Mitochondrial Remediation

Here's another response from Dr Klionsky:

Subject:  Re: Autophagy

>I'd read that autophagy is attracting interest for
>anti-aging research, particularly for the elimination
>of defective/inefficient mitochondria. One main
>identifying characteristic of a defective mito is
>supposed to be the low charge potential across its
>membrane.
>
>One approach for preserving mitochondrial vigor I'd
>read about involves protecting mitochondrial DNA from
>being damaged by reactive oxidative species produced
>within the mito, by targetting them with a drug like
>Mito-Q or Mito-VitE. These agents apparently mate an
>anti-oxidant with a large lipophilic cation, in order
>to directly target mitochondria for anti-oxidant
>delivery.
>
>Similarly, the low membrane potential of defective
>mitochondria presents the opportunity for possible
>targetting with mitotoxic agents in order to
>selectively eliminate them and leave behind a
>population of relatively healthy mitochondria.
>
>However, I am curious as to whether autophagic tagging
>of defective mitochondria might not be a superior
>approach over mitotoxic agents. The idea would be to
>somehow exploit that low membrane potential that is
>the hallmark of defective mitos, in order to
>selectively tag them for autophagic destruction.
>
>Do you have any awareness of what governs the tagging
>of defective organelles for autophagotic envelopment
>and destruction? How could tagging be controlled and
>directed to better target defective mitochondria, to
>improve the quality of an aging mitochondrial
>population?

Again, a good question.  We have some idea how peroxisomes
are tagged, but not mitochondria.  With peroxisomes the change in
nutrient conditions causes Pex3 to leave the organelle, and this
uncovers Pex14, which appears to be the direct tag.  One paper has
identified Uth1 as a mitochondrial tag but I do not think the data
are convincing.  But even if this was the tag it would probably not
help in regard to improving mitochondrial turnover.
It is thought that damaged mitochondria might be recognized
and preferentially removed by autophagy, for example to prevent the
need for apoptosis.  But again, nothing is known about how a damaged
mitochondrion is detected.  It seems to me that the issue might not
be in better tagging but in being able to activate autophagy at will
(and then shut it off at will) to drive routine elimination of
cytoplasm.  Autophagy declines with age so being able to induce it
might provide the beneficial aspects including removal of part of the
mitochondrial population.  True, this would not target specifically
defective mitochondria unless the autophagic system is able to select
them out (which is the hypothesis after all).

So as previously discussed, triggered spurts of autophagy would be a useful way to keep our cells in shape. But how often is often enough?
Imagine triggering an autophagy spurt every night by taking a pill before you go to bed. But how long does it take to regenerate fresh mitochondria? What are the relevant timelines here?

Hmm, I still want to know more about why autophagy is declining with age. Any further insights, people?

It seems to me that the issue might not
be in better tagging but in being able to activate autophagy at will
(and then shut it off at will) to drive routine elimination of
cytoplasm.  Autophagy declines with age so being able to induce it
might provide the beneficial aspects including removal of part of the
mitochondrial population.  True, this would not target specifically
defective mitochondria unless the autophagic system is able to select
them out (which is the hypothesis after all).

Isn't this the whole point, ie that cellular autophagy fails to detect/eliminate mutant mitochondria, thereby giving them a selective advantge over their wild type counter parts?

The tagging idea is interesting. The key is of course designing a tag that (a) can identify and attach to the mutant mitos and (b) triggers the autophagy response.

Hi Don, I wonder if it might be possible to have a modified tag tailored to homing in on the defective mitos -- perhaps by making the tag as a moiety attached to a lipophilic anion. An anion would be mainly repelled by the high charge potential on a good mito, but would not be as effectively repelled by the low membrane charge on a defective mito.

It's okay if our lipophilic anion-mated tag doesn't perfectly select for bad mitos over good ones, just as long as it has sufficient bias to help us alter the status quo. If the destruction of bad mitos can even be marginally boosted over the destruction of good ones, then enough autophagy-induced turnover will eventually purify the mito population.

