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The below has two authors who appear to have not much
interest in the energy, and restriction thereof, in the role
mitochondria
and oxidation play in brain diseases. The role of similar
mechanisms for
brain aging and CR nevertheless may be evident.

Insight
Nature 443, 787-795(19 October 2006) |
doi:10.1038/nature05292; Published
online 18 October 2006

Mitochondrial dysfunction and oxidative stress in
neurodegenerative diseases
Michael T. Lin1 and M. Flint Beal1

Many lines of evidence suggest that mitochondria have a
central role in
ageing-related neurodegenerative diseases. Mitochondria are
critical
regulators of cell death, a key feature of
neurodegeneration. Mutations in
mitochondrial DNA and oxidative stress both contribute to
ageing, which is
the greatest risk factor for neurodegenerative diseases. In
all major
examples of these diseases there is strong evidence that
mitochondrial
dysfunction occurs early and acts causally in disease
pathogenesis.
Moreover, an impressive number of disease-specific proteins
interact with
mitochondria. Thus, therapies targeting basic mitochondrial
processes, such
as energy metabolism or free-radical generation, or specific
interactions of
disease-related proteins with mitochondria, hold great promise.

... Mitochondria and ageing
By far the greatest risk factor for neurodegenerative
diseases such as AD,
PD and ALS is ageing, and mitochondria have been thought to
contribute to
ageing through the accumulation of mitochondrial DNA (mtDNA)
mutations and
net production of reactive oxygen species (ROS).

Although most mitochondrial proteins are encoded by the
nuclear genome,
mitochondria contain many copies of their own DNA. Human
mtDNA is a circular
molecule of 16,569 base pairs that encodes 13 polypeptide
components of the
respiratory chain, as well as the rRNAs and tRNAs necessary
to support
intramitochondrial protein synthesis using its own genetic
code. Inherited
mutations in mtDNA are known to cause a variety of diseases,
most of which
affect the brain and muscles - tissues with high energy
requirements. One
hypothesis has been that somatic mtDNA mutations acquired
during ageing
contribute to the physiological decline that occurs with
ageing and
ageing-related neurodegeneration.

It is well established that mtDNA accumulates mutations with
ageing,
especially large-scale deletions2 and point mutations. In
the mtDNA control
region, point mutations at specific sites can accumulate to
high levels in
certain tissues: T414G in cultured fibroblasts, A189G and
T408A in muscle,
and C150T in white blood cells3. However, these
control-region 'hot spots'
have not been observed in the brain4. Point mutations at
individual
nucleotides seem to occur at low levels in the brain5,
although the overall
level may be high. Using a polymerase chain reaction
(PCR)-cloning-sequencing strategy, we found that the average
level of point
mutations in two protein-coding regions of brain mtDNA from
elderly subjects
was 2 mutations per 10 kb6. Noncoding regions, which may be
under less
selection pressure, potentially accumulate between twice and
four times as
many7.

The accumulation of these deletions and point mutations with
ageing
correlates with decline in mitochondrial function. For
example, a negative
correlation has been found between brain cytochrome oxidase
activity and
increased point-mutation levels in a cytochrome oxidase gene
(CO1)6.
Moreover, somatic deletions can be clonally expanded in
individual neurons,
and high levels of such deletions correlate with cytochrome
oxidase
deficiency on a cell-by-cell basis in the substantia nigra,
perhaps
contributing to the age dependence of PD8, 9. However,
although the
cell-by-cell correlation provides strong circumstantial
evidence,
correlations do not prove that somatic mtDNA mutations cause
age-related
pathology.

Recently, several groups have addressed the issue of
causation using a
clever approach to generate mtDNA mutations experimentally
(for a review see
ref. 10). MtDNA replication is carried out by mtDNA
polymerase- (POLG),
which has 3'-to-5' exonuclease (proofreading) activity in
addition to its
5'-to-3' polymerase activity. If the proofreading activity
of POLG is
eliminated and the polymerase activity preserved, mtDNA
mutations accumulate
because of uncorrected errors during replication. In mice
with such
proofreading-deficient POLG (mtDNA-mutator mice), mtDNA
mutations accumulate
to high levels in all tissues. By 8 weeks of age, homozygous
Polg-/- animals
had 9 point mutations per 10 kb in cytochrome b. By
contrast, normal mice
had less than 1 mutation per 10 kb. This marked increase in
mtDNA mutations
resulted in decreased respiratory enzyme activity and ATP
production. To
begin with, the mice appeared normal, but by 25 weeks of age
began to
exhibit pathology frequently seen in human (although not
necessarily murine)
ageing, including weight loss, alopecia, osteoporosis,
kyphosis,
cardiomyopathy, anaemia, gonadal atrophy and sarcopaenia.
The median
lifespan of such mice was 48 weeks (none lived beyond 61
weeks of age) -
much shorter than the typical murine lifespan of 2 years.

