Fight Aging!

This picture in the link below nicely illustrates how the nuclear and mitochondrial ribosomes cooperatively construct the mito double membrane
https://pbs.twimg.com/media/DCw4bqxWsAAmV0V.jpg

Leo Nijtmans recently cracked the construction code complex I. Unfortunately hard allotopic expression (all mito genes or protein) is a pipe dream, mitochondrial aren't built like that. I asked Leo to present the recent ATP6-ATP8 paper published by the SENS folks at last weeks EuroMit conference to his many sceptical collegues because I think the paper is great work and shouldn;t be ignored by them. Leo got back to me with many criticisms including a few things that should probably be adressed or otherwise further clarified which can be summarized as follows
1. What are the specific tricks they did to improve the allotopic expression (compared to earlier work).
2. Why did they only use the ATP8 mutant for the allotropic (stable?). Why not also wt cells, rho zero, or NARP (8993) cybrids?
3. I think that it would be elegant to use classical import assays to show the import of the allotopic gene. Perhaps also Prot K treatment to show that the proteins are really imported.
4. Blue native gels show co-migration with HMW complexes (not necessarily CV). It would have been good if they had also used other approaches to show the incorporation of the allotopic protein. e.g co-IP or complexome profiling could be informative.
5. I was also surprised that there was no low molecular weight species of A8 (unincorporated in the complex) in the BN gels.
6. Also why does the stable expressed allotopic gene does not show a unprocessed species (uncleaved presequence).

Some of the good news is that the SENS Research foundation won't have to worry about researching allotopic expression of the ND1 gene as Genesight will be pursuing this commercially:

"Because ND4 mutations account for more than 75% of the LHON population in North America and Europe, we chose to first focus on this specific mutation. We have demonstrated the feasibility of using the MTS technology platform for the treatment of LHON due to the ND4 gene mutation in animal studies. We plan to use our MTS technology platform to address other LHON mutations and have already initiated a research program for our next potential product candidate, GS011, which targets the ND1 gene mutation."

@John Hewitt - I don't follow your logic from presenting the fact that a Dutch researcher Leo Nijtmans saying that he would like to see a few more details determined to be 100% sure that the SENS paper on ATP6 and ATP8 is not an artifact... to then somehow concluding that expressing all mitochondrially encoded proteins allotopically is a 'pipe dream'?

Do you have any facts or knowledge that you didn't mention to back up your assertion?

@john hewitt You can ask that directly to them: http://www.sens.org/contact-us They always replied to me in the same day.

@Antonio, already did, were much less than appreciative of my comments. The top brass their delegated my inquiries, threatened me against further talking daring to inform them, and then began spouting directives about how to even address them. I like what they are doing, however the rest of the field seems unreceptive, I quote: 'Also most of the people I spoke to (at Euromit) had enough of the offensive attitude the allotopic adepts had with everyone criticizing their work. There was definitely not a lot of attention to this work.'

@Jim, if you've been reading anything about mitochondria for the last 5-10 years, as I'm sure you have, you are already well aware that the elemental units of both you and me are our mitochondria. The fact that theystill translate their own proteins is not a sideshow of eukaryotic life, it is the main event. You can read for yourself if still unclear, for example, here (later published in the journal 'Inference') http://neurowritings.blogspot.com/2016/09/the-future-of-neuroscience.html

Are you saying a SENS was delegating your inquiries... tho whom?!
They have one communications guy and then there are the researchers and Aubrey. There is no one to delegate your questions to - they can only go up the chain of command.

Or are you talking about the people at the conference?
I can imagine those being unperceptive. Synthetic biology and genetic engineering are unfortunately not popular topics in Europe. Or anywhere in the West currently. As sad as it is, this we live in a society of neo-Ludites and that includes our scientists as well.

@anonymouse, what I am saying is this,
When we all learned that free radical generation was not a bug but rather feature, a critical regulator of all things mitochondrial, what did the SENS consortium do? Did they acknowledge this new information, absorb it, and modify their outputs accordingly, or did they double down in the allotopic war on radicals?
'Nearly all of your analogies and criticisms are invalid, your analysis of the issues is pitifully broken' Aubrey to me.
Has SENS, and for that matter the academic 'neo-luddite' researchers (who incidentally have been great at figuring out how mitoribosomes work hand-in-hand with nuclear ribosomes to sequentially piece together each subunit of each complex), managed to draw any distinction between allotopically expressing one gene vs allopotopically them ALL? For that matter, have they managed to draw any distinction between 'replacing mtDNA' vs 'duplicating mtDNA into the nucleus" in both 'soft AE' ( which I am 100% certain can be made to work to some unregulated degree for one gene to help some very sick patient), and 'hard AE', or total nuclear backup copies, which will never work for the simple reason that life as we know it is the mito-nuclear hybrid dynamic.
The answer is no, they have not, furthermore, I will tell you why you are not making these distinctions: because as soon as you get regulated nuclear full alloptopic bliss your mitochondria are done, they will not persist many generations. DUPLICATING mtDNA in the nucleus is REPLACING mtDNA in the steady state.

