Transfer mtDNA to nucleus?

Started by olaf.larsson , Jun 10 2004 01:03 AM

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#1 olaf.larsson

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Posted 10 June 2004 - 01:03 AM

DNA in the mitochondria is subjected to a huge free radical damage compaired with nuclear DNA. How about taking mtDNA and fuse mitochodria targeting sequences infront of the genes, change the alternative genetic code and insert the sequence into nuclear DNA?
What would happen? A organism with a slower rate of aging could be the result.

Edited by wolfram, 16 June 2004 - 01:07 PM.

#2 Cyto

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Posted 10 June 2004 - 03:17 AM

Targeting sequences for the mitochondria can be learned about through the Bcl-2 family of anti-apoptic factors, the problems with transmembrane domains is the hydrophobicity in which can cause aggregates. There is some evidence as to chaperone's which escort such proteins to the mitochondria thus forcing movement and avoidance of cytotoxic inclusion bodies. So we may see a need for some molecular chaperone's. And to reference another example: the r-endoplasmic reticulum forms aggregates if the GRP78/GRP94/GRP170 protein array doesn't "cover" hydrophobic folding domains.

I wouldn't say this if I thought the genes were not heavily transcribed.

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#3

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Posted 10 June 2004 - 09:38 AM

So you want to preserve the integrity of mtDNA by sending it in to the relatively safer, oxidatively speaking, domain of the nucleus using a nuclear targeting motif. Ok, but how is the mitochondrion meant to undergo binary fission during cell division? It has its own DNA for a reason. In fact mitochondria have their own ribosomes and tRNA. If the reason for sending the DNA to the nucleus is to keep it safe, then you need a mechanism to bring it back in to the mitochondrion when it is needed to replace the damaged one. Preferably a periodic mechanism. But why make things so difficult and complex. Firstly, aside from the oxidative environment of the mitochondrion, another reason why DNA is so susceptible to damage is that it is not folded with histones and protected like genomic DNA in chromosomes. It exists as a circular structure and unlike nuclear DNA it has 1000 copies of its DNA per cell. One benefit is that it does not undergo recombination with each generation, yet its DNA is more susceptible to damage. In fact, DNA repair activity in mitochondria is significantly lower than that of nuclear DNA, which is very interesting and has a lot of evolutionary implications.

I think you would be better off inserting DNA repair enzymes into the mtDNA to express increased mtDNA repair enzyme. I don't know if this has been done. --- Nothing on Pubmed, although there are studies on drug delivery targeted directly to the mitochondrion.

Considering that mitochondrial failure as a result of mtDNA damage results in common diseases such as heart failure, cancer and diabetes you are definitely on the right track insofar as looking for therapeutic targets.

#4 olaf.larsson

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Posted 12 June 2004 - 01:08 PM

Yes mitochodria have their own ribosomes and tRNA, but I see no use for the protein synthesis machinery when if all the protein coding genes would be in nuclear DNA instead. Im not sure how mitochondria divide themselves, but if there are any proteins needed in mitochodria division they also could be imported.

"I think you would be better off inserting DNA repair enzymes into the mtDNA to express increased mtDNA repair enzyme. " Projects like this are not really science they are biological engeneering projects. People are not used to think about biology as engeneering thats maybee they have mental blocks against projects like this.

#5 manofsan

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Posted 12 June 2004 - 05:35 PM

But I wonder why evolution hasn't shifted everything into the nucleus by now? Is it possible that Nature hasn't had enough time, or is it that some things have to stay on the mito side?

Does DNA have to be present in the mito to guide its replication? I thought DNA is temporarily out of action while cell division is occurring. I can see why you'd need ribosomes to build proteins locally at the mito.

But what you said about periodically refreshing from the nucleus makes sense. Actually, why not just refresh at the point of cell replication. A mito-DNA refresher for each cell generation should be good enough, seeing how often cells themselves replicate.

So there needs to be a modified cellular/mitochondrial replication process, in which the mitochondrial DNA is refreshed from the nucleus. Is there anyway that retrovirus technology could be used for this? Is it possible to have a purely intra-cellular virus that only penetrates mitochondrial membranes, but doesn't pass out of the cellular membrane?

What is the difference between the mitochondrial membrane and the cellular membrane? Can I assume that the mito membrane is weaker?

Hehe, in a wierd way it brings to mind the image of "sperm" that is emitted by the nucleus which then fertilizes and refreshes the mitochondria. Sort of an internal fertilization process, lol.

