
inhibitors. Complex I inhibitors in the environment may modify the
progression of PD in humans as shown by increased frequency of PD in
workers chronically exposed to pesticides 69.

A similar situation exists in AD, where another part of the electron
transport chain, complex IV, shows decreased activity in the brain and
other tissues 70, while a complex IV inhibitor, azide, replicates
symptoms of cognitive decline in rats 71 as well as increased
oxidative stress and deposition of phosphorylated tau protein, one of
the histopathological hallmarks of AD. The recent discovery of a human
model of AD in AIDS patients receiving HAART (highly active
antiretroviral therapy) points further to the importance of
mitochondria in AD pathogenesis. Nucleoside anti-retrovirals which
were originally designed as inhibitors of the viral DNA polymerase
also inhibit the mitochondrial DNA polymerase, polymerase ¦Ã 72. During
prolonged administration there is accelerated accumulation of mtDNA
mutations 73, including deletions and substitutions, producing
cognitive dysfunction and AD-like pathology 74, 75, in direct parallel
to the MPP+-evoked parkinsonism.

For both PD and AD there is ample research in an in vitro model as
well, the cytoplasmic hybrid, or cybrids. Cybrids are produced by
fusing cells previously deprived of their endogenous mtDNA (so called
¦Ñ0 cells) with mitochondria from patient samples, resulting in a
hybrid with a constant nuclear background and a transplanted
mitochondrial gene complement. In this way the influences of the
mitochondrial genome on the physiology of a cell can be separated from
nuclear factors, and direct comparisons can be made between the
functional properties of mitochondrial DNA from various sources.

Cybrids generated with mitochondria harvested from PD patients exhibit
complex I deficiency similar to patient cells, showing direct
inheritance of the biochemical abnormality through mitochondria 76,
77. Since this complex I deficiency is stable over tens of cell
doublings, any protein or membrane components transferred with mtDNA
become diluted and removed, leaving only mtDNA as the carrier of
information causing complex I dysfunction. In addition to complex I
deficiency, PD cybrids exhibit a number of other physiological
derangements found in PD patient tissues, including abnormalities of
calcium homeostasis 78, increased antioxidant enzymes, increased ROS
production 79, and protein aggregation in the form of Lewy bodies, the
histopathological hallmarks of PD 80.

Alzheimer's cybrids replicate complex IV dysfunction 76 and other
features of AD brain, such as increased ROS output 76 and deposition
of ¦Â-amyloid 81.

There is ample literature on the mitochondrial
localization/interaction of nuclear gene products causing familial
forms of PD 10, 82, 83, 84 and AD 85, 86, 87, 88, 89, 90, 91, also
reviewed in section 5.1. Certain mitochondrial haplogroups (K,
possibly J) confer protection from PD 92, 93, while others (JTWIX) 57
are associated with higher risk. In AD, haplogroups K and U reduce the
impact of the ApoE4 allele 94.

These data suggest, although certainly do not prove, that
mitochondrial dysfunction is the initial event in the pathomechanism
of both PD and AD, with protein aggregation abnormalities being
important secondary, downstream events. The recent discovery of
protein aggregates serving a protective role as way stations for
misfolded proteins 19 shifts focus from the accumulation of protein
aggregates to the adaptive roles these proteins may play in the
homeostasis of the cell in the face of mitochondrially generated
stress.
  
It is worth noting that aside from cybrids there are no other models
of AD or PD where material from sporadic patients is sufficient to
replicate histopathology of the disease ¨C the AD models based on
mutated versions of APP use material from familial cases, and
therefore have somewhat limited relevance to the majority of AD.

3.3 Diabetes and hypertension

There is also mounting evidence for mitochondrial involvement in type
II diabetes, and hypertension. In the case of diabetes, between 3 and
5 % of cases can be traced to specific mitochondrial DNA mutations 95,
and using more sensitive statistical testing as much as 22.5 % of
cases can be ascribed to maternal inheritance 34. A number of mtDNA
homoplasmic and high-level heteroplasmic mutations have been shown to
correlate with diabetes, including the MELAS A3243G mutation which is
found in about 1 to 2.8% of DM in the general population. Another
mitochondrial mutation predisposing to DM is the T16189C variant 96,
97, 98, 99, 100. There is evidence for the role of mitochondrial
dysfunction in the elderly in the etiology of insulin resistance 101.
Mitochondrial activity is also impaired in insulin-resistant offspring
of patients with type II diabetes 102, 103, and in their healthy
relatives 104. Since impaired insulin resistance is the best predictor
of developing diabetes, this indicates that mitochondrial dysfunction
is present in the earliest stages of diabetes development. Work in a
mouse model of insulin resistance, the conditional insulin receptor
knockout MIRKO mouse 105 indicates that the mitochondrial
abnormalities are independent of insulin resistance and are sufficient
to cause most of the gene expression abnormalities associated with DM,
again pointing to mitochondrial dysfunction as the cause rather than
the effect of insulin resistance. Mitochondrial dysfunction in HIV
patients on HAART induces diabetes 106. Finally, the mechanism by
which alloxane and streptozotocin cause diabetes in animal models is
through increasing oxidative stress, underscoring the susceptibility
of pancreatic ¦Â cells to mitochondrial insults 107.

In the case of hypertension, elevated oxidative stress is
well-established as a contributing factor 108. A model of
mitochondrial causation is again observed in patients receiving HAART,
where accelerated aging of the cardiovascular system is present 106.
There is a significant maternal component in the inheritance of
hypertension, estimated at 55% 34. Specific mtDNA mutations have been
found in African American men with hypertension 109. Recently, a large
kindred with maternal inheritance of hypertension,
hypercholesterolemia, and hypomagnesemia caused by the mtDNA mutation
T4291C was found 110.  Elevated ROS production is seen 111 in
hypertensive patients and diminished ETC activities 112 are found in
an angiotensin-II model of hypertension.

