41 replies to this topic

[manofsan]

And here's some more good news:

http://www.betterhum...ID=2005-02-23-3

So here, mitochondria are being targetted for gene delivery and also protein delivery. Let's see how it works for ataxia and also aging.

Any comments on the merits of this approach and its utility?
Pro's? Con's?

Any comments on how this compares to MitoQ?
MitoQ of course mates the anti-oxidant with the large lipophilic cation, but this article talks about direct DNA replacement.

I'm more in favor of DNA replacement than in mere repair of defects or anti-oxidants. After all, no repair job can be 100%, but direct substition of a gene will bring in a perfect copy. To me, that's a better option. What do you all think?

[manofsan]

I want to further ask about epigenetic regulation in regards to mitochondrial DNA. Are mitochondrial genes epigenetically regulated? Or is the mode of gene operation in the mitochondria simpler, with them always being turned on?

The reason why I ask, is that if you're inserting fresh gene copies into mitochondria, then how do you ensure that these newly introduced copies properly integrate themselves into the normal operation of the mitochondria?

Can anyone shed more light on this for me?

Anyway, this mitochondrially-targetted protofection, if it works, seems like a winning idea.

[reason]

Rafal (from the article) is a fairly active transhumanist - you could certainly ask him directly.

http://www.fightagin...ives/000247.php
http://www.ncbi.nlm....t_uids=15377877
http://www.healthsys...l.cfm?drid=1151

Reason
Founder, Longevity Meme
reason@longevitymeme.org
http://www.longevitymeme.org

[manofsan]

Hiya Aubrey,

Indeed I will ask him then, since my curiosity's quite piqued by this stuff. But when you call him an active transhumanist, I worry about a person's objectivity. When someone is enmeshed in a cause, then there is a danger of over-optimistic reporting of results. As usual, rigorously peer-reviewed conclusions are the best ones. Wouldn't want to see another 'Cold Fusion' round of wild euphoria.

I saw his post to you from May2004, it looked intriguing. Hopefully they have made more progress towards the goal since then.

[Cyto]

Aubrey is ag24, Reason is Reason [lol]

[reason]

"Active" in transhumanist circles means "is alive, occasionally posts to mailing lists" ... he's a very sane fellow, solid scientist.

Reason
Founder, Longevity Meme
reason@longevitymeme.org
http://www.longevitymeme.org

[jaydfox]

No mention of what to do with the existing faulty DNA. Would the two sets of sub-complexes be competing to form complete complexes? Wouldn't be much of a tune-up if you can only do it once, with each successive tune-up being significantly less effective than the previous.

For that matter, what would a mitochondrion do with the excess DNA material? I suppose that when the mito divides (about every 1-3 months in humans I think), it might just have less copies to make until the DNA is diluted to normal levels. If that's the case, however, then if the mutant DNA has any replicative advantage, the tune-up will only be good for a few years at most, as clonal expansion will wipe out the "protofected" DNA.

Still, interesting questions to be answered if this group gets more funding to do lifespan studies in their "transgenic animal models". In other words, I hope they don't just test that the condition is cured and then stop the experiment, but that the condition remains cured for an extended period of time (to allay fears of clonal expansion of mutant mtDNA), leading to a somewhat normal lifespan.

[manofsan]

Hi guys,

You know, no matter how many times I hear you all mention the "mutation replication advantage" I still don't buy it. A healthy mito will be able to produce energy at optimal levels, meaning an overall healthy vigorous cell. A mito that is defective will be agglomerating the lipofuscin, and building up towards a runaway chain of unhealthy oxidative reactions. I don't see how non-optimally functioning mitochondria can out-breed optimally-functioning mitochondria. If that were somehow the case, then we'd all be living longer with our mutated mitos.

Even if you buy the "short term advantage" argument, then it still leaves the reality that over the long term the repaired or undamaged mitos would win out over the long run. Those cells which had more defective mitos would show the conventional age-related deterioration and die out, while those which had predominantly repaired/undamaged mitos would live on.

As far as eliminating faulty DNA is concerned, I'd say that this protofection thing or some other delivery vehicle should be adaptable to delivering mito-toxic agents that select for the defective phenotype of low charge gradient.

As long as we can use phenotype selection, we should be able to kill off enough mutants.

[jaydfox]

You should read de Grey's paper:
de Grey ADNJ. A proposed refinement of the mitochondrial free radical theory of aging. BioEssays 1997; 19(2):161-166. PDF link.