But how would you eliminate the existing mitochondrial DNA in order to make way for the new replacement DNA from the nucleus? I would say that don't allow the mitochondrial DNA to replicate in the first place. I bet that even just using that basic tactic of mito-DNA replication denial, you could employ a Darwinian algorithm to naturally select for cells who can pull off the substitution correctly, because anyone who can't won't survive.

Comments?

#6

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Posted 13 June 2004 - 05:28 AM

The function of the nucleus is to safely store and make available for transcription via an enormous amount of regulatory mechanisms selected segments of the genomic archive. The function of mitochondria is to produce ATP. These are fundamentally different organelles from every level of consideration. It is simpler to increase mtDNA repair function rather than a convoluted mechanism of mtDNA shuttling from nucleus to mitochondria. With respect, read up on the differences between the two. A great staring point is at http://www.ncbi.nlm....TOC&rid=mcb.TOC which is the entire Molecular Cell biology text book online. You can browse and learn lots of interesting facts about cells and their inner workings as we presently know them. Particularly chapter 17 in "Protein Sorting". You can then see the differences in trafficking between the nucleus and mitochondria.

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#7 olaf.larsson

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Posted 13 June 2004 - 09:43 AM

In the latest version of Molecular Biology of the Cell its written that some yeast cells could live without having any mitochodrial genome. The growth replication of the mitochodria is done enitirely by proteins imported from nucleus. Now I have a question: Are you sure that mitochodrial proteins would not be imported if they were in the nucleus and had the right sequence? Has anyone tried it?

This could be a nice experimental approach: Take a mitochodrial protein gene add import sequence, modify the alternative genetic code, fuse the gene with a sequence for a fluorescent protein, insert the gene in nucleus. If we are lucky we will see that the mitochodria will be fluorenscent.

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Posted 13 June 2004 - 03:58 PM

Unfortunately, we are unable to precisely localize the point of insertion of any DNA that gets inserted into the genome. This is the reason why a moratorium on gene therapy has been placed. The danger is in disrupting a key DNA segment with repercussions as serious as cancer. Once we have the technology to precisely insert a DNA segment where we want it will herald the era of true gene therapy. Until then we can only insert genes via constructs such as plasmids which do not need to enter the nucleus for transcription of the genes they encode. I know it would be great to shift out of the oxidative environment the 37 genes that mtDNA encodes for. It does not sound like much compared to the 30,000 or more genes that are in nuclear DNA. The fact that the nucleus routinely encodes and transports proteins for the mitochondria makes this even more tantalizing. Another point to note is that mitochondrial DNA is read differently in mitochondria, i.e. the codon UGA in nuclear DNA means stop, whereas in mtDNA means Tryptophan. Furthermore each cell has several thousand copies of mtDNA. Also with mtDNA transcription is continuous. Thus you have multiple genes continuously transcribed on multiple DNA strands continuously. This is a specialized system of a huge amount of production of a small number of components in continuous fashion. Better off to increase mtDNA repair.

Here is a basic comparison of mtDNA with nuclear DNA from the online version of HMG2 http://www.ncbi.nlm....d=hmg.table.653

Edited by prometheus1, 14 June 2004 - 03:46 AM.

#9 olaf.larsson

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Posted 14 June 2004 - 07:31 AM

Why are you continuing saying there are 37 genes in mitochodria prometheus? There are only 13 protein coding genes. There is no need for mt-tRNAs or ribosonal parts if there are no protein coding genes in the mitochodria.

I must say Im sceptical to the idea of improving dna repair in mitochodria the idea of exporting the 13 protein coding genes to the nucleus is so much more beautifull to me.

Ofcourse gene therapy is not yet good enough to try on people but one could first try on mouse or drosophila embryos to see if the concept works and gives longer and more healthy life.

#10

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Posted 14 June 2004 - 08:59 AM

For clarification:

From Molecular Cell Biology 4th Ed

"The human mitochondrial genome contains 37 genes: 28 are encoded by the heavy strand, and nine by the light strand. Of the 37 genes, a total of 24 specify a mature RNA product: 22 mitochondrial tRNA molecules and two mitochondrial rRNA molecules, a 23S rRNA (a component of the large subunit of mitochondrial ribosomes) and a 16S rRNA (a component of the small subunit of the mitochondrial ribosomes). The remaining 13 genes encode polypeptides which are synthesized on mitochondrial ribosomes."