These data show that mitochondrial dysfunction is an important event
in the pathogenesis of DM, and hypertension, and are compatible with
the interpretation of mtDNA dysfunction as a contributing primary
event, rather than an epiphenomenon or secondary event in
pathogenesis.

3.4 Cancer

When Otto Warburg originally observed that cancer cells utilize
glycolysis almost to the exclusion of oxidative metabolism for energy
production, he could not have anticipated that mtDNA is mutated in the
vast majority of tumor masses studied to date 113.  While nuclear
mutations are clearly indispensable for the evolution of a neoplasm
and are the focus of most oncological research, mtDNA is now starting
to be seen as more than just an epiphenomenon 114.  The exploration of
its role can be approached from two perspectives: the observed
biochemistry of tumor tissues and the analyses of evolutionary
mechanisms which allowed the formation of multicellular organisms.

The biochemical side of the equation is demonstrated by reliance of
tumor cells on glycolytic metabolism, which is widely observed in many
neoplasms 115. Since mtDNA codes for enzyme subunits indispensable for
oxidative phosphorylation (OXPHOS), mutations damaging it will leave
the cell dependent on glycolysis.

The evolutionary perspective was explored in a series of studies by
Pfeiffer and Bonhoeffer 116, 117. In organisms capable of living both
in a single-cell and a multicellular form, such as the slime mold
Dictyostelium, switching from OXPHOS to glycolysis initiates a switch
to single-cell mode, where competition for glucose prevents cell
cooperation.  On the other hand, because oxidative metabolism provides
a significantly more efficient means of energy utilization, in
substrate-limited states a cell can afford to cooperate with its
neighbors, thus enabling multicellularity.  These observations and the
fact that mitochondria are found in one form or another throughout the
eukaryotic lineage 118 have prompted some to hypothesize that
endosymbiosis and the gain in oxygen utilization were a necessary step
in the formation and success of multicellularity on Earth 119, 120.

It would be thus expected that depriving mammalian cells of their
ability to use OXPHOS will shift them to a low-cooperativity, less
differentiated state with a higher mitotic potential. Indeed, this is
what is observed: cells devoid of their mtDNA have higher propensity
to metastasis than their mtDNA-possessing cohorts as well as gene
expression profiles reminiscent of a less differentiatied state 121,
122.  MtDNA dysfunction in classical mitochondrial diseases leads
otherwise quiescent cells, such as cardiac muscle to have basal
activity of cell cycle components typical for dividing cells, between
G2 and M 123. Furthermore, cybrid cells containing mutations in mtDNA
increase tumorigenesis when transplanted into nude mice 121, 124, 125.
 The increase in tumorigenic potential can be attenuated by allotopic
expression of the wild-type mitochondrial gene 125.  These
observations paint a picture of many neoplasias as an evolutionary
process of cell adaptation with mitochondria playing a prominent role.

Two additional elements needed to build a coherent, mechanistic
explanation of mtDNA involvement in cancer are the role of
mitochondria in apoptosis 23, 126, and their role in production of
ROS. Loss of mitochondrial function limits apotosis 126. Increased
production of ROS, such as may be caused by mtDNA mutations, leads to
nuclear DNA mutations, telomere attrition and genomic instability 127,
128, decreased nuclear genomic maintenance 129 and favors metastasis
130.

The chain of events leading to neoplasia can be thus described as
follows: As nuclear and mtDNA mutations accumulate, ROS production
increases, accelerating accumulation of mutations. Many cells, sensing
a rise in ROS levels, act to limit ROS production by down-regulating
electron transport and shifting to glycolysis through master
regulators such as HIF-1¦Á 131 and VHL 132, 133, 134. Shifting to
glycolysis by utilizing these master regulators also initiates global
gene expression changes towards a less specialized, more
dedifferentiated state 135, 136.  This adaptation initially limits
mitochondrial ROS production but some ROS generation is shifted to
redox reactions that can be carried out on the plasma membrane 137.
Plasma membrane ROS are potent mediators of cellular motility and
metastasis 138, 139. Finally, the burden of ROS, lack of oxidative
metabolism, shift in global gene expression to a more dedifferentiated
state, limitations of apoptotic ability of mitochondria and increasing
nuclear mutations enable a cell to escape the cell death program and
proliferate despite its neighbors.

3.5 Animal models of mitochondrial aging 

The close causal relationship between accumulation of mtDNA mutations
and age-related functional loss is also evident in a mouse model of
hypermutated mitochondrial DNA induced by a mistake-prone version of
the mitochondrial DNA polymerase, pol ¦Ã 51.These mutants exhibit
accelerated accumulation of mtDNA mutations and an accelerated aging
phenotype of osteopenia, kyphosis, alopecia, weight loss and
cardiomegaly.

Recently, utilizing allometric relationships, Wright et al. 140
compared degenerative rates between five species with varying maximum
life span potentials.  They found that mitochondrially generated
oxidative stress was the primary regulator of degenerative rates in
retina and brain despite the varying influences of nuclear genes and
species specific background.  These findings and the significant role
mitochondria play in the intrinsic apoptotic pathway help point to
mitochondria as primary rheostat for the cell¡ªintegrating various
signals while producing a cell's energy.

To summarize: many of the specific manifestations of aging have been
shown to be related to mitochondria by multiple lines of evidence -
matrilineal inheritance, transmission of pathology with mtDNA in
cellular models, direct evidence of mitochondrial abnormality in the
form of oxidative stress, as well as replication of aging phenotypes
by mitochondrial toxins, and by some mitochondrial mutations.