That building respiratory complexes are actually like building fine swiss watches not making a bark and leather gumbo in an Irish famine pot; that mitochondria will lose their powers more quickly and surely than a blind cave fish lose his eyes and melanin genes when put out of the sun's influence; and that an American auto industry fully replaced by alloptopic expressed cars from Japan would kill off the entire industrial complex and quickly America itself -- all analogies which Aubrey says are ignorant analogies, are in fact perfect metaphors; I first most clearly articulated it, and have the balls to say it again.
The reason I linked above to the neuroscience based article is because when you understand that neurons are devices custom built for sorting mitochondria, and furthermore, that axons and dendrites first evolved when the machinery for ejecting and transmitting mitochondria across cells (transexudation) was in place, you can begin to appreciate that the road ahead for mitochondrial modificationis not in reducing them to mitoplastic shells, but rather more likely the reverse, ie. adding more genes back to the mitochondria. Look what happens with the nervous system and mitochondria in cancer: https://medicalxpress.com/news/2017-03-nervous-tumor-growth.html

@john hewitt: The SRF target is mitochondrial mutations period; doesn't matter how they happen. That's the strength of allotopic expression as an approach, it is agnostic of cause of mutational damage and the dynamics of mutational replication.

I'm not sold on the idea that allotopic expression in adults will cause some form of decline in mitochondria; not clear whether you are talking about impact to dynamics, or microevolution, or? But it should be a fairly straightforward process to work through that in yeast, given the basic tools. Run a yeast lineage with allotopic expression for a few thousand generations and see what happens. Sounds like a useful experiment for someone to run; raise the funds and go do it if you feel strongly on the topic.

"you are already well aware that the elemental units of both you and me are our mitochondria"

What???

"The fact that theystill translate their own proteins is not a sideshow of eukaryotic life, it is the main event"

Seriously, what??? That fact is simply due to the mitochondria's origin as symbiotic prokaryotes.

"The top brass their delegated my inquiries, threatened me against further talking daring to inform them"

Who delegated to who? And who threatened about informing to who? I can't understand what you are talking about.

"mitochondria will lose their powers more quickly and surely than a blind cave fish lose his eyes and melanin genes when put out of the sun's influence"

99% of the mitochondria's proteins are already encoded in the nucleus, so that catastrophic scenario has not much sense. At least, certainly you need MUCH MORE solid evidence to prove that claim.

@Reason That's why I support the SENS effort to try to get allotopic expression, it should work a bit for someone very sick people with mutants. What Iam arguing is the extension to this ludicrously uncritical mantra of allwe need to do is copy all the genes to the nucleus, which I think some are already now wisely denying was the expressed sentiment.

I shouldn;t say 'threatened'eith, how the heck to you edit comments here, the SENS people merely strongly expressed the fact that they think I am disrespectful and should never under any terms ever even thinjk of writing to them again.

Who did you talked to?

@antonio, the experiments to test this in yeast, as Reason suggests are wise, but they are for you to do to convince yourself, not for me, I have no such need. While you are at it, see if the mitos can still do their one essential job in all eukaryotes, ie, synthesizing iron sulfur clusters, and of course also check on other things like fatty acid processing and steroidogenesis in those guys.

Well that is an interesting theory and you are free to pursue it just like Aubrey is free to pursue what he believes in.

The point of allotopic expression of mitochondria is to provide a backup, not to replace the function of the mitochnodria.
You are making quite the assumption in proposing we'll lose our mitochondria if the mitochondria doesn't retain any genes.
But even if you are correct there are more than enough eukaryotes with only rudimentary mitochondria (as few as 3 genes remaining) and they don't seem to be bothered much by it.
Neither are the Monocercomonoides found last year to lack any mitochondria bothered by it all that much as well.

The only way to get proof is to do the science. Theories only go so far.

@john hewitt

Can you answer my question? You are accussing an unspecified person of an organization of unpolite/unfair behaviour. If you don't specify who he/she is, that person or the organization can't defend themselves. It's like throwing the stone and hiding the hand.

I asked technical questions several times to SRF and obtained a thoughtful and detailed answer from Michael Rae in the same day. I also asked about fundraising and merchandising and obtained a reply in the same day by Jerri Barrett.

