Bay Area Aging Club – Session I: Model organisms & model systems

Subhash Katewa (Kapahi lab, Buck Institute) talked about the metabolic adaptations that occur in flies whose lifespan is being extended by dietary restriction (DR). Katewa is studying translational control in DR using a method called translational profiling, which uses the number of ribosomes bound to each mRNA as an index of translational activity (more ribosomes = more translation). He found that DR increases translation of messages that encode a variety of mitochondrial functions; this observation led to some interesting findings about the differential turnover of triglycerides in DR vs ad libitum flies.

Adam Freund (Campisi lab, Buck Institute) spoke about the sources of age-related inflammation, focusing on the senescence-associated secretory phenotype (SASP). Freund has elucidated mechanisms of SASP control that intermediate between the most upstream events in senescence (DNA damage) and its downstream effects (secretion of inflammatory factors). I have it on good authority that he has a completed manuscript on the subject, hopefully to be publshed soon, so I won’t say more about his story here. (Mr. Freund happens to be my baymate.)

Dario Valenzano (Brunet lab, Stanford University) is studying the genetic architecture of longevity in a short-lived fish Nothobranchius furzeri, the shortest-living vertebrate that can be reared in captivity. As a graduate student, Valenzano developed a system of biomarkers for tracking the progress of aging in skin, brain and other tissues – not only physical markers like the senescence-associated beta-galactosidase but also behavioral markers that change over the lifespan. He is now proceeding to map the longevity-associated genes in N. furzeri and testing the sufficiency of the genes he finds. Early results indicate that short-lived and long-lived fish are dying from different causes, as evidenced by a bimodal distribution of death rate vs. age.

Adolfo Sánchez-Blanco (Kim lab, Stanford University Medical School) described the “molecular odometer” for aging in the worm C. elegans. He began with the observation that lifespan is variable, even among clonally identical individuals kept under identical conditions. With genetics and environment taken out of the picture, what makes some individuals live longer than others? In order to address this question, SB had to develop a molecular marker (e.g., promoter activity of some gene) that measures physiological age (as opposed to chronological age), and then determine whether the expression level of that marker in individual worms is predictive of lifespan. SB has identified several such genes whose expression at middle age strongly predicts remaining lifespan. He is now actively looking for interventions that abolish the correlation between marker expression and longevity: if the marker gene’s activity is serving to overcome the life-shortening effect of some stress, then removing that stress will not necessarily abolish the variability in the marker, but will eliminate the correlation between marker levels and lifespan. (This is a subtle but important logical issue; I would have thought that one should look for interventions that drove the population distribution of marker levels toward the favorable side of the distribution. It was clear from questions that a lot of audience members had trouble with this logic, and I’m still not sure I understand it myself.)

(next session)

 

SIRT3: fighting cancer in the mitochondria

Yesterday we learned that the most well-characterized mammalian sirtuin, SIRT1, is involved in the control of behavior in response to food availability. SIRT1 is just one of seven sirtuins in mammalian genomes, each of which has a characteristic expression pattern, subcellular localization, and physiological importance.

Today we’re going to talk about another member of the sirtuin family, SIRT3, which has been known for a while now to localize to the mitochondria. Now, Kim et al. have shown that the SIRT3 protein acts as a tumor suppressor:

SIRT3 Is a Mitochondria-Localized Tumor Suppressor Required for Maintenance of Mitochondrial Integrity and Metabolism during Stress

The sirtuin gene family (SIRT) is hypothesized to regulate the aging process and play a role in cellular repair. This work demonstrates that SIRT3−/− mouse embryonic fibroblasts (MEFs) exhibit abnormal mitochondrial physiology as well as increases in stress-induced superoxide levels and genomic instability. Expression of a single oncogene (Myc or Ras) in SIRT3−/− MEFs results in in vitro transformation and altered intracellular metabolism. Superoxide dismutase prevents transformation by a single oncogene in SIRT3−/− MEFs and reverses the tumor-permissive phenotype as well as stress-induced genomic instability. In addition, SIRT3−/− mice develop ER/PR-positive mammary tumors. Finally, human breast and other human cancer specimens exhibit reduced SIRT3 levels. These results identify SIRT3 as a genomically expressed, mitochondria-localized tumor suppressor.