4. Mitochondrial theory of aging

4.1 Objections to the mitochondrial hypothesis of aging

The primary obstacle in the acceptance of the mitochondrial theory of
aging and age-related diseases was heretofore the lack of a set of
mutations which would be detectable in all aged adults at levels
sufficient to explain the physiological derangements. Many authors
point to the estimate of a total of 0.1% mutated mitochondrial genomes
derived from searches for known pathological mutations performed in
healthy aged adults  64, 65, 141, 142, as evidence that mtDNA could
not be the clock of aging.

The disease-correlated homoplasmic mtDNA mutations in AD and PD are
found in a very small percentage of patients. Similarly, mtDNA
mutations in diabetes and hypertension are present in a minority of
patients. Thus, while this particular type of mtDNA mutation could
explain some features of aging in a few families, it would not be
widely applicable, just as a nuclear gene AD or PD mutation found in
one kindred cannot explain these diseases in general.

The changes in the D-loop 38, 143, the common 4977-bp deletion, and
other deletions increase in frequency with aging in the elderly 144,
145. However, the D-loop changes, while accumulating to high levels,
are found in a minority of patients, even in centenarians. Deletions
are typically found at levels < 1% (except in a minority of sections
of muscle fibers where they reach significant levels 60) and fail to
show a clear correlation with diseases of aging. In some experimental
paradigms (caloric restriction) it is possible to reverse the
accumulation of the common deletion in some tissues to a youthful
level, with only partial reversal of the phenotype 146 which militates
against this mtDNA change as the inexorable clock of aging. Thus,
these forms of mtDNA damage are still insufficient to keep the
mitochondrial theory of aging alive.

What is perhaps equally troubling, there appears to be only a modest
maternal inheritance effect in AD 147, and PD 148, 149, and a rather
moderate effect in DM 34, 55. Hypertension was found to have a major
maternal effect of 55% in one study 34 (maternal effect is here
defined as the percentage of cases attributable to a maternally
inherited factor). Low or nonexistent levels of maternal inheritance
would be expected if aging was caused by a random accumulation of
mtDNA mutations, but then other (nuclear, environmental) factors would
be needed to explain the inter-individual variation in the patterns of
aging. Random accumulation, however, would also predict a general
decline in ETC activity and would be difficult to reconcile with data
from cybrids in PD and AD, where specific elements of the ETC are
impaired in all tissues. Conversely, if aging is caused by inherited
focal mutations capable of generating specific ETC activity losses,
then strong maternal inheritance would be expected.

Finally, there are reports of a lack of significant decline in ETC
activity in isolated muscle mitochondria from aged humans 150, 151.
These studies were performed on mitochondria from homogenates,
therefore they may exhibit selection effects due to the possible loss
of more fragile mitochondrial sub-fractions, and directly contradict
other studies 152, 153 but they seem to be incompatible with the
notion that mtDNA damage contributes to aging by lowering the overall
ability to perform oxidative phosphorylation.

4.2 Microheteroplasmic mitochondrial theory of aging

These major deficiencies of the mitochondrial theory of aging are
addressed by the recent discovery of mitochondrial microheteroplasmy.
As indicated in section 2.4, microheteroplasmy affects the vast
majority of genomes in adults, with at least 90% of genomes in every
aged adult predicted to have at least one amino-acid changing mutation
per genome. Even if some fraction of these mutations is innocuous, the
total level is on par with levels of deleterious mutations sufficient
to cause severe phenotypes in classical mitochondrial conditions. It
is no longer necessary to postulate the existence of "amplification"
of the impact of rare age-related mutations by hypothetical
mechanisms, since the mutations actually present may suffice to cause
dysfunction by the same pathways involved in e.g. MELAS 154.

The mutations are present in all tissues examined so far and in every
individual, making them the suitable substrate for a ubiquitous clock.
Mutations levels are lowest in the neonate, and smoothly increase with
age, exactly as would be expected from a time-measuring quantity (or
perhaps more precisely, an integrator of damage over time). There are
differences in the rate of accumulation between various regions in the
brain, with the substantia nigra exhibiting a level of about 200
mutations per 106 bp (200/106) even in the youngest age group (<10
years old), while the frontal cortex starts at a level of about 45/106
and gradually, in the oldest age group (~ 75 years), approaches
SN-specific levels 28. This is consistent with oxidative stress being
the chief source of mutations, since the substantia nigra has an
unusually high level of ROS generation related to the synthesis of
dopamine in the pigmented SN neurons 155.

As shown in 24, 25 and 29, there are narrow regions in some complex I
genes, most notably in ND5 and ND2, where the presence of mutations
correlates very closely with the PD phenotype, and indeed is
sufficient to classify samples as PD or control with good accuracy in
prospective studies (15 out of 16 samples). High predictive value of
these mutations is not inconsistent with them being the prime
determinant of the development of PD. Some of the mutations found in
PD patients at low levels, usually 1 ¨C 2 %, have been previously
characterized in classical mitochondrial conditions. Thus the ND5
mutation at codon 145, A12770G, causes a severe childhood neurological
disorder, MELAS, if present at a level of 48 % 156. This is consistent
with the PD-correlated mutations having a gene dosage effect
(analogous to many classical Mendelian disorders, such as
phenylketonuria), where a higher level of the mutation may cause a
severe childhood phenotype and a lower level may cause a late-onset
dysfunction.

The above observations begin to address the major objection to the
mitochondrial theory of aging: the lack of evidence for the presence
of a sufficiently high load of potentially deleterious mutations, in
all humans. The dearth of mutations in previous studies of mtDNA is
due to the methodology used for their detection which lead to
erroneous conclusions, much like reliance on the light microscope
might lead one to deny the existence of viruses.