Yes, there are only 13 protein coding genes. These are related to the respiratory function. However, the rate and regulation of transcription is another matter. Remember, there 1000's of copies of mtDNA in each cell. Why? If we look at other biological systems where this type of genetic arrangement exists it suggests that an enormous amount of gene expression is taking place.

I'm all for re-engineering mitochondria, as next to telomeres, they are the next weakest link in the cell. I am unconvinced of the merit, however, of shifting such an enormous amount of protein expression to the nucleus and the subsequent trafficking back to mitochondria.

If you can explain to me how it would be of more benefit to have the arrangement you propose rather than increasing DNA repair in the existing mitochondrion I would be delighted to agree with you.

#11 olaf.larsson

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Posted 14 June 2004 - 03:21 PM

The idea of exporting the genes the nucleus is more beautyfull to me. But improving dna repair in mitochodria could be nice to. Now I suggest we could start with trying to find out if it could be like this that germ cells could have special mitochondria repair mechanisms that other cells dont have. If they have it could be relativly easy to introduce this repair mechanisms to other cells then germ cells.

#12 kevin

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Posted 14 June 2004 - 03:59 PM

Quote Prometheus1:
"I am unconvinced of the merit, however, of shifting such an enormous amount of protein expression to the nucleus and the subsequent trafficking back to mitochondria."

I tend to agree with that statement and think that protecting the DNA with upregulation of endogenous antioxidants is the first line that should be worked on as it is likely the easiest to accomplish.

From http://www.pressbox....ailed/9446.html

Dr. Richard Cutler and his colleagues are applying the idea that simple genetic changes could have a large impact on lifespan, by investigating how to up-regulate many of the genes involved in controlling "oxidative stress" status -- the extent to which cells and tissues are constantly bombarded with free radicals, toxic molecules produced as a side-effect of using oxygen. These genes include the thioredoxin redox regulating system and the antioxidant response element (ARE) controlling an array of "phase II" proteins (proteins that are produced by cells when they sense elevated oxidative stress). Dr. Cutler is developing transgenic and pharmaceutical means to enhance expression of such systems to achieve a dramatic decrease in cellular oxidative stress. If this works they will then test the mice for lifespan enhancement

Some related links for those interested with time to read them.. flies seem to be popular..

[*]Induced Overexpression of Mitochondrial Mn-Superoxide Dismutase Extends the Life Span of Adult Drosophila melanogaster - Genetics-2002
[*]Effects of Overexpression of Copper-Zinc and Manganese Superoxide Dismutases, Catalase, and Thioredoxin Reductase Genes on Longevity in Drosophila melanogaster
[*]Thioredoxin Peroxidase Is Required for the Transcriptional Response to Oxidative Stress in Budding Yeast

I found this interesting paper which indicates that the researchers have found a nuclear gene, POS 5, in yeast which functions as exclusively to maintain the stability of mitochondrial DNA via NADH kinase dependent mechanism, so at least in this case, the protection of the mitochondrial genome could feasibily be increased by upregulating this product if it has a homologue in the human genome. (something I'd be interesting in finding out.. )

http://ec.asm.org/cg...nt/full/2/4/809

#13

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Posted 14 June 2004 - 05:23 PM

Precisely Wolfram! We need to observe why germ line and stem cells are not as vulnerable as somatic cells.

Lets see what we know of the advantages of germ and stem cells over somatic cells:

1. Telomerase positive - these cells have no upper limit on the times they can divide, and are less quiescent and more proliferative. The advantage insofar as DNA repair in this case is that they can take advantage of global genomic DNA repair that occurs during the S phase of mitosis whereas cells that are not dividing seem to be limited to transcription coupled repair (repair associated only with genetic sequences that involved the transcription of genes).

2. Selective segregation of DNA template - in stem cells, the original template strand is retained with each subsequent division decreasing the likelihood an error during copy.

3. Back to telomeres - other mechanisms associated with telomere maintenance aside from telomerase have been implicated in DNA repair. Thus any cells that want to be division limited will also lack this DNA repair mechanism.

As you can see I haven't mentioned differences specific to mitochondria. That's because I haven't been able to reference any studies which could mean that either I have missed some or there aren't any.