Again, I am not trying to burst anyone's allotopic bubble, just wake a few folks up as to what we are talking about here, namely, the wholesale unemployment of mitochondria. To the extent that mitochondria are still semi-autonomous (if transient) machines as we know they are to some extent by the way the body treats them, (dangling them as immunogenic lures in the bloodstream to let their bacterial formylated peptides, unmethylated CpG islands in the mtDNA, and cardiolipin membrane products attract blood born agents of inflammation like mast cells, platelets which also excrete mitochondria whole and inside vesicles), then seriously folks, what do you expect will become of them once we put them on the nuclear dole?

What we should be spending time on is modifying the mitochondria themselves using the new methods we have, like figuring out how to do the necessary end joining and DNA repairs that techniques like CRISPR-casetc. Would require (since mitos don't have much endogenous repair), and particularly transplanting them whole like reason posted a few days ago that the CHinese are doing.
Unfortunately the China group won't respond when I emailed them to say that their 2 hour immunogenicity test is woefully inadequate to properly evaluate for the above-mentioned immunogenicity factors

@antonio, sorry yes, matt O'Conner from the ATP6-8 paper unfortunately could offer me nothing of substance scientifically when I asked him several questions. After several days of getting proper permission to be interviewed by me I gave up and talked directly to Aubrey. He responded at length and as I said, basically claimed I understand nothing and should talk to Mike. Mike was only interested in how I should properly address him and unfortunately that is all I got. He seems quite knowledgeable and I asked him several questionsbut he is still stuck on that issue.

Ok, so he replied to you several times and finally got tired of arguing with someone that hasn't enough knowledge. I would not consider that unpolite.

About the burden of proof. It's you who made the extraordinary claim, so it's you who need to provide the proof, nor the other people. As I said, 99% of mitochondrial proteome is already encoded in the nucleus. As Anonymoose said, other eucharyotes encode even more of the proteome without any problem. So it's you who is making the claim that is a priori highly unlikely (but not impossible) so it's you who need to justify it. And, since it's you who sustain the extraordinary claim, it's quite unpolite to say that the rest of the people reject it because they are just repeating some kind of mantra and being self-delussional.

@antonio, ah geez, we got a climate change 99% settled science fanatic here itseems.
I never claimed to have all the knowledge, so don't beat me up for that mon frer your most high excellency, but I have enough sense to understand how mitochondria are built and function based on what we now know from the current literature. If you and SENS radicals want to ignore it and ban me go ahead, if not then show some respect and lets collective admit the points I mentioned above and figure out a strategy for mitochondria in normal but aging people.

@johnhewitt, I'm pretty firmly with Aubrey on this one. I think your understanding of the role and position of mitochondria in the cell is confused; comments like:

"neurons are devices custom built for sorting mitochondria, and furthermore, that axons and dendrites first evolved when the machinery for ejecting and transmitting mitochondria across cells (transexudation) was in place"

expose critical misunderstanding of multiple topics in a single fell swoop. Additionally, the analogies that you claim are "perfect metaphors" are neither good metaphors nor good analogies.

If you've been persistent in trying to promote these views, I can see why you would run into less than polite resistance.

@Dennis Thanks for taking the time to share your insight and earnestly share your advice.
You are absolutely correct to note those ideas have never been seen in science, and neither you nor anyone else is likely to accept them as blasted here. I can only suggest you read the link to the neuro post I put above and then tell me what you think after that.

@Dennis, sorry, my interest here is to solve this mitochondria issue, but yest the neuro stuff is critical. That article was highly edited and published and then reviewed fairly critically recently on Hacker news where a lot of people (likely folks who don't know much of what I would call hard neuroscience like, I would guess Aubrey and maybe even yourself?) expressed much the same as you just did, ie, several 'quakery' comments, but alas, if you read, some rather favoreable and optimistic.

@ Dennis here: https://news.ycombinator.com/item?id=13088772

I can tell you what I've been told when I've proposed mitochondrial transplantation as an alternative therapy - the mutant mitochondria in old cells out-competes normal mitochondria - not unlike the mutant cells from the previous article posted today. So mitochondrial transplantation will not help as long as that is the case.

So Aubrey is sticking with allotopic expression because that should in theory allow the cell to properly react to it's damaged mitochondria - apoptosis - instead of the cell continuing to operate which has negative effects for all cells in the vicinity.

I think this is why Aubrey disagreed with you strongly - you seem to have the wrong idea about MitoSENS - all it's meant to do is ensure the cell will enter apoptosis as it should even if the mitochondria is damaged. There really is no alternative to it the way I see it, since apoptosis is driven by the mitochondria - if the mitochondria is damaged the cell will malfunction.

@Antonio, correct I accept that I am making extraordinary claims, however that fact does not make me the more impolite party here, and certainly I have made none more extraordinary then the claim you can put all the genes in the nucleus and still have a mitochondria.