It's not a long paper, and it lays a pretty decent case for why defective mitos would have a selective advantage at least some of the time. The reason for the long delay before an accumulation of such damage is twofold:
1) such mutations, which have a selective advantage, must be pretty rare early in life, since their accumulation doesn't really get going until middle age.
2) Once such a mutation comes along, it still takes scores of mito division cycles to overwhelm the entire cell, and if each division takes three months, that could mean ten or twenty years' worth of delay from the initial mutation to appearance of a defective cell. If the selective advantage is only very slight, it might even take 100 or more mito cycles, meaning 25 years or more in delay.

And you would think that such cells would just die out without mitos to generate power, but de Grey lays out a decent case in another couple papers that those cells fall back on a "backup" mechanism whereby they can generate power from glucose and/or other chemicals (even fats), but at the expense of pumping massive ROS out into the body, where it oxidizes something else (presumably cholesterol, LDL, but I haven't read enough to see if this theory has panned out any new research in the last few years).

All in all, it's a heck of a lot better of a theory than the vicious cycle theory which I had been hearing much about when I first got into the anti-aging community. It's got some weak links, such as why evolution never overcame what seems like a rather trivial barrier (creating a better selection mechanism against defective mitochondria), but it explains the data and studies, or at least the data/studies he cites.

[jaydfox]

As far as eliminating faulty DNA is concerned, I'd say that this protofection thing or some other delivery vehicle should be adaptable to delivering mito-toxic agents that select for the defective phenotype of low charge gradient.

If de Grey's theory is correct, that would kill cells which have been taken over by mutant mtDNA (because they would lose even the backup option for making energy). Cells in a heterokairotic state (some mitochondria have mutant DNA, others do not) should see the most benefit. Cells that are in a homokairotic, heterochondrous state (all the mitos are the same, and have some mutant and some normal DNA) will also have all their mitos killed, and hence the cells will die; that, or none of the mitos will be killed, and the damage will continue to accumulate.

Nevertheless, there should be a good deal of benefit in many organs, especially for people in late middle age (but probably not extreme old age). de Grey raises the possibility that such an approach (i.e. killing bioenergetically deficient cells) might not be good for muscles though (and neurons, I suspect, but that's my speculation). But hormone therapy and exercise might mitigate the negative effects for muscle tissue.

[jaydfox]

Why kill the entire cell when we can just kill the mutant mito?

If there are no non-mutant mitos left in a given cell, that will kill the cell. Clonal expansion of mutant mitos leaves some cells with very few, if any, wild-type mitos. Of course, only about 1% of cells reach this state, even in extreme old age, but for certain tissues the sudden death of 1% of cells would be quite significant (the brain especially), and in muscles, while 1% of the mass might be affected, a much larger percentage of the fibers are affected (because fibers are partially mutant and partially normal), which means you could kill much more (5%, 10%, 20%?) of the muscle fibers.

All of this is predicated on de Grey's interpretations of the mosaic of COX-negative cells and the apparent clonal expansion of mutant mtDNA (I haven't read any opposing views to his, but I assume there are others which might be as valid and which might not forecast such doom and gloom).

As for cells which have a mix of wild-type and mutant DNA, this will depend on the distribution. If the cell has healthy and mutant mitos, we can kill the mutants and leave the wild-type to proliferate and rejuvenate the cell. If, however, all the mitos in the cell contain a mix of mutant and normal DNA, then either you kill the cell or you leave the mutant DNA, with no in-between option. Unless we can replace or remove the mutant DNA from individual mitos.

But, there's a good chance the percentage of cells in such states will also be very small, because of clonal expansion (they'd rapidly be overtaken by mutants), so again, perhaps only another 1% or less might be affected. There's no solid data on this number, though, or there wasn't as of a few years ago.

At any rate, it seems more likely that we'd just ablate the defective cells, rather than try to restore them, since more cells will be fully mutant than partially mutant (or so I've read, without much supporting evidence, due to lack of studies. But I'm reading papers that are a couple years old, so maybe there's new data to support or break that claim?).

[jaydfox]

If there are no non-mutant mitos left in a given cell, that will kill the cell.

Not if the treatment only targeted mutants.

Maybe you missed my point. If every mito in a cell is mutant (due to clonal expansion), then the treatment, which targets only mutants, will kill all the mitos in that cell. The cell will die without the mitos, unless de Grey is grossly wrong about the PMOR (Plasma Membrane [NADH-]OxidoReductase), which I don't think he is.

Actually, let me rephrase, as I think I figured out where we talking past each other. Manofsan suggested using a mitotoxin that is targeted specifically to mutant mitos. It was this to which I was referring. If we just try to repair mutants, then this won't be a problem.