The low degree of maternal inheritance of age-related diseases is
consistent with two explanations which are not mutually exclusive:
nuclear/environmental contributing factors, versus germline- and
early-acquired mitochondrial mutations. Since the attention of the
scientific community has been so far concentrated on nuclear heredity,
and only tentative explanations for AD, PD and other conditions have
been formulated, we will analyze the other explanation.

As mentioned in section 2.2, mitochondria in germline cells
continuously accumulate mtDNA mutations, and undergo a stringent
culling to remove any cells with high overall mutation loads (which is
responsible for the eventual exhaustion of viable germline cells in
females). Yet, even a very stringent selection is likely to allow a
certain small number of mutated mtDNA genomes to be present in the
ovum. This assertion is backed up by observations in PD, where a
generalized complex I dysfunction is present in all tissues, implying
the presence of relevant mutations from the very earliest stages of
development ¨C zygote or earlier. If the responsible mutations arise de
novo in the germline, their transmission will not be detectable by the
observation of matrilineal inheritance.

According to Chinnery et al. 157, the load of mtDNA mutations arising
in a cell may change relatively quickly due to random drift. This
could explain how even a single mutation present among the roughly 200
000 mtDNA copies in an ovum could over time become established at a
measurable level throughout the whole organism.

These observations help address the other objection to the
mitochondrial theory of age-related disease: the modest degree of
matrilineal inheritance of age-related diseases, coupled with specific
rather than generalized dysfunction of the ETC. An outline of the
postulated changes in mtDNA mutation levels in ontogeny is shown in
fig. 1.

The last objection mentioned in the previous section is addressed by a
model of mitochondrial cardiomyopathy where a hypermutable ¦Ã
polymerase was targeted to the heart 158. Essentially normal ETC
activities are present in association with severe cell loss, implying
that cellular dysfunction due to microheteroplasmy does not have to
involve an overall ETC activity loss, reconciling the results of
Rasmussen et al. 151 and others with the mitochondrial theory of
aging. Here, it is the accumulation of misinformation (defective
copies of ETC proteins) rather than simple lack of correct information
(wild-type ETC proteins) that leads to damage.  Further research in
this and other models of accelerated accumulation of microheteroplasmy
159 should provide a detailed description of the pathways leading from
increased ROS production to e.g. upregulation of ¦Â-amyloid production
and apoptosis of neurons (fig. 2).

The basic claims of our hypothesis can be encapsulated in the following summary:

1)	Mitochondrial microheteroplasmy, that is, the presence of multiple
mtDNA mutations each present at a low level (usually affecting less
than 2% of mtDNA copies in a tissue but adding to a total mutational
burden of >90%) is a major contributing factor in age-related
pathology, including PD, AD, DM, hypertension, and possibly other
manifestations of aging.
2)	Mechanistically, microheteroplasmy acts through the accumulation of
dysfunctional copies of mitochondrially-encoded ETC subunits, which
leads to increased production of ROS (a form of toxic
gain-of-function), lowered peak ATP production (loss-of-function
mechanism), impaired oxidative phosphorylation, and in turn, to
apoptosis or impairment of cellular function. The abnormalities
secondary to microheteroplasmy include, but are not limited to,
elevated levels of oxysterols, and other oxidized macromolecules,
accumulation of lipofuscin, intra- and extracellular forms of amyloid,
inclusion bodies such as Lewy bodies, insulin resistance, and others.
3)	Mitochondrial microheteroplasmy consists primarily of a combination
of mutations arising in the germline prior to the formation of the
zygote, and mutations accumulating throughout the lifespan of the
individual. There is also a contribution of mutations inherited from
mother, sufficient to explain the existing matrilineal inheritance
levels in age-related disease 160.
4)	Differences in the location and quantity of germline mutations
(focal microheteroplasmy) are at least in part responsible for the
variation in age-related phenotypes, such as for example the
development of PD in some but not all aged individuals 25.
5)	Age-acquired microheteroplasmy and the drift in germline mutation
load are the factors responsible for the delayed onset of age-related
disorders. The phenotypic manifestations of aging in a cell become
apparent after the total microheteroplasmic mutation burden, both
inherited and acquired, crosses a threshold of incompatibility with
normal function.

Thus we expand on the concepts presented by Kraytsberg et al. 41 and
build on the ideas discussed by Dufour and Larsson 159, who described
the mutations described prior to the clone-sequencing mutation surveys
as "the tip of the iceberg". Mitochondrial microheteroplasmy forms its
base.

4.3 Cellular responses to microheteroplasmy

Increased ROS production, which we propose as the most important link
between microheteroplasmy and aging, leads to extensive antioxidant
responses. Within this category we include not only upregulation of
antioxidant enzyme reserve, but also the increased turnover of damaged
DNA, RNA, protein, and lipids, since oxidatively modified cellular
constituents promote further ROS damage 161.  When ROS production
becomes chronic, the very ability to remove damage becomes compromised
161.  Precisely because microheteroplasmy compromises mitochondrial
function, the machinery required to repair and remove damage fails to
import into mitochondria further exacerbating ROS damage 162. These
responses have been exhaustively reviewed elsewhere 163, 164, 165.

The concept of ROS involvement in aging recently received support from
a study showing lifespan extension in mice expressing catalase, an
antioxidant enzyme, in their mitochondria 166. Interestingly,
expression of catalase outside mitochondria (e.g. in the nucleus) did
not result in significant slowing of aging, indicating that
mitochondrial damage may more important, at least as far as
ROS-related mechanisms are considered.

It is known from cybrid studies that the presence of different
mutations in one cell may result sometimes in genetic complementation
167. ROS generation as a form of toxic gain of function is not likely
to allow for complementation between different mutations present in a
single cell, and should instead result in additive or synergistic
effects.