Not one of you wants to do the thought calculus here. Okay, so I will; so you express ATP6 & ATP8 pretty good allotopically and your mitos still translate the other guys more or less reliably. But now maybe you throw in a little allotopic CYTB for good measure, and maybe some ND4, and since you are at it maybe ND4L since it is nearly the same and you might even do a one off with alternative splicing. After much tweaking you still get some endogenous translation and subunit assembly but what exactly do you have? Incrementaly, as you try to add the rest you have mitochondria with the full translation machinery to express what, one single stubborn protein that won't get itself back into the mitochondria after nuclear expression and gett put into the correct subunit at the right time in the right place and in theright membrane orientation. That mitochondria ain't work, the cell won't it. It is beyond preposterous.

Now mito translation is a special beast altogether, codons and tRNAs are totally optimized for just a few proteins. There are not so much erros, as there are heteroplasmic substitutions my friends, it isquite different from the nuclear where the ration of tRNAs to genes is much lower. The mito tRNAs actually do all sorts of things in the mitochondria. When you remove that last mito gene what do you want to do the tRNA and the 2 rRNAs? You certainly don't need them just for protein translation. For that matter, you don't need MANY of the 1500 natural mito nuclear-encoded genes that are involved in translation regulation. Now what kind of mitochondria, or for that matter cell do you have? Sorry my friends.

@anonymoose, yes, you and others have been told a lot of things, but the reality isn't simply old experiments showing some clonal expansion of bad acting mitos with major deletions. Even now I am sure the SENS folks must admit that. What I can do is post some more modern literature showing what mitos in cells get up to. For example look at this,

Asymmetric apportioning of aged mitochondria between daughter cells is required for stemness
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4405120/

and stuff like this:

How to Modify Mitochondria
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4864607/

Platelets release mitochondria serving as substrate for bactericidal group IIA-secreted phospholipase A2 to promote inflammation https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4260364/
Stimulated Human Mast Cells Secrete Mitochondrial Components That Have Autocrine and Paracrine Inflammatory Actions https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3524249/

http://onlinelibrary.wiley.com/doi/10.1002/bies.201600235/full
Targeting the SASP to combat ageing: Mitochondria as possible intracellular allies?

Mesenchymal stem cells use extracellular vesicles to outsource mitophagy and shuttle microRNAs
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4598952/

"ah geez, we got a climate change 99% settled science fanatic here itseems. I never claimed to have all the knowledge, so don't beat me up for that mon frer your most high excellency, but I have enough sense to understand how mitochondria are built and function based on what we now know from the current literature. If you and SENS radicals want to ignore it and ban me go ahead, if not then show some respect and lets collective admit the points I mentioned above and figure out a strategy for mitochondria in normal but aging people."

Now I perfectly understand why people at SRF got tired of you.

As I said, the name of the game is selection as you intuited here, but it isn't isolated cells succumbing to mutant mitos. You have to look at the new picture where for good and bad open borders reign. Look at it if you don't believe me, look its right here, in every organ, and every cell,precise vectors of mitochondrial transmission: 'Mitochondria have no boundaries' https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5039171/

What others haven't appreciated yet is that the central selector is the nervous system. Polarized neurons, infact polarized from dendriteto axon by mitochondria themselves, linkup in circuits ofmitochondrial transmission as much and likely more so theen they do for raw electrical considerations.

What is SRF?

The question then is not how to allotopically obliterate mitochondria in a flase 'completion' of natural evolution that is almost 99% done -- The nucleus is merely a convenient RAM stash for our mitos. For all the nonsense talk of the microbiome, the irony is that the real microbiome is already front and center, our mitochondria -- but rather not just how to modify and add favorite genes back to mitos, rather where to put the new mitochondria into the bodies circuits, and where to tapthem off. Now as mentioned the blood injection Reason posted has pre-existing flaws and is highluy nonspecific. Some of the papers I just referenced have actually done similar raw injections to mouse tail veins well before the Chinese guys. Rather, we need to put them into their natural distribution system, the nervous system, and perhaps the best place to start is just put them in the ventricles and then do the same slice and dice the other authors did to see where they go.

Putting some more relevant science here, there is this 2016 review of mitochondrial deletions that references a number of papers from the past couple of years.

http://dx.doi.org/10.1016/j.cmet.2016.06.024

Investigations of mitochondrial deletions are very much an ongoing topic, and clonal expansion of deletions is, like it or not, a consensus vision of what is taking place in mitochondria. Similarly, allotopic expression of ND4 by Gensight appears to work by all measures of the situation so far, from cell studies through to quality of sight regained by the patients.