So, the absence of the SIRT3 gene disrupts mitochondrial function and destabilizes the mitochondrial genome, but the causal relationship is unclear: one can imagine a derangement of mitochondrial morphology or physiology causing the genomic instability, but one can also imagine a primary defect in DNA metabolism causing the physiological defects — and one could also imagine both, operating in a vicious cycle.

The significance of the “single oncogene” observation requires a bit of explanation: In a normal cell, simply turning on a single tumor-promoting gene isn’t sufficient to transform the cell, i.e., create a tumor — if a cell detects a hyperphysiological level of a growth factor or internally generated mitogenic signal, it will undergo senescence, a permanent cell cycle arrest that (among other things) prevents mutated cells from turning into cancers.

In the case of the SIRT3 knockout, however, a single oncogene is enough to transform an otherwise normal cell, i.e., the loss of SIRT3 function appears to potentiate the transformation process. Furthermore, this implies that the mitochondria are involved in the decision to undergo senescence. This is not to say that SIRT3 is directly involved in the senescence fate decision: the SIRT3 knockout has broadly deranged mitochondria, and it’s not clear which mitochondrial function is involved in senescence.

While this report does not fully elucidate the mechanism of SIRT3 action, it is clear that oxidation must be central to the issue: reactive oxygen species (ROS) levels are high in the SIRT3 knockout, but increasing the dose of an antioxidant enzyme prevents the single-oncogene transformation. We don’t yet know whether the ROS themselves are causing oncogenic mutations, or the loss of the mitochondrial function in senescence is allowing cells that would have arrested to progress further down the path to cancer. (Could be both, obviously, but it’s likely that one of the two is more important.)

These findings are consistent with what we already know about the connection between mitochondrial ROS, cellular damage and lifespan (ideas that are central to one strategy for developing longevity-enhancing drug compounds by targeting antioxidants to mitochondria), but they raise a question: how much of the “longevity regulation” we owe to antioxidant genes can be attributed to tumor suppression?

Kim, H., Patel, K., Muldoon-Jacobs, K., Bisht, K., Aykin-Burns, N., Pennington, J., van der Meer, R., Nguyen, P., Savage, J., & Owens, K. (2010). SIRT3 Is a Mitochondria-Localized Tumor Suppressor Required for Maintenance of Mitochondrial Integrity and Metabolism during Stress Cancer Cell, 17 (1), 41-52 DOI: 10.1016/j.ccr.2009.11.023

There’s lots more about SIRT3 in a recent post at @ging.

 

Yet another reason to like vitamin C

The hottest thing in stem cells right now is induced pluripotency, i.e., converting somatic cells back into pluripotent cells by introducing a few stem cell-specific genes (or even the encoded proteins). Induced pluripotent stem cells (iPS) harvested from a donor’s skin would be automatically immunologically matched; furthermore, they completely circumvent some of the “ethical” and supply issues raised when using embryonic stem cells.

The process is slow and inefficient — but happily, an inexpensive and ubiquitous compound you might be familiar with can help boost both speed and efficiency:

Vitamin C Enhances the Generation of Mouse and Human Induced Pluripotent Stem Cells

Somatic cells can be reprogrammed into induced pluripotent stem cells (iPSCs) by defined factors. However, the low efficiency and slow kinetics of the reprogramming process have hampered progress with this technology. Here we report that a natural compound, vitamin C (Vc), enhances iPSC generation from both mouse and human somatic cells. Vc acts at least in part by alleviating cell senescence, a recently identified roadblock for reprogramming. In addition, Vc accelerates gene expression changes and promotes the transition of pre-iPSC colonies to a fully reprogrammed state. Our results therefore highlight a straightforward method for improving the speed and efficiency of iPSC generation and provide additional insights into the mechanistic basis of the reprogramming process.

If it really is true that vitamin C (ascorbate) is acting by blocking senescence, then there will be other paths to improving the efficiency of iPS generation, including rapamycin, which also blocks senescence. Compounds other than ascorbate will almost certainly be more expensive, but on the other hand, ascorbate probably won’t prevent all types of senescence. Vitamin C is an anti-oxidant, and it’s a good bet that it prevents induction of senescence by oxidative damage — but that’s not the only way senescence is induced. Combining a number of methods to block senescence will likely have a synergistic effect, helping us get closer to 100% efficiency.

