Complicating the Picture for Aging, Cellular Senescence, and Bcl-xL

Efforts to build rejuvenation therapies that work by selectively destroying senescent cells are very much in the news of late. One class of senolytic drug candidates works by inducing apoptosis, a form of programmed cell death, via reduced levels of Bcl-2 family proteins, such as Bcl-2 itself, Bcl-xL, and Bcl-W, all of which normally act to suppress apoptosis. Senescent cells are inclined towards apoptosis already, so a modest nudge in that direction can destroy a fair proportion of these unwanted cells without causing harm to healthy cells. These apoptosis-related proteins have numerous other roles as well, however, since evolution is very much in favor of reusing the tools to hand. For example, Bcl-xL is also involved in mitochondrial damage protection, the immune response, cellular respiration and DNA repair: quite the portfolio, and all items that are connected to aging in one way or another. I noted an open access paper today that muddies the water considerably on the topic of Bcl-xL, as it shows that more Bcl-xL rather than less (a) reduces incidence of cellular senescence in tissue cultures, (b) extends life in nematode worms, and (c) is found in human centenarians, but not younger individuals.

Ordinary somatic cells, the vast majority of the cells in the body, become senescent when they reach the Hayflick limit at the end of their replicative life span, or in response to damage, or a toxic local environment, or as a part of the wound healing process. Senescent cells cease dividing, and most either self-destruct or are destroyed by the immune system soon afterwards. This behavior has evolved because it suppresses cancer incidence, at least initially, by removing those cells most at risk. Unfortunately not all are destroyed, and those that linger cause harm to surrounding tissues via a potent mix of inflammatory signals known as the senescence-associated secretory phenotype (SASP). Given enough senescence cells, as few as 1% or less of all the cells in an organ, significant dysfunction and inflammation is the result, contributing to the development and progression of age-related disease. It even comes to a point at which the presence of larger numbers of senescent cells raises the risk of cancer and allows tumors to grow more readily. Getting rid of these cells has been demonstrated to improve tissue function and extend healthy life spans in mice, and we are all looking forward to forthcoming human trials of this class of rejuvenation therapy - the first of which will most likely use apoptosis-inducing drugs that work via inhibition of Bcl-2 family proteins.

Given this, how is it that increased levels of Bcl-xL can be associated with longevity and lesser degrees of cellular senescence? The authors in the paper linked below, perhaps wisely, do not speculate all that much and largely restrict themselves to reporting their findings. The lesson we must constantly learn is that biochemistry is complicated. It is a linked system of countless feedback loops, many of which share protein machinery. Nothing can be accomplished in isolation, and there is always a way for very similar interventions to result in diametrically opposed results. Once might speculate that, for example, reduced levels of Bcl-xL are only useful in conjunction with reduced levels of other Bcl-2 family members. Or that drugs like navitoclax are pushing other levers and buttons whose significance is not yet as well understood in this picture. Alternatively, consider that in the short term reduced levels of Bcl-xL could induced senescent cell destruction, and resulting health benefits, but in over the long term, and without any intervention to clear senescent cells, higher levels of Bcl-xL could aid cellular health via other mechanisms such as mitochondrial function, immune function, and DNA repair. It is plausible that better functionality for those line items might reduce the number of cells entering senescence. Or, alternatively, there is the explanation proposed by the authors of the paper involving different paths to apoptosis under different circumstances, not all of which are necessarily desirable. But this is all speculation at this stage, to be confirmed by further research. Regardless of the role of Bcl-xL in natural variations in longevity, it is certainly the case that senescent cell clearance will be a beneficial procedure. Someone who undergoes that procedure will have an incrementally longer life expectancy than someone who didn't - and the plan is to keep doing it as often as needed to keep senescent cell counts beneath the level at which they produce a meaningful contribution to aging and age-related disease.