@Reason Good stuff. Initially the Gensight people offered to answer some critical questions I posed to them, but alas the scientists clammed up when I asked what I thought were basic questions.
The issue I think is how dowe best measure the heteroplasmy we know must exist throught the body, and how do we make valuejudgements on that. Clearly there is a vast and old literature describing mitochondrial differentiation to various morphologies suited to steroidogeneis with tubular cristae, fatty acid degredation, thermogeneis etc, and certainly slimmed down mitochondria with higher or lower membrane potential, radical load, and electron transport paths and directions, would be more or less well suited to specific tasks.

As far as this ND4 business, Nijtman's recent exposition of Complex I assembly sequence suggests that just getting the protein to reach the mitos, TIM-TOM its way in, get folded, and membrane inserted is the easy part, rather you must ingratiate yourself with the compex in the right way. First ND4-containing Po-a sub-subuints are constructed in a precise order, then they are amalgamated with the other sub-subunits.

Someother peripheral considerations for someone wanting to make allotopic expression a routine teechnology
1) Assuming regular old mitochondria will be maintained normally astride nuclear encoded genes (rather than trying to delete or modify the endogenous mtDNA encoded proteins) wouldn't it be wise to restrict this tech (presumably deployed in somewhere like China orBelize) to fully developed humans, men or post-menopausal, non-mitochondria-donating women so we don't try have a situation of trying to develope embryos that lack normal regulation of mitochondrial protein disbursement? Where would be the best place(s) in the nuclear genome to stick extra copies and also how many copies. (I recently talked to Nick Lane regarding the open question of respiratory supercomplexes and stoichiometry and he says he is working on experiments to address that and will let me know later this year).

2) I speculated previously in an article that in some cases, despite coding-recoding issues, mitochondrial ribosomes might translate nuclear RNAs. I have since stumbled upon at least one paper showing that this is in deed the case, at least in some sperm. What does this say about our understanding of the big picture and perhaps the issue of mitochondrially resident or at least associated nuclear ribosomes ?
See http://genesdev.cshlp.org/content/20/4/411.full.pdf

3) Whileperhaps a peripheral issue, if we are correct in assuming that mitochondria are actually the primary targets of radiation damage, particularly in standard cancer therapy, then we should note that these therapies may be considerable less effective, see, http://www.sciencedirect.com/science/article/pii/S0891584913003687

4) 4) Given free range on where to put the mitogenes in the nucleus, would it be prudent to expose them to selection on the female chromsome phase in a place where there are no recombination or retrovirus signatures, ie, most notably, to put them on the non-inactivated X chromosome?

@johnhewitt, what I'm seeing is a lot of assumptions about how things work, assumptions which aren't backed up by anything except two articles which you also wrote. That isn't sufficient evidence for me to ascribe to your point of view.

If you really want to show your ideas correct, if you really want to pursue this approach, here's what I'd do:

1) make a new, specific, testable prediction based on your ideas
2) design an experiment to test it
3) publish a prediction and an experimental design to test that prediction
4) get some lab equipment and run the experiment
5) publish the results and the actual experiment setup
6) ask others to review it
7) repeat, integrating feedback from review if it's available

That's what I'd do, because that's what works. Simply asserting something and writing the equivalent of blog posts isn't going to be sufficient, no matter how right it sounds in your head.

Thanks again @Dennis
You can call them blog posts, that is fine by me. I got $5000 to publish the first article which is decent for a blog. Over the last 2 or 3 years I published several hundred articles on these sites below, for cash too, mostly very technical neuroscience and neurology issues, but several more regarding mitochondria, which sounds better than the academics who PAY the journal to publish I would think and publish just a handful articles. http://neurowritings.blogspot.com/
You can do whatever experiements you want, I am busy doing this, something which is sorely needed it seems.

@Dennis I am not so sure science actually works that way.
Aubrey is certainly a great man, and pioneer, and deserves the success he has and more.
I think where I went wrong was I thought I had done your points 1,2,3, and tried to go right to point 4, rented a 6,000 sq ft shop, furnished and set up a lab, and funded it by making research devices universites all over the NorthEast, basically machining, electronic design, and integration. After 10 years of sweat, ultimatealy I hadlittle timefor experiments, basically failed and went back to your point 1, a little wiser.
Science doesn't advance by the scientific method. It advances by doing what I just did here today.
Someone comes along and puts tired ideas to rest, and replaces them with completely new, bold, and in any event probably most assuredly right, and definitely non-peer-reviewable. Why would I even try to send them to an academic review journal of morons who simply say that is not the consensus.
So there I am, as you say, exposed.

