July 22, 2005
Genetically Engineered Mice Age More Rapidly Due To Mitochondrial Mutations
Tomas A. Prolla of the University of Wisconsin found that mice genetically engineered to undergo more rapid mutation of mitochondrial DNA aged at a much more rapid rate and experienced cell loss, especially in cells that divide rapidly.
Using mice genetically altered to have a deficiency in a protein that proofreads mitochondrial DNA and thus accumulate genetic mutations at a higher rate than unaltered mice, the group led by Prolla found evidence that programmed cell death, known as apoptosis, was greatly accelerated. The altered mice exhibited obvious hallmarks of aging - including graying, hair loss and atrophied muscle and bone - at a pace much faster than the typical laboratory mouse.
"It's like a broken spellchecker," says Prolla. "By introducing a malfunction in the (genetic) proofreading domain, these mutations accumulate much more rapidly."
The new work lends support to one of the two leading theories of how animals, including humans, grow old and die. It supports the theory that apoptosis or programmed cell death underpins aging. A competing theory holds that oxidative stress - the body's reaction to oxygen and the production of reactive, cell-damaging molecules known as free radicals - is responsible for the aging process.
According to the new Science report, markers of oxidative stress did not parallel the accumulation of mitochondrial genetic mutations. Instead, the group found evidence that indicated accelerated cell death, especially in tissues characterized by rapid turnover of cells, occurred as mutations mounted in the mitochondrial DNA.
"We found no evidence of oxidative stress," Prolla explains. In fact, the team noted less oxidative stress in some tissues - the liver, for example - which suggests that accumulated genetic mutations in mitochondria slow metabolism. In turn, that change prompts cells to produce fewer of the reactive free radical molecules.
The symptoms of aging become pronounced with the loss of some critical cells, notably adult stem cells from some tissues and that are essential for replacing cells that die. "If these stem cells are lost, tissue structure and the ability of tissue to regenerate are impaired," Prolla explains. "We have observed that in tissues like bone marrow, intestine and hair follicles."
The altered mice used in the study were created by manipulating mouse embryonic stem cells to produce mice with the defective DNA proofreading protein. The mice develop normally, but age rapidly and develop such things as age related heart dysfunction, hair loss, loss of immune cells, anemia, and loss of male germ cells that lead to reduced sperm production and infertility.
We need constant supplies of replacement cells. Aging of stem cell reservoirs in the body are especially harmful because stem cells divide to produce a very large range of replacment cell types throughout the body.
Prolla thinks an obvious next step would be to genetically engineer mice to have a lower rate of accumulation of mitochondrial DNA mutation.
Prolla suggests that new studies of mice engineered to have fewer than normal mitochondrial DNA defects or improved mitochondrial function may pave the way for strategies to retard aging. "The idea would be to reduce the level of cell death and improve function. If that pans out, then we can begin to think about pharmaceutical interventions to retard aging by preserving mitochondrial function."
Stem cell therapies could replace the stem cells lost due to aging. That would provide enormous benefit. Also, growth of new replacement organs would allow entire aged organs to be replaced by youthful organs. But we also need the ability to send in gene therapy to repair or replace the mitochondrial DNA of aged cells. Look at brain cells. Unless you want to entirely forget the past you don't want to just replace old nerve cells because those old cells hold memories and have been conditioned to perform various tasks.
Mitochondrial DNA is the genetic Achilles Heel of eukaryotic cells (and human cells are eukaryotic because they have specialized compartments such as the nucleus and mitochondria). Eukaryotic cells have mitochondria which are sort of like mini-cells within cells. These mini-cells (usually called organelles) are specialized to carry out the task of breaking down sugar to produce energy molecules (ATP and NADH) used elsewhere in cells to provide energy to carry out just about all cellular tasks. The problem with mitochondria is that they have their own DNA which is exposed to free radicals generated by the breakdown of sugars. Damage to the DNA by those free radicals probably cause the mitochondrial DNA to accumulate mutations at a faster rate than nuclear DNA.
Biogerontologist Aubrey de Grey proposes the development of modifications of the 13 mitochondrial DNA genes to allow those genes to be moved to the cell's nucleus. This would reduce the rate at which the mitochondrial genes get mutated and therefore delay the decline of cellular energy production as cells age. Cells with better protected mitochondrial genes would continue to produce energy for many more years (perhaps decades longer) and therefore general cellular aging would be much slowed. This latest report provides evidence that strongly suggests Aubrey's proposal would help.
Hopefully Prolla's report will be seen by the scientific community as a reason to do the work necessary to genetically engineer mitochondrial genes to move them into the nucleus.
The nice thing about mitochondrial aging, is that in principle it ought to be fairly straighforward to reverse. For tissues which have significant cell turnover, introduction of gentically reengineered stem cells would probably do the trick. In the brain? Well, it appears that mitochondria evolved from cellular parasites, and such parasites are still around... We might genetically engineer a strain capable of "infecting" brain tissue with improved mitochondria. This still leaves the problem of continued intracellular reproduction of compromised mitochondria... But to the extent that mitochondria could be considered alive in their own right, I suppose it ought to be possible to develop a toxin which would kill off the old, damaged ones, while leaving the new ones untouched to take their place.
A more ambitious approach might be to alter the glial cells to generate the ATP needed by the neurons, and supply it to them from the outside. This could free neurons entirely from the dangers of oxidative metabolism. One might even consider a nanotech device that would extend processes into each cell of the body, to recharge their ATP from an abiological source of energy, in order that the entire body could be maintained as an anerobic zone. Oxygen, however we rely on it, IS a toxin!