Human exceptional longevity: transcriptome from centenarians is distinct from septuagenarians and reveals a role of Bcl-xL in successful aging

Centenarians, for example, exhibit medical histories with remarkably low incidence rates of common age-related disorders such as vascular-related diseases, diabetes mellitus, Parkinson's disease, and cancer. Over 80% of centenarians delay their first experience of diseases often associated with high mortality till beyond the age of 90 years or escape these morbidities entirely. Moreover, centenarians may have better cognitive function and require minimal assistance for activities of daily living compared with younger elderly who exhibit normal aging. The Spanish Centenarian Study Group, founded in 2007 as a population-based research program focused on centenarians living in various areas of Spain, previously investigated molecular mechanisms by which centenarians maintain homeostasis and thereby evade age-related morbidities as evidenced by changes in their microRNA (miRNA) expression profiles in peripheral blood mononuclear cells (PBMCs). Our previous analysis of miRNA microarray data ("miRNome") showed that miRNA expression in centenarians (successful aging) exhibited significant overlap with that in young people but not with septuagenarians (normal aging). We thus hypothesized that expression patterns of mRNAs in centenarians versus septuagenarians and young people might provide further insights into what discriminates those with exceptional longevity from normal aging. In the present study we sought to identify expression patterns of mRNAs in centenarians as means to elucidate factors that influence why these individuals live such long, healthy lives. We have identified Bcl-xL as one of these factors that influence longevity in humans.

Analysis of the genes over-expressed in centenarians reveals relations to three genes: Bcl-xL (also known as BCL2L1), Fas and Fas ligand (FasL), all of them involved in the control of apoptosis. Moreover, using Gene Ontology we detected that apoptosis is one of the processes most commonly conserved in centenarians. Fas and FasL are mainly involved in the control of the extrinsic pathway to apoptosis, whereas Bcl-xL inhibits the intrinsic, mitochondrial pathway to apoptosis. Bcl-xL down regulates apoptosis and promotes cell survival by migrating to mitochondrial outer membrane, counteracting mitochondrial permeabilization (pore formation) activity, and inhibiting cytotoxic adaptors needed for activation of caspases that dismantle the cell. We evaluated centenarians' expression of BcL-xL and confirmed that it is indeed up-regulated compared with septuagenarians and young people. To validate the results obtained in the Spanish cohort, we measured BcL-xL expression in another well characterized centenarian population, i.e., that of the Sardinian centenarians. We found that, as in the Spanish cohort, Sardinian centenarians display a higher Bcl-xL expression than septuagenarians and maintain an expression similar to young individuals. The same pattern is shown when measuring Bcl-xL protein expression.

As stated before, BcL- xL is important in the development and maintenance of the immune system. Moreover, immunosenescence (age-related decline of immune function) has been posited responsible at least in part for the well-known increased incidence rates of infections, cancer, and autoimmune diseases that arise in elderly persons who display normal aging. We thus analyzed lymphocyte function in centenarians and showed that leukocyte chemotaxis and NK cell activity were significantly impaired in septuagenarians compared with young people whereas in centenarians these indicators of immunosenescence were similar to the picture noted in young people. Therefore, using centenarian-donated PBMCs, we observed a number of similarities between centenarians and young persons, which were not reflected in cells donated by septuagenarians, in a variety of biological factors suggestive that centenarians may evade the relentless onset of immunosenescence that is seen in normal aging.

The general picture that emerges from our series of experiments is that centenarians have an intact extrinsic pathway of apoptosis thus killing cells that may be damaged by environmental insults but down-regulated intrinsic apoptosis thus sparing cells that have not been exposed to genotoxic or other challenges. Upregulation of Bcl-xL as noted in our gene expression studies suggests that regulation of apoptosis is deranged in septuagenarians (normal aging) yet preserved in centenarians (exceptional aging). Taken together, our results demonstrate that, similar to what we found in microRNA expression, septuagenarians (normal aging) display a cell health impairment which is not so evident in young people or centenarians (exceptional aging). Moreover, they suggest that Bcl-xL may play a major role in healthy aging.

To assess if increased activity of Bcl-xL promotes longevity in vivo we turned to the simple model organism C. elegans. This nematode has several advantages for aging studies: it has a short life span of around twenty days, it shares the main hallmarks of human aging and around 70% of the human genome has a C. elegans ortholog, including the apoptotic pathway that was originally described in this organism. ced-9 is the only C. elegans member of the Bcl2 anti-apoptotic family and thus the ortholog of human Bcl-xL, showing 44% homology and the same protein domains. Among the multiple available ced-9 alleles, ced-9(n1950) is a missense G to A substitution that confers constitutive activity to the CED-9 protein. We hypothesized that this mutation could mimic the increased Bcl-xL levels of centenarians and thus we performed longevity curves of ced-9(n1950) compared to wildtype worms. Interestingly, ced-9(n1950) animals showed a significant increase both in the mean and maximum survival time. Moreover, at 25 days, which can be considered a very advanced age for a worm, the percentage of ced-9(n1050) survivals was more than double compared to wildtype.

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