@anonymooose
yes, 3 genes in mitochondria can be enough, or even zero genes and zero mitochondria, if you want to go through life as a Monocercomonoides. As I mentioned above, synthesis of FeS clusters is the only proven essential reason to be for mitochondria across eukaryotes, unless, and as you point out Monocercomonoides is quite the exception, you actually manage to pull of the entire FeS chain outide of mitochondria by adopting a unique lifestyle and completly co-opting bacterial horizontal gene transfer for the FeS synthesis like Monocercomonoides incredibly managed to do.

Hi John !

Just my 2 cents. I think that you have immense knowledge of the mitochondria, biology and I can at least imagine your frustration of trying to show your points; although, has some have said it is better to go with a less 'me or the highway' approach because it can backfire and doors will close then. I'm not saying you shouldn't believe in what you believe (it's believers,
whom became makers/changers, that changed the world because they believed in what they believe (just like AdG, there are AdGs in the making everywhere; despite everyone not believing it).
Sometimes you must take the road alone. Also, as said, we emitt hypothese and theorize (conjecture) but nothing is 100% sure. The paper results help to lend credibility to making these hypotheses but nothing is certain until you or someone decides to take
on those words; and make it happen (which I see you tried to do by making lab experiments). It makes me think of many recent reserach on CR (calorie Restriction), it was very
great to have more research (so we understand it better); The Problem : they don't act (up) on it - either because of lack of funds or funds limiting the research to only being 'informative' but non-action (on the results found in the study). For example, a CR study finding out that mTOR has a key role - ok - so, then, what ?... The Buck Stops There. Many recent studies are like that. Dead Ends. Although, SENS is quite different in the sense that they are a taking the 'action' approach (of doing something about it and building therapies that will rejuvenate body - instead of doing 'more' research - for - research(ing)). As some have said, we don't know Everything in the body and at cell level - but we know sufficiently enough to (at least) try to Do something about it - and cure Death once and for all.
Not just diseases - but Death (instrinsic aging caused).

I think mitochondrial allotopic expression is another 'weapon' (if you will) to the weaponstash of SENS; and if it can at least curb mutation diseases (which is nearly %100 sure what it will do, I believe) like mitochondrial pathologies (some have more or less mutations) :
MELAS, Diabetes, Atherosclerosis, Progeria HGPS/Werner, Marfan, Trisomy 21 Down's Syndrome (althought that's more nuclear chrosome problem),...
then it is worth it.

For sure, that improving mitochondrial function should have great impact on health and healthspan - how much of intrinsic aging will be impacted,
I'm not sure but for example Centenarians (one study showed) have a fusion of half-dead mitochondrias (mitofusion, not mitofission); they 'stick together' in a huge mass and thus, maintain they adequate ATP levels require to energize the cell. This mitofused 'grouping' of
badly functioning mitochondrais happens only in individuals who live above a 100 years old. It is compensation for the failing mitochondrias in elderly (which makes for more and more reduced energy levels from the mitochondria Complexes failing to do proper respiration from
ROS damage to the Complexes/accumulation of deletions/lesions in mitochondrial DNA).

But then, why do these people Still die at 122 (Maximum human lifespan) - why don't they go on to live 500 years with 'grouped' mitochondrias... clearly, there is more to the picture than meets the proverbial eye and 'physical' eye (pun intended).
I think : it is really a big balance between MaaaNNNyyyYYY factors, from mitochondrias, to cell cycle mechanism (replicative senescence), to mTOR, to nuclear damage (telomeres), nuclear epimutations, nuclear epigenetics (global demethylation), chromosomal decompaction and histone loss, junk (lysosome lipofuscin)/AGEs/crosslinks/ceroid/and waht have you...to mitochondrial dynamics (such as iron fenton reaction causing h2o2/ROS) that damage the mitomembrane bilayer (inner membrane/outer membrane).

I sometimes think that mitochondrias could be the source of extreme lifespan - but only in animals that have short lives : One study in C.elegans demonstrated that mitchondrial lipidome was remodelled by creating a virtually insulin deficient nematode
that grew extremely slowly (reduction of growth, growth is mTOR/IGF, which dictate metabolism, telomere attrition, nutrient sensing and senescence entry...)).
These nematodes lived 200+ days 10fold longer (the C.elegans lifespan is 20 days) and were 'retarded/sluggish/frail - but lived extremely long in half-asleep state'; this mitochondrial lipidome showed that the phospholipid were reordered and the composition changed : Fatty acids were changed in phosphatidylethanolamine and phosphotidylcholine (the major ones).
There was a clear connection between longevity and mitochondrial lipidome changes (in fact, one study said that mitochondrias faced 'selection' pressure on specific genes : lipidomic genes that regulate membrane viscosity and fluidity); that 's becayuse mitochondrias's complex I-V produce the ROS at the membrane potential, which catalyze the lipoperoxidation process (which is magnified) and creates a chain of perodixed-chains (peroxidation of membrane polyunsaturates fatty acids)). This research demonstrated that the PI (peroxidizability index) in mitochondrial membranes equals the MLSP in certain animals' organs - not all of them though (such as the brain) (especially, mammals'livers and hearts). Evolution 'played' in the lipids (changing for monounsaturates vs polyunsatures to treduce polyunsaturization peroxidation susceptbility) - instead of playing in the Complex I-V...it could have, but it didn't (it could have used the AO (alternative Oxidase) to create the necessary ATP, instead of counting on electron-to-Complex I in ETC). One study showed that when drosophila were switched from Complex Respiration in ETC to AO in ETC, they had increased lifespan - that was because there was less ROS produced and less electron loss/leakage - for electron forward flow/transport and backflow was decoupled from Complex I; as such mitochondria Complex I and III did not produce ROS that damages the mitochondria's membranes and mtDNA; for it completely circumvented their usage).