But the reason we even brought up mitotoxins is that I was concerned that inserting new, working copies of mtDNA into mutant mitos won't help, because the mutant DNA will still be there, and will just clonally expand in a few generations again. So, we discussed the idea of killing the mutant mitos and sparing the normal ones, and round and round we went. It's a good idea, replacing mtDNA, and it's half of a solution. The other half is eliminating the mutant mtDNA. Solve that, and we've solved the problem of ROS-production being a hard limiting factor on MLSP, without allotopic expression.

Of course, what we're proposing is a periodic cleanup, and allotopic expression would be a more permanent fix (assuming we fix nuDNA problems as well), so it's not a replacement for allotopic expression, just something that can get to market a heck of a lot faster.

[manofsan]

Well, I'd wonder why nature hasn't naturally moved all mtDNA to nucleus, or is it the coding difference that's a fundamental barrier? I doubt that, since otherwise none of the other genes would have made it to nucleus. It would seem that there is some sort of natural selection pressure which is keeping that remaining mtDNA right where it is in the mitochondria. Moving it will be a nice way to test that theory, but it would seem likely that cells/mitos won't be able to survive if all the mtDNA is shifted to nucleus. Otherwise, why would they have stayed in the mito all this time?

[ag24]

See my paper in the April 2005 issue of BioEssays for an excruciatingly thorough explanation for why some genes remain mitochondrial. I expect it'll go online in a week or two.

[manofsan]

Okay, well in the meantime, I googled the definition of Autophagy:

http://users.rcn.com.../Autophagy.html

http://www.medicalne...hp?newsid=15983

So this seems a little more reasonable than apoptosis where you kill off the whole damn cell. Here, you're just digesting/recycling particular defective components. I like that -- it's more efficient.

But why does this ability decline with age? What exactly is degrading/deteriorating so that autophagic ability is declining?

Is there some kind of degradation of nuclear DNA which is at the root of the decline in autophagy with age? Explanations please?

What things have to be working properly in order for autophagy to be successful?

[manofsan]

Well, I can intuitively see how CR would increase autophagy, since it forces the cell to rely more on recycling because of lesser availability of external resources.

But I'm not sure I why defective mitos are less susceptible to autophagosis. Is it really purely a probability thing -- is there not some process that governs tagging for autophagy?

In our previous discussions on culling and replication, it was generally agreed that it's better to have culling before replication/regeneration, since that lets you replication from a purer starting population. So rather than the overkill of apoptosis, you'd want to use the autophagy for culling, followed by mitochondrial replication.

But how to link the telltale indicators of mitochondrial defects (the low charge potential of the membrane) with the tagging for autophagy?
Instead of exploiting the low charge potential for delivery of mitotoxin, can you use that low-charge potential to trigger the tagging for autophagy?

How does something get tagged for autophagy in the first place? Surely this isn't some arbitrarily random probabilistic thing?

Also, if you're saying that having more and smaller mitos is better than having fewer and larger mitos, for purposes of autophagotic envelopment -- then is this something that is age-correlated, or does that result from inheriting some favorable genes for it?

As far as lipofuscin is concerned, from previous reading on this, it's not clear to me whether this accumulating material is truly non-decomposable or whether it's really accumulating due to diminishment of normal mitochondrial ability to dispose of the stuff.

If the stuff really is a non-decomposable nuisance, perhaps nano-particles or something might have to be resorted to, for sponging the stuff up and getting rid of it. I guess autophagy wouldn't really be a useful solution in that case.

[manofsan]

Hey, I forgot to post the link to a new journal devoted to this topic:

http://www.landesbio...nals/autophagy/

Note that they are offering free subscriptions until Jan1/2006
They do mention aging research as part of the scope of discussion

[manofsan]

Well, here's my email to Dr Klionsky and his response. He's researching the role of autophagy in mitochondrial recycling:

>Hi Dr Klionsky,
>
>Sorry to bother, but I'd read an article in Medical
>News Today about your work on Autophagy, and thought I
>would ask you about it.
>
>Why does Autophagic ability decline with age? What is
>the main culprit? Is it the deterioration of
>particular organelles responsible for autophagy? Is it
>deterioration of nuclear DNA? What would be the main
>reason for the decline in this ability, which seems to
>maintain quality control in the cell.

This is really a good question but I do not know the answer.
On a molecular level it seems like it might have to do with a
decrease in the level of Beclin 1 (the homologue of Atg6).  Beclin 1
is haploinsufficient--a single copy leads to disease so a mutation in
one of the two genes is all it takes for a problem to develop.  But
that just leads me to wonder why each gene does not have a stronger
promoter so that one copy would be sufficient to maintain a critical
level of autophagy.  Perhaps there is some other problem with making
too much of this protein (too much autophagy?) but I don't know of
any studies that have examined this.