In each case the evidence converges to DNA repair. If we are to look at mitochondria specifically, there is one more strategy we can employ to reduce mitochondrial induced apoptosis - upregulation of the superoxide dismutase gene or the BCL2 gene which is another oxidant reducer. These genes are encoded in the nuclear DNA. I haven't been able to find any studies that show that these are upregulated in germ line or stem cell mitochondria. They have transfected cell cultures with these genes for the purpose of overexpression and found that they extended cell lifespan in radiation exposure conditions by keeping the mitochondria healthy.

#14

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Posted 14 June 2004 - 05:30 PM

Thanks Kevin for those references.

I think it would be a good idea if we surveyed all the mitochondria studies and listed all genes associated with their survival. Including DNA repair, antioxidant or any other mechanism. Your help would be greatly appreciated.

#15 olaf.larsson

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Posted 15 June 2004 - 12:38 PM

Thank you for your information Kevin. POS5 seems to have at least three homologs in humans AK023114, BC001709, FLJ13052.
Improvement of various kinds of anti oxidative mechanism has been done before in diffrent animals with some moderate life extension as result, so to do this is not very spectacular. Unfortunatly I dont know about any special mitochodria maintence mechanism in germ line cells, but logic says to me that they must exist. If they wouln´t the ofspring would get old mitochodria from the mother. So the thing to do is to find those mechansism.

If someone here would come up with a good idea here will anyone have the resources to do it practicaly?

#16 manofsan

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Posted 15 June 2004 - 06:21 PM

Wow, some interesting posts since I last posted!

Well, can we do better than nuclear-mito DNA exchange? We have hard-drives too now. Can the mito DNA be refreshed from outside the body itself?

ie. can we infect the body with a retrovirus that will specifically infiltrate the mito rather than the nucleus, in order to replenish genes that are dysfunctional?

Then you don't have to merely rely upon the nucleus as the repository for those vital mitochondrial genes.

Are there any existing precedents in nature that would point to a mitochondrial-specific virus? They are ex-bacteria after all, so shouldn't there be something that will particularly go after for them?

You guys have said that the genes in the mito are free-floating, if I'm correct. So they're not part of any structured chromosomal type units. So any refresher genes to be inserted by a viral delivery vector would only have to be brought into the mito, and wouldn't have to be incorporated into any formal chromosomal structure. So there wouldn't be any danger of disrupting existing genes then.

And hey, if some cells unfortunately don't get infected (ie. mosaicism), then they'll just die sooner anyway, and the modified cells will live on.

Hmm, let me further build on that with my imagination. Could the delivery vector be used to mop up the existing genes in the mito first, before releasing the new replacement genes? Perhaps the exterior capsule of the delivery vector could bind with the existing genes, before opening up to release the replacement genes it contains. I guess I'd want to sweep out the existing mito genes in order to start everything with a fresh slate. If you have surviving pre-existing genes left around as holdovers, they may skew the expression rates by adding to what the refresher genes are doing.

But really, if mito DNA is unstructured compared to nuclear DNA, then isn't this a useful fact which can be exploited for gene replacement purposes? ie. no recombinant-style insertion between existing sequences required, therefore no real danger of gene disruption from insertion, and therefore a much easier time getting the new genes into the game?

Comments, please?

#17 manofsan

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Posted 15 June 2004 - 07:00 PM

Actually, further building on this, could various other non-mitochondrial genes be deposited into the mito, relying upon the mitochondrial ribosomes to express them?

Yes, I realize that the oxidatively active mitchondrion is not as safe an environment as the nucleus longevity-wise, but in another sense it could be seen as safer for insertion purposes, since there wouldn't be any recombinant insertion required, with its attendant danger of gene disruption. The free-floating nature of the mitochondrion's DNA might make it a better target insertion-wise, for a variety of genes that we'd like to see inserted into our "genome"

#18 olaf.larsson

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Posted 16 June 2004 - 01:04 PM

As first experiment I would go for an artificial chromosome in yeast cells with many copies of the mitochodrial genome with target sequences and modified genetic code. As stated earlier this could cause a trafficing problem but it might as well not. Artificial chromosomes are used since long time and not very exotic things.
Insert your artificial chromosome in petit-mutant yeast cells which lack mitochondrial genome and see if they become reversed to pseudo wild-type.

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Posted 17 June 2004 - 06:52 PM

Using non viral gene therapy technology such as plastic coated vesicles containing a plasmid that encodes the 13 mitochondrial genes fused to mitochondrial targeting sequences would presumably complement mitochondrial proteins that could be getting damaged by oxidation. This would be the safest way to go and could be done today in any lab (order the DNA, clone in plasmid and gently mix with plastic vesicle compound).