But then other studies do not see much changes in lifespan by improvement in mitochondrial function; that means mitochondria have a strong percentage 'say' in the maximum lifespan; but in long-lived animals (whom are already 'fine-tuned' by evolution) the
benefits of mitochondrias become lesser). That's why centenarians don'T go live above 122 - even if they have strong mitochondrial changes such as mitofusion to support the grouping/fusing of bad mitos into a big group that 'still' can make it/produce enough ATP.
It's also funny because I've seen studies where the drosophila/fruit fly had Less ATP and Lived Longer;
so it's not all 1:1 thing; there are definately shades of grey. Many of the changes we see with aging, are not always deleterious but 'Adaptive' Compensatory Feedback mecanisms to the 'aging' organism. It's just another 'phenotype', not necessarily worse, just different (aka. 'aged').
With that said, that's what scares me a bit : if we are so 'optimized' in our genes; what more could we gain and perhaps, that damage is actualyl Very Small in humans and so small that it'S like trying to find a needle in a haystack; it could take forever.
When I say 'so small' I mean in the sense that we are an animal whom lives up to 122 YEARS,, that's pretty impressive when we should technically be dead by 25-50 in terms of mass/body size/resting metabolic rate, etc. Evolution found ways : it tinkered our phosholipids in our mitochondrial membranes (towards peroxidation resistance and thus, stop damage (such as MDA/TBARS, Carbonyls, mitochondrial and nuclear DNA 8-oxo-dG lesions, SSBs/DSBs, CML/AGEs, HbA1c, prostane, etc...), what else : it slowed our onset of 'reproduction' (just like in slow growth animals whom have protracted brain development/plasticizing 'remolding' brain over many years until puberty/adult) thus slowed mTOR/replicative senescence/loss of Telomeres - thus protecting chromosomal integrity (chromosome end termini telomeric DNAin the nucleus).
Tehre are many other 'tricks' of the trade.

The point I'm trying to say is taht allotopic expression could turn out incredibly important or much more moot(er). I definately hope it's the former. It's only the combination of the 7 or more therapies (most likely we'll need Way more than 7 ones, I could bet on that) that we will
allow a human near-True Reversal of Aging Bio Rejuvenation. Even then, I hold my optimism when AdG did not say the Price of these therapies (at first)
when they come out (we suppose one day, in the next 20-25 years that's being optimistic it could take longer)); Not just that, the 7 therapies do not cover everything (some things are not even in those therapies) - but they do Cover A Lot, so it could end up Being Enough for Robust Rejuvenation (I hold a slight 'hold' because I'm taking certain 'bottlenecks' could end up not allowing humans to live above 120-130 - even with SENS - that said, if we live 120 healthy that's a pretty good start and we'll take it). Some of these bottlenecks could be other things : such epigenetics (global DNA demethylation/epigenetic transcriptional drifting), Redox (that is something very important I been banging a long time), telomere loss (we could obviate that, not sure how now they wish to stop telomerase altogether to avoid cancer danger),
loss of histones/chrosomome decompaction (loss of Lamin function/Lamina which causes Progeria), replicative senesce/Haayflick limit, I'm forgetting some more, it just keeps on piling. So can we 'fairly' say that everything will be cured with a few therapies, probably not. But, it is a start and others will join in/the more people talk about it (the less Ludites we will be/sound to others).

Just a 2 cent.

@ CANanonymity - I'm optimistic the first 7 SENS therapies will give us enough leeway to discover the next problems and fix them in time, as stated in Aubrey's LEV paper.

In the meantime, yes Humans have surprisingly long lives compared to many animals, but there are some that do a better job - look at crocodiles. They don't stop growing so don't have the problems with spontaneous senescence caused by MTOR signaling to quiescent cells, don't have issues with junk build up (can continue to dilute it), and I expect that the hayflick limit isn't an issue because their stem cells continue to replace exhausted proliferating tissues without going senescent. Not suggesting becoming a giant is the solution to aging in humans, but certainly much to learn here.