Though direct ROS modifications of cellular constituents are a crucial
mechanism by which mitochondrial microheteroplasmy would affect cell
metabolism, other less salient mechanisms are implicated.  Quite
broadly, we divide these responses into three categories: 1) down
regulation of ETC and OXPHOS; 2) energy switching from oxidative
metabolism to glycolysis; and 3) responses to this metabolic shift
(Fig. 2). We already alluded to these processes in the section on
cancer. Here we discuss some of the genes involved in the cellular
responses to microheteroplasmy in AD, hypertension, and diabetes.

In Alzheimer's disease, several investigators have noted an inverse
correlation between A-beta deposition and ROS generation (for review
see 168).  The fact that A-beta is a potent inhibitor of cytochrome
oxidase (COX) may help explain this phenomenon as well as providing a
salient explanation for the eventual cognitive deficit in familial AD
(FAD)88. In FAD, early and chronic overproduction of A-beta would
decrease COX despite otherwise healthy mitochondria 86, explaining the
loss in synaptic function as mitochondria and mitochondrial access are
necessary for maintaining both the high energy fluxes and Ca2+ demands
of continued synaptic function 169.  In the aging brain burdened by
microheteroplasmy and subsequent mitochondrial dysfunction, A-beta
would actually be protective, by limiting ROS production. Furthermore,
despite decreasing COX, A-beta may preserve an immediate downstream
signal of redox status, namely NAD+/NADH ratio 170, 171 by possibly
shifting redox reactions to the plasma membrane and preserving the
activity of NAD+/NADH sensitive processes such as the sirtuins.
Localized mitochondrial A-beta production may provide the primary
mechanism by which this peptide modulates oxidative metabolism 85,
172. This hypothesis is further supported by the fact that ¦Ñ0 cells
that lack OXPHOS and are primarily glycolytic are immune from A-beta
toxicity 90, 173.  In fact, A-beta ceases to be toxic in other cells
receiving their energy through glycolysis alone 91.

Another molecule highly sensitive to redox status that may help
regulate ETC function and thus ROS generation is nitric oxide (NO), a
signaling molecule well-known to hypertension researchers.  NO is a
potent regulator of complexes I and IV 174, 175, 176, 177, 178,
possibly through direct nitrosylation.  In fact, nitrosylation of
mitochondrial proteins would preclude direct oxidative modification of
the nitrosylated residues and as shown by 179 can be removed once in
the cytosol and away from the oxidation-rich mitochondrial milieu.  NO
may act not only locally in the cell, but would predictively signal a
hypoxic response necessitating vascular remodeling in order to supply
the increasingly glycolytic tissue with substrates.  NO also acts on
mitochondrial biogenesis 178.

Upregulation of mitochondrial biogenesis may play a role in responses
to microheteroplasmy and ROS. Increased generation of new
mitochondrial components and increased mitochondrial turnover would
reduce the average amount of oxidatively damaged components. This
effect is demonstrated by the therapeutic usage of PPARG, PGC, and NRF
agonists, such as tioglitazone, piogliazone and others, in diabetes.
By acting on the prime controllers of mitobiogenesis these drugs
increase mitochondrial reserve and may prove efficacious in the short
term. However, there is no reason to assume only functionally intact
mitochondria would be replicated and an indiscriminant increase in
both healthy and compromised mitochondria may actually hasten cellular
damage and increased ROS despite supplying sufficient ATP 180.  This
warrants a warning on cellular and animal models of this disease ¡ª
within the context of aged animals with perturbed mitochondria, the
glitazones may 'paradoxically' increase toxicity 181, 182.

4.4 Focal microheteroplasmy 

To explain the patterns of inheritance of age-acquired disease, the
responsible mutations might have the following characteristics; they
are present in the earliest stages of embryonic development, and are
either inherited from the mother, or, predominantly, acquired randomly
during the quiescent stage of oocyte development (lasting usually
between 15 and 45 years). The total number of mutations per oocyte
compatible with maturation of the oocyte (as opposed to oocyte
apoptosis) is very small, perhaps as low as 10 mutated loci per oocyte
out of the roughly 105 genomes present. Every surviving oocyte will
have a different set of mutations, except for occasional mutations
inherited from the mother. Stochastic processes, such as random
accumulation of mutational hits, are likely to produce non-uniform
distributions if the number of hits is small (1 to ~ 30), that is, the
number of mutations in any two compared areas may differ by a large
factor, while as the number of hits increases, mutation numbers in
different areas approach an average, and the distribution becomes
uniform. Therefore, if the number of mutations per oocyte is indeed as
low as postulated above, in many individuals there will be an uneven
distribution along the genome, for example occurring predominantly in
the most crucial areas of complex I genes, or in complex IV genes, or
in other functional units.  During embryogenesis mutated mtDNA will be
inherited by most or all cellular lineages, leading for example to the
observed complex I deficiency in all tissues in PD, but without strong
maternal inheritance or concordance among siblings. These are the
mutations found in cerebral cortex of almost all cases of sporadic PD
examined so far. There is a low level of concordance for PD in
monozygotic twins 183, and this observation is also compatible with
the above mechanism ¨C each of the monozygotic twins inherits only half
of ooplasm, and no correlation is likely to exist in the distribution
of mutations in their respective mtDNA complements. Maternally
inherited mutations and mutations accumulated after conception are
insufficient to account for these experimental data.