Beyond that, all I have to say is that this is a damn handy result. Nice that it’s something so cheap and readily available.

Esteban, M., Wang, T., Qin, B., Yang, J., Qin, D., Cai, J., Li, W., Weng, Z., Chen, J., & Ni, S. (2010). Vitamin C Enhances the Generation of Mouse and Human Induced Pluripotent Stem Cells Cell Stem Cell, 6 (1), 71-79 DOI: 10.1016/j.stem.2009.12.001

 

Reviewing the reviews (#4, in case you’re counting)

Here’s the latest in our (infrequent and irregular) series of “review roundups” — links, without extensive further comment, to the reviews I found most intriguing over the past few weeks. For the previous foray into the secondary literature, see here.

Remember, each Review Roundup is guaranteed to contain at least one link to a review you will find highly educational, or your money back.

Autophagy:

• Regulation of the aging process by autophagy, Salminen & Kaarniranta

Chaperones:

• Hsps and aging, John Tower (who is capable of giving reviews more colorful titles)

Evolution:

• The Evolutionary Theories of Aging Revisited – A Mini-Review, Ljubuncic & Reznick

Glycation:

• The receptor for advanced glycation endproducts (RAGE) and cardiovascular disease, Yan et al.

Immunology:

• The Role of Stress Factors during Aging of the Immune System, Bauer et al.
• Key Research Opportunities in Immune System Aging, Swain & Nikolich-Zugich

Mitochondria:

• Functions of mitochondria: from intracellular power stations to mediators of a senescence program, V. P. Skulachev

Neurodegeneration:

• A metabolic and functional overview of brain aging linked to neurological disorders, Baquer et al.

Resveratrol:

• Resveratrol and chemoprevention, Goswami & Das

Senescence:

• Cellular senescence, ageing and disease, D. G. A. Burton

 

UCSC Cancer Genomics browser

UCSC, the institution that brought you the industry-standard genome browser, has now launched the UCSC Cancer Genomics Browser:

The browser is a suite of web-based tools to integrate, visualize and analyze cancer genomics and clinical data. This browser displays a whole-genome and pathway-oriented view of genome-wide experimental measurements for individual and sets of samples alongside their associated clinical information.

This site hosts the public UCSC Cancer Genomics Browser. The public site contains a rapidly growing body of publicly available cancer genomic data, including 12 published studies, datasets from the TCGA consortium, and others.

We encourage you to explore these data with our tools. The browser enables investigators to order, filter, aggregate, classify and display data interactively based on any given feature set including clinical features, annotated biological pathways, and user-edited collections of genes. Standard statistical tools are integrated to provide quantitative analysis of whole genomic data or any of its subsets.

I suspect that the Cancer Genomics Browser will provide an indispensable tool for biogerontologists who are seeking to explore the mechanistic connections between aging and cancer. I’m currently trying to think up an interesting way to use the service (and publicly available data) in my own work: e.g., tumors all have to undergo cellular senescence; would it be possible to find some fingerprint of senescence bypass mechanisms by looking at expression data from large numbers of tumors?

 

“When Cells Grow Old”

There’s a good non-technical overview of cellular senescence, with interviews from some of the field’s luminaries, in the most recent HHMI Bulletin:

Adding just four genes can turn adult cells back into embryonic-like cells, able to develop into any cell type in the body, according to Daley’s studies. In culture dishes, cells from a younger postdoctoral fellow in Daley’s group were “youthful and vigorous,” he says; many of them morphed into stem cells. But Daley’s cells were stubborn, refusing to reverse their clocks. It seems as a person ages, cells get increasingly stuck in their ways.

Daley isn’t taking it too personally. “I’m deficient in a lot of things, and reprogramming seems to be one of them,” he says. He plans to use the observation to understand how to reprogram cells most efficiently.

His finding points out an important concept: cells might not sprout gray hair, get achy joints, or forget where they put their car keys, but they do age. Several HHMI researchers are just beginning to learn what happens to cells as they grow old, and they’re making connections between those changes and cancer, deficiencies in wound healing, and other problems that increase in likelihood as a person ages.