If autophagy can't be made selective/preferential towards elimination of bad mitos, then I don't see how it can be used to improve the quality of the mitochondrial population, since autophagy would then fail to affect the status quo, by not altering the balance in favor of good mitos over bad.

But what is the advantage of homing in on defective mitos with an autophagic tag, over homing in on them with some toxic agent -- perhaps an agent that simply destroys their mtDNA? I don't think we care so much about the actual superstructure of defective mitos per se, we are really only threatened by that infernal mutant mtDNA which they house. If you could just go after that bad mtDNA and even just get rid of that, then aren't you nailing the heart of the problem right there?

Further thoughts...

Supposing that autophagy is not naturally selective against defectives, meaning that a selection mechanism like the one described above ends up having to be engineered. Perhaps it might also be feasible to use this preferential targetting merely to selectively "neuter" defective mitochondria by simply eliminating their mutant mtDNA so that they cannot replicate. Then the gradual turnover from natural autophagy would automatically clear away the neutered mutants.

My main reason in mentioning that alternative, is that artificially-selective autophagy seems likely to be a more involved and difficult to achieve as compared to the mere selective neutering of self-replicating organelles like mitochondria.

However, selective autophagy would be ideal, if possible.

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.

If autophagy can't be made selective/preferential towards elimination of bad mitos, then I don't see how it can be used to improve the quality of the mitochondrial population, since autophagy would then fail to affect the status quo, by not altering the balance in favor of good mitos over bad.

Isn't this the whole point, ie that cellular autophagy fails to detect/eliminate mutant mitochondria, thereby giving them a selective advantge over their wild type counter parts?

What is important to note is that aged cells have overall reduced rates of autophagy - not just in the mitochondria that manage to evade autophagy. Therefore if the rate of autophagy were to be increased to youthful levels we should observe a decrease in ROS because dysfunctional and leaking mitochondria would be proportionally reduced from the non evading mitochondrial pool which means it would still be beneficial. Unfortunately, it would mean that giant and other mitochondria that have managed to elude lysosomal or vacuolar degradation may not be eliminated but the rate of healthy mitochondria joining their ranks should be reduced and hence the cell should be able to survive longer.

A treatment that increases the rate of autophagy, even if non-specific to damaged mitochondria, should provide tangible anti-senescence benefits because it will prevent damage that would have occurred if healthy mitochondria were also allowed to exist for too long without being recycled and thus run the risk of joining the other "zombie" mitochondria.

Note that biogenesis does not appear to be compromised with aging and thus efforts designed to accelerate the rate of autophagy do not need to account for a commensurate increase in mitochondrial biogenesis.

Musing:

Would upregulating proteosomal activity be the key to restoring autophagic activity of not only mitos but other defective components of the cell?

http://www.ncbi.nlm....t_uids=15325579

Point Kevin. Not only autophagy but proteasomal function too is generally impaired with age, which has gene regulatory, cell cycle, DNA repair, etc. ramifications. Proteasomal proteolysis, however, is ubiquitin driven in contrast to mitochondrial autophagy which appears to work by lysosomes and vacuoles and the literature suggests that not even putative tagging mechanisms have been identified to date. I would say that increasing proteasomal function (since it is reduced with aging) should also have anti-senescence benefits even though we would have to be more careful since proteasomal activity is associated with so many cell functions.

Say, does anyone actually have access to the autophagy journal that manofsan linked to (edit: at the top of this page)?

Edited by DonSpanton, 29 March 2005 - 10:15 PM.

Hi Don, doesn't the link work for you?
The journal is free until Jan2006.
I've been able to read articles off it, and so you should be able to as well.

Point Kevin. Not only autophagy but proteasomal function too is generally impaired with age, which has gene regulatory, cell cycle, DNA repair, etc. ramifications. Proteasomal proteolysis, however, is ubiquitin driven in contrast to mitochondrial autophagy which appears to work by lysosomes and vacuoles and the literature suggests that not even putative tagging mechanisms have been identified to date. I would say that increasing proteasomal function (since it is reduced with aging) should also have anti-senescence benefits even though we would have to be more careful since proteasomal activity is associated with so many cell functions.