4.5 Microheteroplasmy in stem cells

We postulate that microheteroplasmy accumulation in tissue-specific
stem cells is the primary cause of the exhaustion of the tissue
renewal capacity in advanced age and that there is a dynamic
equilibrium between cell loss and renewal from stem cells, even in
many so-called postmitotic tissues. Consequently, occasional
dysfunctional cells seen in young patients (e.g. the NFT- or
AGE-bearing neurons in the cortex of healthy young humans 184, 185,
186) would not survive for extended periods of time as previously
assumed, instead they might undergo apoptosis and be replaced by cells
freshly generated from stem cells. The fraction of dysfunctional
differentiated cells in a tissue would be then determined by the
relative abundance of functional stem cells, and the longevity of
their differentiated progeny. Both of these parameters are impacted by
microheteroplasmy: accumulation of microheteroplasmy in stem cells,
such as the mutations observed in human colonic stem cells 187,
reduces their proliferative capacity by oxidative stress-related
mechanisms 3. Differentiated cells generated from progenitors with
accumulated mtDNA mutations start out with a higher mutation burden,
produce more ROS, and may be expected to accumulate deadly levels of
microheteroplasmy after a shorter time. We postulate that the
exhaustion of adult neural stem cells found in the mesencephalon 188
and in the subventricular zone 189 is the specific histopathological
mechanism of the loss of function in PD and AD, respectively. Similar
mechanisms may operate in other tissues. Analysis of microheteroplasmy
in stem cells will allow the quantification of their mitotic potential
and prediction of the course of aging of a tissue.

5. Competing hypotheses

5.1 Relationship to competing hypotheses

Our hypothesis helps reconcile a large body of observations regarding
the inheritance and pathophysiology of age-related diseases which is
not adequately explained by other hypotheses. Most importantly, it
allows us to answer the question posed in the second paragraph of this
article: Why do certain forms of dysfunction, such as AD or diabetes,
arise only at an advanced age?

As discussed before, a molecular clock mechanism must be postulated to
explain this phenomenon. Of all known molecular properties of the
constituents of the human body, microheteroplasmy is the one which
fits the bill most appropriately and in the largest number of
conditions. Existing theories of, for example, AD causation do not
provide an answer this question: the aggregation of ¦Â-amyloid cannot
act as the clock, since it is not a process continuously proceeding
through ontogeny 190, and as noted previously, it is reversible in
animals. Similar objections can be leveled against other explanations
of age-related diseases which rely on protein aggregation, protein
modification, toxic influences, and other processes which do not allow
for extended time-keeping (slow and continuous accumulation of change
over decades).

Regarding disease-specific theories, such as the amyloid hypothesis of
AD, and the insulin resistance hypothesis of DM, the microheteroplasmy
hypothesis aims to incorporate them as descriptions of secondary
events in aging. Neither amyloid accumulation nor insulin resistance
are explained at a causal level by previous hypotheses ¨C their
proponents do not provide a description of exactly how these phenomena
are brought about (except in the rare familial cases, where indeed
amyloid mutations or insulin receptor mutations are responsible for
the phenotype). The microheteroplasmy hypothesis describes the
ultimate, physical mechanisms of mtDNA mutations in all aging humans,
and a path from these primary events to the eventual outcomes,
encapsulating these competing narratives in a larger framework.

Mitochondrial microheteroplasmy as the molecular clock of aging fits
very well in the general framework of available knowledge about
pathology of age-related diseases: many of the known causes of
familial, early-onset form of age-related diseases such as PINK1,
DJ-1, parkin, APP, are nuclear genes which have a direct impact on
mitochondria (their products are localized in mitochondria, or cause
oxidative stress by interfering in protein degradation). Their
pathogenic mutations may act to accelerate the accumulation of the
age-related component of microheteroplasmy, or reduce the cell's
ability to cope with the metabolic derangements caused by it. For
example, it is known that wild-type APP is protective against
apoptosis 191. It localizes to mitochondria and interacts with
elements of stress response systems, such as ABAD 192. Mutated forms
of APP are less effective at this task, in part due to shifted
patterns of proteolytic processing 193. The diminished protective
activity of APP results in an earlier onset of cellular loss, leading
to an earlier age of onset in familial forms of AD.

Most of the processes which are observed in aging, such as
inflammation, autoimmunity, protein aggregation, insulin resistance,
and others, may represent adaptive or maladaptive responses to
mitochondrial dysfunction, and as such remain viable and important
research themes 194. Some of them, such as inappropriate inflammation
may through the generation of ROS contribute to the acceleration of
aging.

Microheteroplasmy is consistent with the observed relationship between
the degree of maternal inheritance for a mitochondrial condition, and
the age of onset: the conditions with the earliest age of onset tend
to have a clear matrilineal inheritance and are due to inherited
homoplasmic or high-level heteroplasmic mutations, conditions with a
later age of onset such as PD have a lower degree of matrilineal
inheritance, and are due to a combination of inherited, germline, and
acquired mutations. Different diseases may vary in the exact
contribution of inherited and age acquired microheteroplasmy necessary
to evoke the phenotype. The same phenotype may appear at an earlier
age if the causative mutation is present at higher level, or at a
later age with lower levels ¨C such relationship is observed in MIDD
pedigrees with the MELAS A3243G mutation 50.

Microheteroplasmy may act in concert with inherited nuclear gene
mutations and environmental insults to further modulate the pace and
phenotype of aging. Thus, changes such as the ApoE mutations may
modulate the resistance to mitochondrial dysfunction in AD 195, and
exposure to HAART or pesticides may accelerate the development of AD
or PD pathology 69, 74.