The vesicles could be ingested or injected intravenously or intramuscularly. I don't think anything could really go wrong with this type of therapy aside from having no effect. The plasmid should last for a few weeks ensuring that expression of the 13 mitochondrial proteins is maintained for this period. Selecting the right plasmid would ensure a longer lifetime in the cell and using a multi-copy plasmid would increase expression.

It's more of a question of what tissues can be targeted and how many cells in those tissues will take up the plasmid. Ideally the first targets should be the brain and heart. In fact I would be willing to try it myself. I suspect that the only differences one would actually feel should be a slightly increased energy level and a reduction of inflammation. It all depends whether the availability of fresh mitochondrial proteins will rescue a tired or toxified mitochondrion.

It sounds easy and it is. It's also safe because the plastic coated vesicles are biologically inert and the plasmid will not enter the nucleus.

#20 olaf.larsson

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Posted 18 June 2004 - 12:15 PM

How about trying out this at least on cells before trying a such a new exceptionally speculative treatment on yourself? Who here has access to a lab to try this out in reality?

#21 manofsan

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Posted 18 June 2004 - 10:23 PM

We were having a discussion on Usenet's Sci.Bio.Technology newsgroup with Aubrey De Grey who seems to specialize in the whole mitochondrial aging thing, and he raised some important points.

He pointed out that defective mitochondria have been shown to out-live and thus out-reproduce healthy mitochondria, due to the former having less oxidative activity and thus self-inflicting less oxidative damage. Therefore whatever mitos you heal, will be crowded out by the defectives anyway.

So then came idea of using some kind of tagging in the genemod to identify healed mitos and destroy the rest. Aubrey mentioned that all defective mitos suffer from low proton gradient, which is the hallmark of defectiveness. It was suggested that a drug be developed/discovered that would only select for mitos with low proton gradient to kill them. Perhaps it would be able to only cross low proton-gradient mito membranes. Or perhaps it would be broken down or disabled by the high positive or negative charge in the healthy mito.

Another problem is that even healthy mitos with high proton gradient tend to oscillate widely between highs and lows, so they're not always high all the time. But then on average the healthy mitos have that higher proton gradient relative to unhealthy ones when measured across a wider timespan. So it would be useful to have a slow-acting drug/poison that would take time to accumulate to kill the mitos.
It would also be nice if the drug/poison would be pumped out of the healthy mitos during their high proton gradient periods. The defective mitos would be low gradient all the time, so they wouldn't be able to pump out the poison and would die.

But what kind of drug can do this? I note that high proton gradients are great at pumping out protons, but this occurs via the specialized ion channels that only accomodate protons which are the tiniest things around anyway. So it's doubtful that a drug or even its broken down constituents would be able to cross out of these proton channels.

I dunno what other membrane channels a mito has, so perhaps if the poison-pumpout option is infeasible, then maybe you want the poison to be broken down or neutralized by the healthy efficient mito. The high proton-gradient corresponds to high interior positive charge, high exterior negative charge, and high interior concentration of NADH, high expulsion rate of protons to synthesize ATP. Perhaps if you could weakly block up the outlet of the ATP-synthase-complex channel from which protons emerge, then defective mitos would be effectively stoppered up and poisoned, while healthy mitos with their robust proton expulsion rate could bust off the weak stopper.

What about that recent discussion thread we had on the Antibodies with the Membrane Translocation Sequence? These were being touted as being able to penetrate inside the cell to target interior organelles. I'm wondering if such antibodies could be engineered to selectively destroy unhealthy mitos. Could such antibodies be designed to function as the weak stopper to cap the ATP-synthase complex channel?

I also suggested as an alternative strategy that instead of trying to kill the defective mitos outright, you'd maybe just try to tilt the reproductive balance against the defective mitos and allow the healthy ones to out-proliferate the defective ones. Perhaps that's a less than ideal solution, but it might afford a wider selection of possible agents than if you're trying to kill something outright.

Slowing down an unhealthy mito reproductively could mean disrupting/interfering/impeding its reproductive mechanisms enough to let the healthy ones gain a significant lead in numbers over them. Perhaps an agent that accumulates to the point of interfering with those chemical reactions/processes colligatively.

Comments?