I found this.. and unfortunately will have to get back to it but I thought it worth putting here. I also wonder whether or not it might be worthwhile to see if the pH of lysosomes could be encouraged to become more acid then normal and perhaps make the junk in them more amenable to degradation.

Int J Biochem Cell Biol. 2004 Dec;36(12):2376-91.
Link: http://www.ncbi.nlm....t_uids=15325579

Autophagy, proteasomes, lipofuscin, and oxidative stress in the aging brain.

Keller JN, Dimayuga E, Chen Q, Thorpe J, Gee J, Ding Q.

203 Sanders-Brown Center on Aging, University of Kentucky, Lexington, KY 40536-0230, USA. jnkell0@pop.uky.edu

In order to successfully respond to stress all cells rely on the ability of the proteasomal and lysosomal proteolytic pathways to continually maintain protein turnover. Increasing evidence suggests that as part of normal aging there are age-related impairments in protein turnover by the proteasomal proteolytic pathway, and perturbations of the lysosomal proteolytic pathway. Furthermore, with numerous studies suggest an elevated level of a specialized form of lysosomal proteolysis (autophagy or macroautophagy) occurs during the aging of multiple cell types. Age-related alterations in proteolysis are believed to contribute to a wide variety of neuropathological manifestations including elevations in protein oxidation, protein aggregation, and cytotoxicity. Within the brain altered protein turnover is believed to contribute to elevations in multiple forms of protein aggregation ranging from tangle and Lewy body formation, to lipofuscin-ceroid accumulation. In this review we discuss and summarize evidence for proteolytic alterations occurring in the aging brain, the contribution of oxidative stress to disruption of protein turnover during normal aging, the evidence for cross-talk between the proteasome and lysosomal proteolytic pathways in the brain, and explore the contribution of altered proteolysis as a mediator of oxidative stress, neuropathology, and neurotoxicity in the aging brain.

Publication Types:
Review
Review, Tutorial

PMID: 15325579 [PubMed - indexed for MEDLINE]

Kevin, I can see that by the emphasis on "elevated" you may be suggesting that during aging autophagy could increase rather than as I have mentioned decrease and consequently affect mitochondrial turnover. Admittedly one can draw an ambiguous conclusion from the abstract so I quote from the passage on lysosomal alterations (1):

While the role of proteasome inhibition as a cause of age-related increases in protein oxidation, and specifically increased lipofuscin is becoming increasingly established, the role of lysosomal dysfunction in each of these age-related events is less clear. Gross impairments in multiple aspects of lysosomal proteolysis are general features of aging in most cells and tissues. For example, age-related impairments in multiple aspects of lysosomal proteolysis are observed in most cell types, including impaired regulation of lysosomal pH, impaired lysosomal stability, impaired targeting of proteins to the lysosome, and potentially deleterious alterations in the expression of individual lysosomal proteases. Numerous lines of evidence suggest that cells attempt to compensate for stresses upon the lysosomal-proteolytic pathway via the selective increased expression of individual proteases. For example, some studies have demonstrated that the levels of cathepsin D and cathepsin E are significantly increased during normal brain aging.

It is important to contextualize the message that autophagy is elevated in the aspect of individual proteases and as a compensatory mechanism for an overall decrease in autophagy function.

I also think it is important that one looks towards the possible relationship between the observed decrease in age-related rDNA (see "the third DNA") and the concomitant decrease in ribosome/transcription activity with the decrease in autophagy and mitochondrial dysfunction. Should such a relationship be established, it would imply that keeping rDNA numbers at youthful levels could also aid the mitochondrial problem.



(1) Int J Biochem Cell Biol. 2004 Dec;36(12):2376-91.
Autophagy, proteasomes, lipofuscin, and oxidative stress in the aging brain. (Attached)
Keller JN, Dimayuga E, Chen Q, Thorpe J, Gee J, Ding Q.

Thanks for the comment prometheus.. the abstract is ambiguous about what they mean by elevated levels of autophagy being a result of reduced function.

I'm interested in the cross-talk between the proteosome and lysosome, which I'm sure is also linked to protein synthesis and the rDNA transcription issues you describe.