It’s a great piece for a reader who might be interested in cellular aging but not have the technical background required to tackle the primary literature. I’m definitely going to put it in the list of pages I send friends when they ask what I work on.

In other news, the HHMI Bulletin is actually pretty good. In the past I’ve turned up my nose at “pet” magazines published by institutions, but recently I’ve given several of them a chance and been pleasantly surprised.

 

More on senescence and allograft rejection

Following up on yesterday’s post about the impact of cellular senescence in organ transplant failure: Another group, working in a mouse model, has shown that older donor tissues express higher levels of the senescence marker p16, which is negatively correlated with proliferative capacity.

But the old material doesn’t just start from behind, but also gets worse more rapidly: p16 levels rise dramatically in old grafts following transplantation, much faster than in young grafts, indicating that older cells are more sensitive to the stresses of undergoing transplantation and that they respond to this stress by undergoing senescence. (In contrast, young grafts only seem to express p16 if they are rejected). From Melk et al.:

Effects of Donor Age and Cell Senescence on Kidney Allograft Survival

The biological processes responsible for somatic cell senescence contribute to organ aging and progression of chronic diseases, and this may contribute to kidney transplant outcomes. We examined the effect of pre-existing donor aging on the performance of kidney transplants, comparing mouse kidney isografts and allografts from old versus young donors. Before transplantation, old kidneys were histologically normal, but displayed an increased expression of senescence marker p16INK4a. Old allografts at day 7 showed a more rapid emergence of epithelial changes and a further increase in the expression of p16INK4a. Similar but much milder changes occurred in old isografts. These changes were absent in young allografts at day 7, but emerged by day 21. The expression of p16INK4a remained low in young kidney allografts at day 7, but increased with severe rejection at day 21. Isografts from young donors showed no epithelial changes and no increase in p16INK4a. The measurements of the alloimmune response—infiltrate, cytology, expression of perforin, granzyme B, IFN-γ and MHC—were not increased in old allografts. Thus, old donor kidneys display abnormal parenchymal susceptibility to transplant stresses and enhanced induction of senescence marker p16INK4a, but were not more immunogenic. These data are compatible with a key role of somatic cell senescence mechanisms in kidney transplant outcomes by contributing to donor aging, being accelerated by transplant stresses, and imposing limits on the capacity of the tissue to proliferate.

In searching for a mechanistic relationship between aging and transplant failure, the authors focus on the loss of regenerative capacity rather than an active role for senescent cells. The latter type of hypothesis will likely gain credence as the field digests the idea that senescent cells secrete immunologically active and potentially deleterious molecules into the extracellular milieu.

 

You can’t teach an old kidney new tricks: cellular senescence in organ transplants

Cellular senescence likely evolved as a defense against cancer: Damaged cells stop dividing, preventing cells that have sustained oncogenic mutations from proliferating and forming tumors. One down side is that the proliferative capacity of the organ goes down; another is that persistent senescent cells secrete factors that damage the extracellular matrix and contribute to age-related decline in tissue function.

Because senescent cells accumulate with age, senescence has been considered a biomarker of aging — that is to say, a measurable feature of a tissue that would allow us to calculate its “biological” or “physiological” age without necessarily knowing the chronological age of the donor. This makes abundant sense: tissues with more senescent cells would have lower proliferative capacity and higher levels of deleterious secreted factors; given two samples of the same chronological age, one could argue that the one containing more senescent cells was “older” in some meaningful way.

This idea is central to a recent paper demonstrating that senescence correlates negatively with the efficacy of a transplanted tissue. Grafts from older donors have poorer outcomes than those from younger donors, but some older tissues work just fine — what’s the difference between an old graft that works and an old graft that doesn’t? The answer, in part, is the level of expression of senescence markers: Higher expression of senescence-related genes predicts a poorer outcome in a transplant. In other words, controlling for chronological age, physiological age is a negative correlate of transplant efficacy. From McGlynn et al.:

Cellular senescence in pre-transplant renal biopsies predicts post-operative organ function

Older and marginal donors have been used to meet the shortfall in available organs for renal transplantation. Post transplant renal function and outcome from these donors is often poorer than chronologically younger donors. Some organs, however, function adequately for many years. We have hypothesised that such organs are biologically younger than poorer performing counterparts. We have tested this hypothesis in a cohort of pre-implantation human renal allograft biopsies (n=75) that have been assayed by Real Time-PCR for the expression of known markers of cellular damage and biological ageing, including CDKN2A, CDKN1A, SIRT2, and POT1. These have been investigated for any associations with traditional factors affecting transplant outcome (donor age, cold ischaemic time) and organ function post transplant (serum creatinine (SC) levels).