#22

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Posted 19 June 2004 - 02:46 AM

Selective destruction of mitochondria? I am troubled by this. We are not talking about selectively destroying cells, but about selectively destroying mitochondria organelles, of which there are anywhere between 50 - 1000 per cell. Not impossible, but is it necessary at this stage when we have not even attempted addressing the root cause, DNA damage, by enhancement of DNA repair/protection mechanisms.

In the sci.biotechnology discussion Aubrey said:

There is a killer problem with transfecting new DNA into mitochondria,
namely that mutant mitochondria have a selective advantage over normal
ones in non-dividing cells (which are the ones that accumulate mtDNA
mutations). Thus, even if your gene therapy rescues the function of
90% of the mitochondria in a cell, it'll be as bad as ever in a year
or so because the successfully rescued mitochondria will have lost out
to the few that didn't get your DNA.

I think this is what has spawned the discussion about selective destruction of malfunctioning mitochondria. Namely, the premise that even if you rescue mitochondrial function via gene therapy you cannot reduce the formation of mutant mitochondria that will eventually diminish normal mitochondrial function. Thus the inquiry on how to identify abnormally functioning mitochondria via proton gradients etc., as well as the interesting ways of delivering antibodies into the mitochondria to modulate their function.

It can be very productive to brainstorm but I think if we are looking for a solution for today, this argument misses the boat. Lets rewind for a moment. What is the root cause?

The very basis for mitochondrial mutation is ineffective repair of damaged DNA. Once the DNA is damaged the mitochondrion can then either signal apoptosis or it can continue to function as a mutant. A subset of some mutants may have a selective advantage over wild type mitochondria. They may even have an abnormal proliferative capacity that eventuates in the cell being engulfed by dysfunctional mitochondria. I say so what. If we address the root cause of the problem using enhanced DNA repair we diminish the likelihood of DNA damage.

This is a solution that can be implemented with technology available today. It is cost effective and it is safe. In my opinion, overexpresison of DNA repair enzymes either in the nucleus or mitochondria delivered by non viral vectors is also extremely safe. This is doable, real and cheap technology.

It will not make you live forever. It will, all other things being equal, fundamentally extend lifespan and quality of life based on the degree of pathology that can be deterred due to cell death via mechanisms associated with DNA damage. Further technological advancements will in time bring about more precise ways of addressing cell function. In the meanwhile all the experimental evidence, and I draw your attention to the recent research that I reviewed in (with some tongue in cheek sociopolitical commentary) http://www.imminst.o...9&t=3808&hl=&s=, on the manifestation of the aging phenotype as a result of DNA damage. In any case, the consensus of relevant literature converges on DNA damage being the prime inducer of aging due to cell malfunction and death.

It would be good to get Aubrey to comment at this point.

#23 treonsverdery

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Posted 19 June 2004 - 03:15 AM

I think it would be a good idea if we surveyed all the mitochondria studies and listed all genes associated with their survival. Including DNA repair, antioxidant or any other mechanism. Your

are the mitochondria or ribosomes of this 40 000 year lifespan shrub different http://www.imminst.o...T&f=48&t=513&s=
sequence that shrub I tell you
[COLOR=blue]

ive wondered if micr rnas that are used to

#24

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Posted 19 June 2004 - 09:54 AM

Yes it would be a good idea. We would find that there are as yet many undiscovered mechanisms of regulation. apoptosis, etc. Your idea at looking at the 40,000 year old shrub is especially good. For instance more studies should be done on mammals such as long lived turtles.

Even more tantalizing examples are the radioresistant extremophile bacteria deinococcus radiodurans, radioresistant lepidopteran insects and cancer cells that develop radioresistance. These are examples of an extraordinary and rare ability to repair DNA damage after exposure to extremely high radiation levels that will kill other organisms.

Studies so far have shown the presence of unusual reducing proteins and increased number and type of DNA repair proteins as the mechanisms behind the radioresistance.

In all, it simply further supports the argument for enhancing innate DNA repair and protection mechanisms via gene therapy as the most viable method of antiaging.

Evidently the most vulnerable component of the cell is the mitochondrion. Considering there can be as many as 1000 in the cell at any one time and considering the apoptotic pathway is directly linked to mitochndrial function, there are correspondingly as many as 1000 "suicide" triggers active. Before I hear calls for, "lets inhibit the apoptotic pathway!", Which is actually quite easy and could be done by intrabodies or RNAi we need to appreciate that this could lead to cancer, so it is as potentially dangerous as unregulated genomic integration of exogenous DNA.