Accumulation of change in the nuclear genome is a well-established
hypothesis of aging. The shortening of telomeres 196, mutations in
oncogenes 197, mutations in the promoters of some nuclear genes
involved in mitochondrial metabolism 198, have all been shown to occur
in aging. Their involvement in cancer and perhaps cellular senescence
(the loss of mitotic potential in culture after many passages) appears
to be supported be a large body of evidence. At present there are
insufficient data to determine the relative importance of these types
of somatic genetic change versus microheteroplasmy, but at least in
PD, microheteroplasmy is sufficient for classification of patient
samples 29, and we postulate that this is the dominant influence.
Indeed, the mechanism by which microheteroplasmy may lead, through
increased ROS production and diminished ETC activity, to the
observable and universally present features of aging, such as
apoptosis 199, failure of stem cell renewal 3, accelerated telomere
attrition 127, accelerated nuclear gene mutation 198, have been
outlined in some detail, while as noted before, nuclear genome somatic
mutations have been tied mostly to neoplasia. Therefore, we would
venture that microheteroplasmy is more important in aging.

It is likely that some features of aging may result from processes
independent of DNA ¨C for example, from accumulation of advanced
glycation end-products in avascular tissues with very slow protein
turnover and from other possible primary processes 200, or processes
at the tissue and organ level. More research will be needed to
elucidate their relative impact.

5.2 Unresolved issues

The relative importance of microheteroplasmy vs. large deletions
present at low levels (up to 20%) is unclear. Clearly, both forms of
acquired mutational burden are present in tissues and play a role 201.

The question of how much the accumulation of microheteroplasmy is
accelerated by the ROS generated as a result of microheteroplasmy
(which would form a vicious circle of mutation and ROS generation) is
not answered by the data we review here. While there are indications
that ROS do contribute to mtDNA mutational load 202, 203, this process
does not in our opinion represent the central premise of the
mitochondrial hypothesis of aging, as stated in 65 . The crux of the
mitochondrial hypothesis of aging is mtDNA as a clock, not the
(perhaps varied) processes accelerating or decelerating its movement.

Alternatives to ROS as the link between mtDNA mutations and aging
cannot be excluded at this point, as shown by the hypermutable ¦Ã
polymerase mouse model where cardiomyopathy is observed in the absence
of elevated ROS production 204. This model is in some ways different
from microheteroplasmy accumulation in regular aging, since the
defective enzyme is activated at birth, only in the heart, and leads
to detectable increases in deletion levels in one week and to dilated
cardiomyopathy with microheteroplasmic mutation levels of 140 per
million bp in four weeks 52. Additionally, cardiomyopathy can in these
animals be prevented by the administration of cyclosporin A and is
associated with vigorous antiapoptotic activity 205. Given that the
microheteroplasmy accumulation in the aging human occurs over a period
two orders of magnitude longer and age-related cardiac dysfunction is
not remediable with cyclosporin A, this mouse model may fail to
capture important physiological features, such as the elevations of
ROS production and the predominantly pro-apoptotic state observed in
cybrids from aged and AD humans 206. Additional research is needed,
especially involving models without tissue specificity and with
mutation accumulation rates closer to physiological.

Phenotypic threshold is the level of heteroplasmy above which an
abnormal phenotype becomes apparent. It is unclear what is the
phenotypic threshold of microheteroplasmy, although it is most likely
not substantially higher than the levels of about 200 to 250 mutations
per million bases found in the substantia nigra 28, since there
appears to be a plateau of accumulation not exceeded even in the
oldest samples. It is also likely that the threshold differs between
various cell types, giving rise to the graded appearance of aging
changes in various organs.

A related question pertains to the apparent absence of effective
mechanisms of mtDNA damage repair. Why is the accumulation of
mutations in mtDNA not better controlled by appropriate homeostatic
mechanisms, given the postulated major impact of these mutations on
health and longevity? One of the answers to this questions is
suggested by the "disposable soma" theory 207. Accordingly, the
energetic cost of more effective mtDNA repair mechanisms sufficient to
delay aging would reduce the resources available for procreation, and
reduce fertility in young age. In the environment of evolutionary
adaptiveness most humans died of causes unrelated to aging, such as
infection, predation, homicide and accidents, well before being able
to benefit from  delayed aging and prolonged fertility, therefore a
reduction in early fertility would decrease overall fitness and would
be selected against. A detailed analysis of the energetic costs of
mtDNA repair would be needed to address this issue.

Another issue is the question of possible replicative advantage of
mutated vs. wild-type mtDNA, and the resulting clonal expansion of
mutations within individual cells. Some research shows a possible
replicative advantage for defective mtDNA 208 and this process was
proposed as the basis for a modified mitochondrial theory of aging
209. Results of clonal sequencing of mtDNA from single neurons and
glia 27 seem to be more consistent with the absence of replicative
advantage for deleterious microheteroplasmic mutations, since most
mutations were found at low levels in each cell, rather than the
near-homoplasmic levels predicted by this hypothesis, however, the
data appear to be still insufficient to resolve this very complex
issue.

Finally, if microheteroplasmy is involved in cancer development, then
the absence of increased frequency of cancer in models of mtDNA
dysfunction would need to be explained. Classical mitochondrial
conditions are not associated with notable increases in cancer. It is
possible that the early mortality in these conditions prevents
patients from accumulating the nuclear mutations needed for neoplasia.