Linear regression analyses indicated a strong association for SC with pre transplant CDKN2A levels (p=0.001) and donor age (p=0.004) at six months post transplant. Both these markers correlated significantly with urinary protein to creatinine ratios (p=0.002 and p=0.005 respectively), an informative marker for subsequent graft dysfunction. POT1 expression also showed a significant association with this parameter (p=0.05).

Multiple linear regression analyses for CDKN2A and donor age, accounted for 24.6% (p=0.001) observed variability in SC levels at six months and 23.7% (p=0.001) at one year post transplant. These data indicate that allograft biological age is thus an important novel prognostic determinant for renal transplant outcome.

Note that the authors measured the levels of senescence-associated gene expression, rather than the number of cells in the tissue that were senescent. This is a subtle but important point, given what we know about the biology of senescence: Gene expression is an imperfect marker for the extent of senescence within a tissue: A given amount of RNA could mean a small number of cells with intense expression or a larger number of cells with lower expression.

Distinguishing between these alternatives may be clinically important, depending on whether the increased senescence is causing the poorer transplant performance — and if so, why? Regeneration is likely necessary for optimal organ performance after a transplant; since senescent cells are permanently nondividing, the presence of a large number of them would predict poorer regenerative capacity. Alternatively (though not mutually exclusively) the protein factors secreted by senescent cells might have deleterious consequences on the function of the tissue itself, and this might interfere with transplant efficacy.

Figuring out which is more decisive — the number of senescent cells in a tissue, or their aggregate production of secreted factors — will become very important if we’re someday able to selectively destroy senescent cells. In the “increased numbers” case, such treatments wouldn’t help (the cells are equally unable to divide whether they’re alive or not), but in the “increased secretion” case, anti-senescence therapeutics could make the difference between success and failure in organ transplants of many kinds.

 

Telomerase activity in dyskeratosis congenita: TERC alone improves cell survival, but is there a trade-off?

In the inherited disorder dyskeratosis congenita (DC), which is linked to deficiencies in telomerase acticity, the principle cause of mortality is bone marrow failure; premature cell death and/or senescence, a consequence of catastrophic telomere shortening, are the likely mechanism(s) underpinning the clinical observations. 

Mounting evidence now indicates that impaired telomerase activity impacts the stem/progenitor compartment, resulting in premature exhaustion of the haemopoietic progenitors, and reduced number of peripheral blood progenitor cells. Mutations in the various subunits of the telomerase complex have been reported, with the RNA subunit, TERC, shown to be the limiting factor for telomerase activity and telomere length in DC patient fibroblast strains transfected with the catalytic subunit, TERT. This has led Kirwan et al. to study growth and telomere dynamics in TERC-transduced primary T-lymphocytes and B-LCLs from DC patients:

Exogenous TERC alone can enhance proliferative potential, telomerase activity and telomere length in lymphocytes from dyskeratosis congenita patients

Dyskeratosis congenita (DC) is an inherited multi-system disorder characterised by muco-cutaneous abnormalities, bone marrow failure and a predisposition to malignancy. Bone marrow failure is the principal cause of mortality and is thought to be the result of premature cell death in the haematopoietic compartment because DC cells age prematurely and tend to have short telomeres. DC is genetically heterogeneous and patients have mutations in genes that encode components of the telomerase complex (DKC1, TERC, TERT, NOP10 and NHP2), and telomere shelterin complex (TINF2), both important in telomere maintenance. Here, we transduced primary T lymphocytes and B lymphocyte lines established from patients with TERC and DKC1 mutations with wild type TERC-bearing lentiviral vectors. We found that transduction with exogenous TERC alone was capable of increasing telomerase activity in mutant T lymphocytes and B lymphocyte lines and improved the survival and thus overall growth of B-lymphocyte lines over a prolonged period, regardless of their disease mutation. Telomeres in TERC-treated lines were longer than in the untreated cultures. This is the first study of its kind in DC lymphocytes and the first to demonstrate that transduction with TERC alone can improve cell survival and telomere length without the need for exogenous TERT.