Thus target the mitochondrion with gene therapy technology designed to increase mtDNA repair and protection in addition to nuclear DNA.

Edited by prometheus1, 19 June 2004 - 10:09 AM.

#25 olaf.larsson

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Posted 21 June 2004 - 02:56 PM

The malaria parasite Plasodesma is the one that has least protein coding genes in mito. Plasmodesma it has only 3 protein coding genes in their mitochodria: Cytochrome b, Cytochrome oxidase subpart 1 and subpart 3. All other known organisms also have this three genes in their mitochodria. If you are going to see if its possible to transfer genes from mito to nucleus start with this becouse all other mito genes nature has managed to trandfer to nucleus.
If this genes are possible to transfer all genes are!

#26

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Posted 21 June 2004 - 11:16 PM

wolfram, it is possible to transfer genes to the nucleus but for reasons stated above it may not be a sound strategy. But seeing as you are so committed to this idea, there is a company, Chromos, that has developed a mammalian artificial chromosome that can incorporate additional genes. Ensuring that the 13 genes that you wish to replace also have mitochondrial targeting sequences completes the picture. Experimentally, it is very simple to run, you could test for the localization of your new genes in mitochondria by fusing a GFP sequence and examine living cells microscopically. Then induce accelerated aging by UV and see if your cells with the added chromosome stay alive longer. Of course you would still have the chance of a mutant mitochondrion taking over so you would have to suppress mtDNA from being transcribed which again is easy enough to do using siRNA. Then you fire UV at these cells and see how they go.

What do you think you would observe?

#27 manofsan

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Posted 23 June 2004 - 11:59 PM

But how can you "repair" damaged DNA thru mere enzymes? Damaged DNA represents loss of information, and how can mere enzymes restore DNA code information which has been lost? It's like me trying restore lost files on my computer, simply by doing a defrag. In cases of very minor damage it could work, but for significant damage why would it?

#28

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Posted 24 June 2004 - 09:14 AM

There is a variety of damage that the DNA molecule can experience and subsequently repair:

Per cell in nuclear DNA:

single strand breaks - most prevalent - estimated rate to be 55,000 per day
depurinations - 10,000 per day
guanine methylation - 3,000 per day
cytosine deaminations - 200 per day

oxidative damage
- 8-hydroxydeoxyguanosine - 140,000 per day
- hydroxymethyluracil - 600 per day
- thymine glycol - 300 per day
- thymidine glycol - 70 per day

Mitochondrial DNA receives about 16-fold more oxidative damage per nucleotide than nuclear DNA.

(Bernstein & Bernstein 1991)

Note that this is not an exhaustive treatise on the topic as there are more types of DNA damage. But each of the DNA damage types mentioned above are repairable by enzymes because they only involve one strand. Thus the complementary strand acts as template for the repair. Double stranded breaks are also repairable so long as they only involve ligation of each strand. On the other hand, DNA damage involving the loss of one or more complementary nucleotides from each strand is are far more rare and can only be repaired by physical recombination with another DNA molecule. In humans this is only presently possible with sperm and ova.

One must appreciate that part of the frenetic activity that is occurring in a cell at any one time is associated with ensuring the integrity of the nuclear and mitochondrial genomes as they come under attack by the metabolic by products, toxins or radiation. The balance between spontaneous DNA damage the ability of a cell to repair it will directly influence its lifespan. The premature aging condition, Werner's syndrome, occurs because of DNA repair enzyme impairment. This results in abnormally high level of DNA damage, triggering accelerated aging which in all other respects is similar to normal aging (Thomson et al 2003).

Consequently, by increasing the rate of DNA repair we should observe an decrease in rate of aging. A recent study confirms that overexpression of 2 mtDNA repair enzymes reduced oxidative stress in mitochondria.

There are many examples in nature where there are DNA repair systems with greater efficacy, particularly radioresistant bacteria. Some human tumor cell lines also show greater resistance to radiation. Cell division provides a very high quality DNA repair system. In fact cells that are unable to participate in cell division are at a considerable risk as they do not have access to this mechanism of DNA repair. Some of our most important organ cells - neurons and cardiomyocytes (heart muscle cells) tend not to divide at all.

With an increased rate of DNA repair not only is the general rate of aging being slowed but also the likelihood of cancer which is an added benefit (the assumption being that there are no oncogenes active). Admittedly this is not aging panacea since it does not take into account the problem of accumulated debris in old, non-dividing cells. However I am convinced that boosting stem cell production will take care of older cells so long as they can be encouraged to migrate to sites of apoptosis.

