Rapamycin, complex I and mitochondrial ROS
Despite plenty of controversy there is ample support for the mitochondrial free radical theory of aging. Here, I want to highlight some recent findings implicating complex I in mtROS production and aging.
Two recent papers (2, 4), Fok and Miwa, both find subtle reductions of specific complex I subunits after rapamycin treatment using *omic approaches. Miwa using proteomics and Fok using transcriptomics. Miwa et al. also showed decreased complex I and superoxide production after calorie restriction and reductions of complex I in a longer vs a shorter-lived strain. These changes were most pronounced in the matrix arm of complex I. Fok (4) studied chronic rapamycin feeding using transcriptomics ("Illumina Mouse Ref8 microarray") and found:
"The majority of the transcripts that change in complex I and II transcripts decrease while complex III, IV, and V transcripts that change increase in chronic Rapa-fed female and Rapa-2 male mice."
I describe the changes as subtle, because often they are not seen on simple western blots against isolated subunits (ref. 7, albeit in muscle tissue).
The strongest comparative study of mitochondrial ROS production and lifespan, from the Brand lab, also implicated complex I as the source of ROS (3). Furthermoe, a more recent bioinformatic study found that amino acid changes correlated with lifespan are overrepresented in mitochondrially encoded complex I subunits (1). At this point no one knows what the latter means, but it does point to complex I.
Kennedy 2013 (5) found no accumulation of oxidative stress induced transversions in human mitochondria (random mutation capture technique). In a related paper (6) it was shown that also in aging Drosophila no such mutations accumulate. What could be the reasons for this discrepancy? Efficient removal of mutations, relevance of mtDNA deletions over mutations, mutagenesis during critical time points (embryogenesis?)
Non-ROS mediated effects of complex 1 on lifespan are conceivable instead, although, this is inconsistent with the Brand study.
1. "It appears that ACL [AA correlated with lifespan] are more frequent in MEPs [Mitochondrially encoded proteins] involved in complexes I and V of the ETC."
Exp Gerontol. 2014 Aug;56:53-8. doi: 10.1016/j.exger.2014.03.009. Epub 2014 Mar 21.
Identification of amino acids in mitochondrially encoded proteins that correlate with lifespan.
Mariadassou M1, Pellay FX2.
2. Miwa, S., Jow, H., Baty, K., Johnson, A., Czapiewski, R., Saretzki, G., ... & von Zglinicki, T. (2014). Low abundance of the matrix arm of complex I in mitochondria predicts longevity in mice. Nature communications, 5.
"During succinate oxidation, H(2)O(2) production rates were generally lower in the longer-lived species; the differences arose at complex I of the electron transport chain during reverse electron transport."
Aging Cell. 2007 Oct;6(5):607-18. Epub 2007 Jun 27. Low rates of hydrogen peroxide production by isolated heart mitochondria associate with long maximum lifespan in vertebrate homeotherms. Lambert AJ1, Boysen HM, Buckingham JA, Yang T, Podlutsky A, Austad SN, Kunz TH, Buffenstein R, Brand MD.
4. PLoS One. 2014 Jan 7;9(1):e83988. doi: 10.1371/journal.pone.0083988. eCollection 2014. Mice fed rapamycin have an increase in lifespan associated with major changes in the liver transcriptome. Fok WC1, Chen Y2, Bokov A3, Zhang Y4, Salmon AB5, Diaz V6, Javors M7, Wood WH 3rd8, Zhang Y8, Becker KG8, Pérez VI9, Richardson A10.
5. PLoS Genet. 2013 Sep;9(9):e1003794. doi: 10.1371/journal.pgen.1003794. Epub 2013 Sep 26. Ultra-sensitive sequencing reveals an age-related increase in somatic mitochondrial mutations that are inconsistent with oxidative damage. Kennedy SR, Salk JJ, Schmitt MW, Loeb LA.
6. PLoS Genet. 2014 Feb 6;10(2):e1003974. doi: 10.1371/journal.pgen.1003974. eCollection 2014.
Oxidative stress is not a major contributor to somatic mitochondrial DNA mutations.
Itsara LS1, Kennedy SR2, Fox EJ2, Yu S3, Hewitt JJ4, Sanchez-Contreras M5, Cardozo-Pelaez F6, Pallanck LJ3.
...further work will be required to determine whether superoxide contributes to the frequency of mtDNA deletions, and to assess the influence of other forms of ROS, such as hydroxyl radicals, on mtDNA mutation frequency.
How can we reconcile our current findings with the body of work supporting a role of ROS in mtDNA mutations? Such work includes direct sequencing of mtDNA , , and a study showing that catalase expression in mouse mitochondria reduced the frequency of mtDNA mutations . One possible explanation for at least some of the conflicts involving sequencing is that PCR amplification of DNA that contains 8-oxo-dG lesions results in G:C to T:A transversion mutations during the amplification process. Sequencing of material containing amplification-induced mutations could thus result in an overestimation of the true G:C to T:A transversion frequency. Further compounding this potential problem is the finding that oxidative modification of mtDNA can occur during mtDNA isolation . By contrast, the RMC assay is immune to this type of artifact because TaqI is capable of digesting oxidized DNA, and thereby removes oxidized molecules prior to mutation detection by qPCR , . Given that the study involving catalase ovexpression also used RMC to monitor mtDNA mutation frequency, we cannot presently offer an explanation for this conflict. However, it is important to emphasize that the findings from studies of catalase overexpression do not necessarily represent a direct conflict with our current conclusion because our findings leave open the possibility that ROS makes a small contribution to the mtDNA mutation frequency. Nevertheless, further studies will be required to explain the sizeable magnitude of the effect of catalase overexpression on the mtDNA mutation frequency, given the small contribution of ROS to mtDNA mutations suggested by our work.7. Ye, L., Widlund, A. L., Sims, C. A., Lamming, D. W., Guan, Y., Davis, J. G., ... & Baur, J. A. (2013). Rapamycin doses sufficient to extend lifespan do not compromise muscle mitochondrial content or endurance. Aging (Albany NY), 5(7), 539.