[manofsan]

So I'm wondering what would happen if Beclin1 levels were boosted either thru overexpression or by somehow injecting it.

But this still doesn't zero in on the mitochondria explicitly. Rather than boosting autophagy in general, you'd want to simply boost autophagotic destruction of bad mitos. In which case, you'd mainly want to boost the tagging of mitochondria for destruction.

Perhaps you'd want to generate a statistical profile of which organelles typically suffer the most degradation. You'd want to tweak your tagging activity to closely mirror that profile.

[kevin]

Autophagy would have to be coupled with biogenesis I would think and perhaps increasing autophagy via overexpression would not be enough to increase mitos.

[manofsan]

Well, the phrase we'd use from chemical engineering is "dynamic equilibrium" -- in this case, balancing a high rate of destruction with a high rate of replication. Perhaps you'd only want to do this in occasional spurts though, to reduce any adverse side-effects.

So whenever you find your overall mitochondrial population quality is suffering,
you initiate some kind of faster-destruction-and-replacement mode. Then once the mitochondrial quality has been restored upto acceptable levels, you go back to normal mode.

But you'd really want to focus that autophagic destructive tagging on the defective mitos, seeking out that low membrane charge potential that you've all been talking about.

[Lazarus Long]

I am going to open a separate thread on this idea but I want to interject a thought/question.

Could the mitochondria be *synthetically* manufactured utilizing nanotech to meet our specification of the optimized mito and then inserted into the cell?

This is not exactly what SENS is suggesting but it *is* an engineering alternative that takes advantage of the areas of overlap between nanotech manufacturing and genetics that could use the information we have, find some target parameters and apply these in a very different manner.

I will go open that other thread for the follow up because I am hoping to invite a few of the serious nanomedical researchers to that one for debating the perspective.

[jaydfox]

Laz, interesting you should bring this up. I have somewhat of a contradiction in my goals and values, which leads me to be personally conservative even though I support progressive approaches to problems. For example, I doubt I'll be uploading any time soon, even if it were offered.

There are many who talk about moving to a non-biological body, to decrease their chance of untimely demise due to biology's inherent fragility.

I, on the other hand, would like to remain in the flesh, but optimize that flesh. For example, you brought up nano-engineered synthetics mitos. I think that our current mitos can be engineered to effectively never accumulate DNA damage. Sure, damage will happen that goes unrepaired, but A) we can reduce the rate of net (unrepaired) damage, B) we can increase turnover and optimize the tagging process such that, effectively, no mtDNA mutations will accumulate, and those that do occur should be eliminated within a small number of mito biogenesis cycles.

(For Prometheus, I should point out that until we understand the tagging process, increased turnover isn't guaranteed to fix the problem, and there's a small chance it might make it worse by increasing the rate of clonal expansion... I strongly agree that turnover is important, but I think the tagging mechanism is more important than the turnover rate. Alas, only experiments will tell for sure...)

Similarly for the other types of damage, I strongly believe that we'll get to the point this century where we can effectively prevent all accumulation of all relevant damage (leading to pathology, age-correlated or otherwise).

On top of that, I believe we'll increase the efficiency and effectiveness of many of our systems, leading to for example increased strength and endurance, controllable pain systems (since pain has its usefulness, I wouldn't eliminate it, but put it in our control; genetic tests have shown that some people are natural wimps when it comes to pain, due to neurotransmitter levels, while others are natural stoics. I suspect I'm one of those genetic wimps, based on my tolerance levels for just about any kind of pain).

To this, I would add limited types of augmentation, such as a BMI for interfacing with the net (I'm thinking along the lines of neural nanonics, for Peter F. Hamilton fans...), as well as possibly artificial muscles for increased strength, and fancy bone materials for increased strength and toughness and reduced brittleness. But as for my core biology, I suspect I'll remain biological at least into the next century.


But, I strongly want to see the synthetic versions pursued, and I'm interested in how uploading will work, or at least a full body prosthesis run by a person's transplanted brain and spinal cord. While I'm hesitant to upload now (based on my understanding of the relevant science), I am also confident that the problem will be solved in some capacity suitable enough for my liking (if for no other reason than to market this solution to holdouts like me who want it, but have reservations).

Anyway, a little off topic, but your comment about nano-engineering (I like the term geneering, again from Hamilton's work...) synthetic mitos got me thinking about it more...