"SIRT3 is one of the seven mammalian sirtuin homologs of the yeast Sir2 gene, which mediates the effect of caloric restriction on life span extension in yeast and Caenorhabditis elegans."
Apparently the effect of caloric restriction (and probably also intermittent fasting) on life span is due (at least partially) to its effect on mitochondria, as mediated by sirtuins. And we need not wait for the Biotech Singularity -- we can restrict our diets anytime we want.
I think gene therapy in the brain is going to be a hard problem to solve. How to deliver genes to so many cells? How to avoid delivering too many gene copies to each cell?
Consider the number of cells that must have gene therapy done to them to restore mitochondrial DNA function:
The adult human brain is believed to consist of at least one hundred billion neurons (nerve cells) and probably five to ten times as many neuroglial (functional support) cells.
How to avoid damaging the cells? How to deliver genes into all of the hundreds of billions of cells? How to avoid delivering too many copies per cell? How to avoid an immune reaction to the gene therapy delivery vehicles. All the viral packages used to deliver gene therapy cause immune responses.
If we deliver the genes into the nucleus then we can get the nucleus to make mRNA to make enzymes and other proteins for all the mitochondria in the cell. That'll fix all the mitochondria in the cell (though some mitochondria might also still make defective proteins that cause problems - still that is a small problem in comparison).
Also, the amount of DNA that needs to be delivered is more than just one gene. The mitochondrial DNA (mtDNA) is about 15,000 letters. The version we'd make to put the genes into the nucleus would probably be longer than that. We might need to deliver 30,000 or 40,000 base pairs to each cell's nucleus.
This strikes me as an incredibly hard problem. Brain rejuvenation strikes me as the hardest part of total body rejuvenation.
That's why I wasn't proposing gene therapy of brain cells, but instead their infection by a modified intracellular parasite. Such an organism ought to be capable of more complex behavior than a virus, such as getting around tissues on it's own, or secreting a marker protien that shows up in the cell membrane of infected cells, and refraining from reinfecting cells already bearing such markers. Or removing itself from an infected cell when that cell becomes infected with the next revision of the parasite. (We WILL need ways to update these improvements, after all!) And it gets around the whole issue of how to avoid messing up functional genes, as is often seen in cells genetically modified by retrovirus.
T. Gondii might even be a good candidate for such an engineering project: http://www.futurepundit.com/archives/2003_09.html
Admittedly, more complex to design than a retrovirus vector, but potentially MUCH more controlled.
Preventing mitochondrial damage may be fairly simple, though - Bruce Ames has done some intriguing work showing mitochondrial damage can be ameliorated by feeding old rats the normal mitochondrial metabolites acetyl carnitine (ALCAR) and lipoic acid (LA) at high levels [6-8]. The principle behind this effect appears to be that with age increased oxidative damage to protein causes a deformation of structure of key enzymes, with a consequent lessening of affinity (Km) for the enzyme substrate . The effect of age on carnitine acetyl transferase binding affinity can be mimicked by reacting it with malondialdehyde (a lipid peroxidation product) . Feeding the substrate (acetyl carnitine) with lipoic acid, a mitochondrial antioxidant, restores the velocity of the reaction, Km for acetyl carnitine transferase, and mitochondrial function . In old rats (vs. young rats) mitochondrial membrane potential, cardiolipin level, respiratory control ratio, and cellular O2 uptake are lower; oxidants/02, neuron RNA oxidation, and mutagenic aldehydes from lipid peroxidation are higher [6-8]. Ambulatory activity and cognition declines with age [6,7]. Feeding old rats acetyl carnitine and lipoic acid for a few weeks restores mitochondrial function; lowers oxidants, neuron RNA oxidation, and mutagenic aldehydes; and increases rat ambulatory activity and cognition (as assayed with the Skinner box and Morris water maze) [6-8].
From http://mcb.berkeley.edu/faculty/BMB/amesb.html. So rather than going the starvation route, this may be somewhat amenable to nutritional supplementation, & LA and ALCAR are readily obtained at Whole Foods. GT sez, check it out.
"That's why I wasn't proposing gene therapy of brain cells, but instead their infection by a modified intracellular parasite. Such an organism ought to be capable of more complex behavior than a virus, such as getting around tissues on it's own, or secreting a marker protien that shows up in the cell membrane of infected cells, and refraining from reinfecting cells already bearing such markers. Or removing itself from an infected cell when that cell becomes infected with the next revision of the parasite. (We WILL need ways to update these improvements, after all!) And it gets around the whole issue of how to avoid messing up functional genes, as is often seen in cells genetically modified by retrovirus."-by Brett Bellmore
That's similar to an idea I've had for quite some time, this of course is an idea for a time that has yet to come(far better tools & knowledge is necessary), the use of mod'd cells as delivery agents for the new genetic material(eventually not only mits, but even the entire nucleus itself could be replaced should such a technique prove viable.). In order to quickly bestow vast enhancements optimally one may need to be able to precisely and safely deliver or change gen. info on multiple chroms and even the use of art-chroms may be desired, doing this in billions of cells may be required for some tissues(e.g. brain.) or certain circumstances(e.g. extreme radiation doses, provided the organism's innate means to suspend activity, or it's externally done so.), and would most certainly be extremely challenging without the carrying capacity and functional-versatility & specificity that's been observed in the cells of complex multi-cell organisms.
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