@CANanonymity Thanks for the encouraging comments, your analysis of the lipid situation seem quite insightful. The one ref Iput above, this one, https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4260364/ does a nice job on some of those issues, particularly regarding how the body still treats vascular and extracellular systemic mitos as bacterial invaders; 'hydrolysis of the mitochondrial membrane by sPLA2-IIA yields inflammatory mediators (ie, lysophospholipids, fatty acids, and mtDNA) that promote leukocyte activation.'

also again thanks to @Reason for consistenting providing the great content, and knowledgeable commenters that make this forum great, glad I found it.

2 observations:
1 - Doesn't anybody have a full name, what's going on?
2 - If there is a simple setting to toggle to add the ability to edit comments, even like phys.org does, for a limited time, like say 20 minutes, or longer like Lubos Motl's excellent physics blog 'The Reference Frame blog' that would add some much needed professionalism, especially me, althoughlikely some others as well, who in the interest of promptness are acustomered to firing off a reply and then going back immediately and correcting the grammer.

As folks have already evidently deduced for themselves, John's approach to us was less than a lesson in scholarly collegiality or mastery of How to Win Friends and Influence People. It's doubly unfortunate that he would wait until after going through several exasperating rounds with both myself and Dr. de Grey before passing on some legitimate questions from his interlocultor "Leo" about SENS Research Foundation's successful allotopic expression report, by which time bridges were sufficiently scorched that no one wanted to set foot on them again.

For the record, here are Dr. O'Connor's replies to the questions John forwarded to us from Leo, which are entirely reasonable and evince familiarity with the underlying laboratory methods:

Leo asked: What are the specific tricks they did to improve the allotopic expression (compared to earlier work).

Dr. O'Connor replies: Not much other than a lot of trial and error of different MTSes, promoters, and plasmid backbones. We don't know why so many attempts (on our part and others') failed and these succeeded. We don't know why some promoters, MTSes, and delivery methods failed. So far we've only had success with the CMV promoter and a dox inducible promoter (the later not shown in the manuscript). We do have a new trick coming soon a journal near you.

Leo asked: Why did they only use the ATP8 mutant for the allotopic (stable?). Why not also wt cells, rho zero, or NARP (8993) cybrids?

Dr. O'Connor replies: We wanted to focus on these particular cells for this story to clearly demonstrate the rescue of a complete null without any complications from partial loss of function or competition with endogenous protein. The ATP6 deficiency in these cells obviously complicated that plan, but we did express exogenous ATP6 along with the endogenous ATP6 and saw an incremental affect. We have done ATP8 in WT cells many times and it works, just didn't seem relavent to this publication. We have now done ATP8 in some point mutant cells and it seems to be working using the same strategy. We took a shot at rho zero and it didn't work. I'm not sure if we (the intern) did that was doing it wrong or if it just doesn't work because of the crosstalk with the other complexes that we show in the manuscript. We'll revisit rho0 again when we have something new to try.

Leo asked: I think that it would be elegant to use classical import assays to show the import of the allotopic gene. Perhaps also Prot K treatment to show that the proteins are really imported.

Dr. O'Connor replies: We did some of that and it worked well, but it would have been a lot of work to do it perfectly and thoroughly enough for a manuscript and we show so much data that it wasn't necessary.

Leo asked: Blue native gels show co-migration with HMW complexes (not necessarily CV). It would have been good if they had also used other approaches to show the incorporation of the allotopic protein. e.g co-IP or complexome profiling could be informative.

Dr. O'Connor replies: I think our BN westerns are way more convincing than any co-IP would be. You can show anything you want by co-IP. We get good separation of the different complexes, perfect alignment with known CV proteins, incorporation of a completely foreign epitope tag with zero background signal, and re-assembly of CV in the presence of our rescue constructs. Then we show much of the same thing using the in-gel ATPase activity assays. Damn we're good.

Leo asked: I was also surprised that there was no low molecular weight species of A8 (unincorporated in the complex) in the BN gels.

Dr. O'Connor replies: Would have run off the bottom of the gel. You see the scale there? A8 is tiny. But the spirit of the question is appreciated. What percentage of A8 is being incorporated into CV? We think it is a very high percentage (in part see #6 below), but we haven't tried to answer that question directly.

Leo asked: Also why does the stable expressed allotopic gene does not show a unprocessed species (uncleaved presequence).

Dr. O'Connor replies: Yeah, in the stable mutant cells it's basically 100% as far as we can tell. We see a lot more unprocessed protein in transient overexpression experiments, especially with ATP6.