It remains a question as to how many years one could add to lifespan once a systemic administration of selected DNA repair enzymes for nuclear and mitochondrial DNA takes place. How cells that have already become senescent would behave. Would the reduced oxidative stress revert gene regulatory mechanisms to pre-stress state? That remains to be experimentally seen. It would be indeed a great day when we can switch senescent regulatory genes in the cell to non-senescent state in-vivo.

I am convinced, however, that as soon as such a treatment of systemic administration of additional DNA repair enzymes is made, the aging process will dramatically slow. Whilst I am not suggesting that one can become more youthful, one would age at a considerably reduced rate proportional to the degree of increase in DNA repair.

You can see why I find Aubrey de Grey's solution of moving the 13 mitochondrial genes into the nucleus so worrying. Aside from all the technical difficulties with accomplishing such a feat, the reward would be very small even if implemented exactly in the way he conceived it without any unforeseen issues with the biology (such as the self-admitted problem of protein hydrophobicity in transferring from nucleus to mitochondria). The obvious reason is that the nucleus is also prone to damage, though not as much, as I have indicated above.

Here is the shock: the treatment of systemic DNA repair enhancement can be available today.

Edited by prometheus, 24 June 2004 - 11:48 AM.

#29

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Posted 27 June 2004 - 01:40 PM

I noticed no one took the bait of the last sentence.

Anyway, here is another study that supports the mitochondrial DNA repair hypothesis for aging:

Premature ageing in mice expressing defective mitochondrial DNA polymerase

Point mutations and deletions of mitochondrial DNA (mtDNA) accumulate in a variety of tissues during ageing in humans, monkeys and rodents. These mutations are unevenly distributed and can accumulate clonally in certain cells, causing a mosaic pattern of respiratory chain deficiency in tissues such as heart, skeletal muscle and brain. In terms of the ageing process, their possible causative effects have been intensely debated because of their low abundance and purely correlative connection with ageing. We have now addressed this question experimentally by creating homozygous knock-in mice that express a proof-reading-deficient version of PolgA, the nucleus-encoded catalytic subunit of mtDNA polymerase. Here we show that the knock-in mice develop an mtDNA mutator phenotype with a threefold to fivefold increase in the levels of point mutations, as well as increased amounts of deleted mtDNA. This increase in somatic mtDNA mutations is associated with reduced lifespan and premature onset of ageing-related phenotypes such as weight loss, reduced subcutaneous fat, alopecia (hair loss), kyphosis (curvature of the spine), osteoporosis, anaemia, reduced fertility and heart enlargement. Our results thus provide a causative link between mtDNA mutations and ageing phenotypes in mammals.

Original link.

For the Methuselah Mouse people: instead of using a knock-in mouse homozygous for a faulty polymerase they could have used a knock-in with either a stronger promoter or more efficient polymerase that would have resulted in decreased mitochondrial DNA mutations. Sorry, no prize for guessing what the effect of decreased mitochondrial mutations would have on lifespan.

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#30 olaf.larsson

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Posted 28 June 2004 - 10:41 PM

Promoteus and how exactly are you going to improve DNA polymerase which is a divice far more advanced than anything a human ever have constructed?
To disrupt the function is easy but to improve it? HOW?

Here is a way to improve mito and nucleus polymerases, repair and proofreading:
The idea is that the organism doesn' t have better polymerases and repair functions then is needed for them to survive under the conditions they live so to get better copy and repair functions equilibrum must be shifted to a more harsh and mutagenic enviement.

A way to improve proofreading etc. I can think about could be to grow small organsism (yeast/drosophila/C. Elegans) or imortalized cells under many generations in diffrent mutagenic conditions. Slowly increase the dose of mutagens so that 90% of the cells/organisms die whith each insrease of the dose. After many generation there should have acculmulated mutations thar give reisistance to further mutations!!!!!!!.
Screen the cells for mutation-resistant cells for mutations in genes that are known to participate in dna replication and repair. Once you have found all beneficial mutations which protect from various mutagens in for example imortalized mouse cells you could insert them all in one mouse and get a mutagen resistent super mouse. Which hopefully will live long also.

(If you (Promotheus or someone else) make any money from my idea I want 10% and I also want to stand as co-author of your work.)