6. Predictions of the microheteroplasmic mitochondrial theory of aging

A hypothesis without testable predictions would be incomplete. What
follows is a number of corollaries of our main claims from section

6.1 Age-related diseases

1)	The microheteroplasmic mutations correlated to PD (focal
microheteroplasmy 25) will turn out to consist primarily of mutations
arising in the germline de novo, prior to conception, with some
contribution from maternally-inherited mutations (mutations present in
mother before differentiation of oocytes). Identical sets of focal
microheteroplasmic mutations will be found in all tissues of each
patient, while the age-acquired mutations (which do not correlate with
PD or other specific manifestations of aging) will differ between
somatic cells of various lineages from the same patient. There will be
some differences between patterns of focal microheteroplasmy in
different PD patients but a substantial overlap will exist as well.
These regularities will be detectable by clonal sequencing of cells
and tissues from PD patients, and to a lesser extent in their maternal
relatives, and offspring.
2)	Since in AD there is a focal ETC dysfunction transmissible through
mtDNA, in direct analogy to PD, we postulate that in AD there is also
focal microheteroplasmy. There are narrow regions in mtDNA where an
increased mutational burden will correlate with AD. The relevant
regions are most likely to be found in the three mitochondrial complex
IV genes, although other focal microheteroplasmic regions, e.g. in
tRNA genes, are also possible. Clonal sequencing (but not direct PCR
sequencing) aided by data analysis methods outlined in 25 will detect
these mutations in all tissues and may be sufficient for DNA-based
diagnosis and prognosis.
3)	Since there is a fair level of maternal inheritance in DM, and
hypertension, as well as clear evidence of mitochondrial dysfunction
in the sporadic forms of these diseases, we postulate that there is
focal microheteroplasmy specific for these conditions, in addition to
the already mapped disease-correlated mutations such as the MELAS
mutation. Since there is no evidence for the specific involvement of
particular elements the ETC, at present we cannot predict the exact
location of these mutations.
4)	Mutations from the regions of focal microheteroplasmy will have
much more serious phenotypic effects than other microheteroplasmic
mutations, or homoplasmic pathogenic mutations. While some homoplasmic
mutations are compatible with survival of the ovum and prolonged
post-natal survival, the PD or AD-related microheteroplasmic mutations
will be likely to cause severe early pathology or death if present at
or near homoplasmy.
5)	Analysis of cybrids from patients with PD, AD, DM, hypertension,
and possibly other specific aging phenotypes will detect focal
microheteroplasmy

6.2 Normal aging

1)	The accumulation rate of microheteroplasmy is modulated by the
levels of ROS production. In caloric restriction, with its observed
reduced ROS production and slowing of aging, there will be slowing of
microheteroplasmy accumulation as well.
2)	If microheteroplasmy accumulation in tissue-specific stem cells is
the primary cause of the exhaustion of the tissue renewal capacity in
advanced age, then analysis of microheteroplasmy in stem cells will
allow the quantification of their mitotic potential and prediction of
the course of aging of a tissue.
3)	Menopause is due to microheteroplasmy in the germline. With aging,
the fraction of ova with faulty mtDNA will increase. Certain
chromosomal abnormalities, such as trisomy 21, will be shown to
protect the ovum from microheteroplasmy-induced apoptosis, which will
explain why these abnormalities are more common in ova from older
women (where microheteroplasmy is more severe). Many of the phenotypic
features of Down's syndrome, especially the accumulation of amyloid at
an early age 210, will be shown to derive from the increased level of
microheteroplasmy inherited thanks to the interference of trisomy with
ovum apoptosis, rather than to direct gene dosage effects from the
trisomic chromosome.
4)	Replacement of mitochondrial genomes in cultured cells 211, by
combining nuclear and mitochondrial DNA of various ages, will
delineate the relative contributions of nuclear vs. mtDNA to the
symptoms of aging.

6.3 Therapeutic opportunity

1)	Therapeutic interventions targeted towards some secondary responses
to microheteroplasmy may be harmful: This is most likely the case with
removal of amyloid in sporadic AD (although the same intervention may
be beneficial in FAD, where microheteroplasmy does not play a leading
role), and possibly in stimulation of mitobiogenesis in diabetes,
which may lead to acceleration of mtDNA attrition even as short-term
improvements in insulin resistance are observed
2)	Replacement of mitochondrial genomes will reverse many symptoms of aging.

7. Conclusion - All roads lead to Rho (¦Ñ)

Microheteroplasmy is orders of magnitude more common than most of
forms of mutational burden examined so far, making it the likeliest
candidate for the substrate of aging. Our hypothesis builds a bridge
from the known causes of mtDNA mutation, through their immediate
effects, to the observed final outcomes of aging, placing the specific
features of aging, such as protein aggregation, in a larger
biochemical framework. The hypothesis also explains the inheritance
patterns and the delay in the onset of aging. Most importantly, our
hypothesis reconciles the two fundamental aspects of aging -
information and energy loss, by relating them to the unique cellular
component, mitochondrial DNA.

8. Acknowledgements

We would like to thank Drs. James Bennett, Russell Swerdlow and Aubrey
de Grey for comments and valuable suggestions that greatly helped in
our work.

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Figure legends:

Fig.1 MtDNA mutation accumulation throughout ontogeny. Wild-type mtDNA
is shown in green, mutations inherited from maternal somatic
precursors in red, mutations acquired in the germline in blue,
mutations acquired from embryonic to adult life in purple. Maternal
mutations may appear in multiple oocytes and may be shared between
siblings. Germline-acquired mutations are not shared between siblings.
Post-conception mutations accumulate progressively in stem cells
during aging, reducing the functional reserve of differentiated cells
generated later in life. ROS generation plays a role in oocyte
selection, depletion of stem cells, and shortened survival of
differentiated cells.

Fig.2 An outline of the mechanism of microheteroplasmy in aging. The
initial event of mtDNA mutation results in ROS generation, which leads
to direct damage to lipids, proteins, and nucleic acids, including
mtDNA. Adaptive and maladaptive responses of the cell ensue, here the
processes most relevant to AD are highlighted. OXPHOS suppression by
A-beta acting on ABAD reduces the magnitude of ROS overproduction but
eventually limits energy production in the cell, leading to
accumulation of abnormal proteins and oxidized lipids. Finally,
mitochondrial dysfunction leads to apoptosis.
Fig.1 

 
 
Fig.2