A second, independent mtDNA-mutator mouse showed a similar
marked increase
in mtDNA mutations, progeric features and early mortality.
Notably,
neuropathology was not reported in either mouse model,
although detailed
examination was not carried out. Humans with POLG mutations
exhibit
parkinsonism, ophthalmoplegia and myopathy (see below), and
the reasons for
the differences between mice and humans with such mutations
are not yet
known.

In both mtDNA-mutator mouse models, markers of apoptosis
such as activated
caspase 3 were increased at times coinciding with tissue
degeneration,
suggesting that apoptosis mediates deleterious effects of
somatic mtDNA
mutations. Interestingly, tissues from the mtDNA-mutator
mice did not show
increased levels of lipid, protein or DNA oxidation,
hydrogen peroxide
production, or sensitivity to oxidative stress. Thus, the
effects of mtDNA
mutations in these mice do not seem to be mediated through
ROS production.

Net production of ROS is another important mechanism by
which mitochondria
are thought to contribute to ageing. Mitochondria contain
multiple electron
carriers capable of producing ROS, as well as an extensive
network of
antioxidant defences (Fig. 2). Mitochondrial insults,
including oxidative
damage itself, can cause an imbalance between ROS production
and removal,
resulting in net ROS production11. The importance to ageing
of net
mitochondrial ROS production is supported by observations
that enhancing
mitochondrial antioxidant defences can increase longevity.
In Drosophila,
overexpression of the mitochondrial antioxidant enzymes
manganese superoxide
dismutase (MnSOD)12 and methionine sulphoxide reductase13
prolongs lifespan.
This strategy is most successful in short-lived strains of
Drosophila, and
has no effect in already long-lived strains. However, it has
recently been
shown that overexpression of catalase experimentally
targeted to
mitochondria increased lifespan in an already long-lived
mouse strain14. The
authors of this work generated transgenic mice
overexpressing catalase
targeted to peroxisomes, nuclei or mitochondria. The
mitochondrially
targeted construct provided the maximal benefit, increasing
median and
maximal lifespan by 20%. Hydrogen peroxide production and
oxidative
inactivation of aconitase were reduced in isolated cardiac
mitochondria, DNA
oxidation and levels of mitochondrial deletions were reduced
in skeletal
muscle, and cardiac pathology, arteriosclerosis and cataract
development
were delayed.

In humans, a recent study of gene expression in the brain
suggests that
oxidative damage has a major role in the cognitive decline
that accompanies
ageing15. Transcriptional profiling of postmortem frontal
cortex samples
from individuals aged from 26 to 106 revealed that after the
age of 40 there
was a decrease in the expression of genes involved in
synaptic plasticity,
vesicular transport and mitochondrial function, followed by
increased
expression of stress-response, antioxidant and DNA-repair
genes. In the
brain, the age-downregulated genes suffered markedly
increased oxidative DNA
damage compared with the age-stable or age-upregulated
genes. Promoter
regions were particularly affected, perhaps because they
contain G/C-rich
sequences that are sensitive to oxidation, or do not undergo
transcription-coupled repair. In SH-SY5Y cells, promoters of
the same
age-downregulated genes were both more sensitive to
hydrogen-peroxide-induced damage and less able to undergo
base excision
repair of such damage than promoters of age-stable or
age-upregulated genes.

To investigate whether impaired mitochondrial function could
predispose
these age-downregulated genes to DNA damage, small
interfering RNA (siRNA)
was used to reduce the expression of mitochondrial F1-ATPase
2.5-fold in
SH-SY5Y cells, approximating the reduction seen in the aged
human cortex.
This resulted in significantly increased promoter DNA damage in
age-downregulated genes, which was partly reversed by the
antioxidant
vitamin E. These findings support the idea that
mitochondrial dysfunction
contributes to the damage of vulnerable genes in the ageing
brain. The
vulnerable-gene promoters are both more sensitive to
oxidative stress and
deficient in repair, and mitochondrial dysfunction could
potentially
exacerbate both by increasing ROS or decreasing the
availability of ATP,
which is necessary for repair. ...