To assess the impact of TERC-transfection on telomerase activity, the authors established primary T-lymphocyte cultures from four DC patients (two with different TERC mutations; two with different dyskerin mutations) and four heathy subjects. T-cells are difficult to maintain for prolonged periods; therefore, B-lymphoblastoid cell lines (B-LCLs) derived from DC-patients, previously shown to grow poorly when compared to those from healthy volunteers, were utilized for the study of growth dynamics.

In the primary T-cells, telomerase activity in the transfected cells was quite similar between the mutant and wildtype, with all wild-types showing a non-significant increase. Of the DC cells, both TERC mutant samples showed a significant increase, but neither of the DKC1 mutants did (one sample actually saw a decrease). This observation is intuitive, given the respective mutations.

In the B-LCL lines, TERC mRNA was expressed in the transduced cells, although not to a significant level in one each of the wild-type and DKC1 lines. Regardless, telomerase activity was increased in all lines, and to a greater extent than in the T-lymphocytes. (However, this comparison was not completely valid, as activity was assessed at 7 days in the T-cells and 98 days in the B-LCL lines; why not also examine activity at 7 days in the B-LCLs?)

Consistent with the increased telomerase activity, mean telomere length was increased in all but one wild-type line 98 days post-transfection, and there was greater net growth and increased live/dead ratios in the mutant lines overexpressing TERC.

Based on their findings, the authors suggests that TERC alone can rescue the phenotype of DC by increasing telomerase activity, extending telomeres and prolonging cellular lifespan. As TERT, the catalytic unit, was not overexpressed, it would appear that the cell contains spare endogenous capacity for telomerase activity. 

This may be a crucial observation as TERT has recently been shown to have roles other than extending telomeres. During times of cellular stress TERT is reversibly excluded form the nucleus in a dose-dependent manner; this extra-nuclear TERT co-localizes with mitochondria, protecting mtDNA and maintaining mitochondrial function. If over-expressing TERC somehow sequesters this “spare” TERT, what then happens if the cell is “stressed”?  While the maintenance of telomere length is important, mitochondrial damage can lead to increased ROS production thus initiating a detrimental positive feedback loop. In this model system then, there may be a critical trade-off between telomere dynamics and protection of mitochondrial function. Further studies will clearly be required to establish if such a trade-off is worthwhile. 

 

AGE modification of chaperones in senescence

Advanced glycation endproducts (AGEs) have been implicated in age-related disease and aging itself, convincingly enough that significant effort has gone into finding compounds that can “break” or reverse them.

Among many unresolved questions: Are specific proteins AGEylated, and if so, which proteins are being modified within cells? Unterluggauer et al. report that in both senescent fibroblasts and senescent endothelial cells, the chaperone Hsc70 is modified by AGE:

Identification of Hsc70 as target for AGE modification in senescent human fibroblasts

Cellular senescence is known as a potent mechanism of tumor suppression, and cellular senescence in vitro also reflects at least some features of aging in vivo. The Free Radical Theory of aging suggests that reactive oxygen species are important causative agents of aging and cellular senescence. Besides damage of nucleic acids and lipids, also oxidative modifications of proteins have been described as potential causative events in the senescence response. However, the identity of protein targets for post-translational modifications in senescent cells has remained unclear. In the present communication, we analyzed the occurrence of oxidative posttranslational modifications in senescent human endothelial cells and dermal fibroblasts. We found a significant increase in the level of protein carbonyls and AGE modification with senescence in both cell types. Using 2D-Gel electrophoresis and Western Blot we found that heat shock cognate protein 70 is a bona fide target for AGE modification in human fibroblasts.

If a major chaperone is modified by AGEs, and the modification is deleterious to its function, this could dramatically decrease the efficiency of protein folding, which (given that chaperones are proteins too) could dramatically decrease the number of chaperones, which…

Garbage catastrophe, anyone? (Then again: senescent cells persist in culture for a long time, implying that they have some way to deal with the admittedly hypothetical protein-folding death spiral implied in the previous paragraph.)