What Bioethicists Don't Do...

Probably because C.S. Lewis and Star Trek are so much easier...

Via the invaluable and tireless Reason at Fight Aging, comes this interesting news from Dr. Rafal Smigrodski...

Today our team confirmed our previous preliminary data showing that we can achieve robust mitochondrial transfection and protein expression in mitochondria of live rats, after an injection of genetically engineered mitochondrial DNA complexed with our protofection transfection agent. A significant fraction of cells in the brain is transfected with this single injection even though we so far did not optimize the dose. This achievement has important implications for medicine: protofection technology works in vivo, and should be capable of replacing damaged mitochondrial genomes.

Now why exactly is this interesting? Should you even give a damn?

Well for starters, it's beginning to look as though mitochondrial failure is implicated in a number of our unpleasant biological failure modes. Yet, as with so many other of our systemic breakdowns, there hasn't been a great deal we could do about it. That may be changing.

Here's Dr. Smigrodski...

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.

Here is an extremely pared-down version of the above paper...

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...We postulate that microheteroplasmy is sufficient to explain the pathomechanism of several age-associated diseases, especially in conditions with known mitochondrial involvement...

The genetic properties of microheteroplasmy reconcile the results of disease...with the relatively low levels of maternal inheritance in the aforementioned diseases, and provide an explanation of their delayed, progressive course.

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. Another element crucial to maintenance of complexity is
integrity of genetic information...

Interestingly, many progerias primarily affect stem cells and their generation of oxidative stress, preventing the repopulation of tissues damaged with age. 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...a number of biochemical processes 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...

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...

...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...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...

The accumulation of mutations in somatic DNA is not a new concept... According to the mitochondrial theory of aging, first proposed by Harman in 1972, mitochondrial genomes accumulate mutations as a result of damage from reactive oxygen species...informational loss
in mitochondrial genomes would predict an exponential decline due to
entropic forces...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...and the individual differences in its accumulation confer a proclivity to develop specific age-related conditions...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...thus acting as the principal component of the molecular clock of aging.

Mitochondria are intracellular organelles found in almost all
eukaryotes, derived from -proteobacteria and possessing their own
genome...They are involved in many biochemical processes, most
notably oxidative phosphorylation, and in apoptosis, or programmed
cell death...

In each cell there are usually between 1,000 and 100,000 copies of mtDNA, on average 4,900 mtDNA genomes per nuclear genome. 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.

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.

However, as noted above, there are many conditions where this
cleansing mechanism fails...

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.

With additional refinements it is possible to increase sensitivity to about 10% heteroplasmy 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) but even there the sensitivity is usually not better than 3%...

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.

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 indicate that microheteroplasmy is present in all tissues, at all ages examined, and in all subjects...

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...

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

As alluded to in the introduction, this finding has important
methodological implications [since] 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.

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.

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. 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 ...

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

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...

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.

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...

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...

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)...


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...

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 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...

2) Mechanistically, microheteroplasmy acts through the accumulation of dysfunctional copies of mitochondrially-encoded ETC subunits, which leads to increased production of ROS...lowered peak ATP production...impaired oxidative phosphorylation, and in turn, to
apoptosis or impairment of cellular function...

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.

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...

5) Age-acquired microheteroplasmy and the drift in germline mutation load are the factors responsible for the delayed onset of age-related disorders...

Increased ROS production, which we propose as the most important link
between microheteroplasmy and aging, leads to extensive antioxidant
responses...When ROS production becomes chronic, the very ability to remove damage becomes compromised.

Precisely because microheteroplasmy compromises mitochondrial
function, the machinery required to repair and remove damage fails to
import into mitochondria, further exacerbating ROS damage...

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.

Interestingly, expression of catalase outside mitochondria (e.g. in the nucleus) did not result in significant slowing of aging, indicating that mitochondrial damage may be more important, at least as far as ROS-related mechanisms are considered.

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...

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...

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...

...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...

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...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...

So after all that you have a better idea why the following news is so very encouraging.

...protofection technology works in vivo, and should be capable of replacing damaged mitochondrial genomes...

To my mind, this is the sort of thing that doctors should be doing with their time. You know, healing the sick, discovering cures?

Leave Plato and Montaigne to the Bioethical Mandarins.

posted by Justin on 07.10.05 at 01:36 PM







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» Human Innovation: Fixing your mitochondria from DownsizeDC.com
Hundreds of millions of years ago small psuedo-bacteria invaded more complex cells, beginning eons of bio-economic trade and eventual symbiosis – the permanent joining of two organisms into one. These invisible invaders are inside you right now, all th [Read More]
Tracked on July 25, 2005 3:32 PM



Comments

That's half of it. Now we just have to figure out how to prosper.

triticale   ·  July 11, 2005 1:24 AM

This post is too scientific for me. The other post was more mythic. The 2 hemispheres of my brain? Wanda vs. Dawn?

"Plastics." "Colors" or "Spectrums"?

"The vertical members betoken the male, upright societal principles, providing overall structure and a firmly clasping support to the horizontal female elements, or shelves."

Transcendental Science?

C. S. Lewis and Aldous Huxley both died on November 22, 1963, the same day Lee Harvey Oswald (a Communist) assassinated President Kennedy. Conspiracy? Orange Lodges?

The style of it all!

We need somebody who knows a lot more about science than I do to comment on this post.


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