A glutamine a day keeps senescence away

Cellular senescence is regarded as a tumor suppressor mechanism: damaged cells permanently leave the cell cycle (preventing tumor initiation), and also secrete factors that trigger both tissue repair and inflammation in the vicinity. This is probably good at first but bad later on: persistent senescent cells also secrete growth factors and metalloproteases that degrade the tissue microenvironment and encourage nearby preneoplastic cells to progress into full-blown tumors. Thus, senescence has been implicated in late-life cancer and age-related decline in tissue function.

The “damage” in question is usually genotoxic in nature: telomere shortening, indicating that a cell has undergone many rounds of potentially mutagenic cell division, or high levels of DNA damage such as that resulting from ionizing radiation or exposure to chemical clastogens. Oncogene expression probably also induces senescence via DNA damage, by triggering over-firing of replication origins and generating broken ends and weird chromatin structures that are interpreted as damage.

Now it appears that falling cellular ATP levels may also result in cellular senescence. Unterluggauer et al. report that inhibition of glutaminolysis (preventing cells from generating ATP from glutamine, an unglamorous and occasionally overlooked pathway that is nonetheless an important energy source in many cellular lineages) results in increased senescence in human vascular endothelial cells (HUVECs):

Premature senescence of human endothelial cells induced by inhibition of glutaminase

Cellular senescence is now recognized as an important mechanism of tumor suppression, and the accumulation of senescent cells may contribute to the aging of various human tissues. Alterations of the cellular energy metabolism are considered key events in tumorigenesis and are also known to play an important role for aging processes in lower eukaryotic model systems. In this study, we addressed senescence-associated changes in the energy metabolism of human endothelial cells, using the HUVEC model of in vitro senescence. We observed a drastic reduction in cellular ATP levels in senescent endothelial cells. Although consumption of glucose and production of lactate significantly increased in senescent cells, no correlation was found between both metabolite conversion rates, neither in young endothelial cells nor in the senescent cells, which indicates that glycolysis is not the main energy source in HUVEC. On the other hand, glutamine consumption was increased in senescent HUVEC and inhibition of glutaminolysis by DON, a specific inhibitor of glutaminase, led to a significant reduction in the proliferative capacity of both early passage and late passage cells. Moreover, inhibition of glutaminase activity induced a senescent-like phenotype in young HUVEC within two passages. Together, the data indicate that glutaminolysis is an important energy source in endothelial cells and that alterations in this pathway play a role in endothelial cell senescence.

The authors provide good evidence that endothelial cells rely heavily on glutaminolysis, and that removal of this energy source both drastically reduces cellular ATP levels and results in a “senescent-like” growth arrest. They then show fairly convincingly that this arrest is very similar to the arrest induced by telomere shortening, DNA damage or oncogene expression (i.e., cellular senescence) — in particular, by demonstrating that the arrested HUVECs express a panoply of senescence-associated gene expression and cytological markers. No word, as far as I could tell, on the reversibility of the arrest upon resumption of glutaminolysis (irreversibility is a hallmark of senescence); I mention this because growth arrest is a fairly obviously sensible response to an energy deficit, but it’s not clear why it ought to be permanent.

The reason I’m interested in this paper is that it might point toward a unifying principle underlying two major subjects within the field of biogerontology — cellular senescence and sirtuins — which both receive a great deal of individual attention but so far have not been demonstrated to have much to do with one another. Sirtuins such as SIRT1 are regulated by cellular energy state (in particular, by the NAD+/NADH ratio); if it turns out that perturbations in the cellular energy budget are an important means of senescence induction, it might be interesting to take a closer look and see whether sirtuin signaling might influence the establishment of cellular senescence.

 

Blog on senescence: Ageing Research

Holy smokes! Over at the (relatively) new blog Ageing Research, Dominick Burton is engaged in nothing less than a comprehensive review of cellular senescence and its (putative) role in organismal aging. He started from first principles (with definitions of aging and a discussion of some early theories regarding the process), and has moved from there through a historical overview of senescence to a discussion of the modern understanding of senescence and aging. He’s now focusing on some of the molecular mechanisms underlying senescence in vitro.

It’s more than a blog — it’s more like a slowly evolving review that grows in bite-sized chunks. That’s a good thing for readers who want to learn about the most modern aspects of the field but might require additional background information to get up to full speed. Heck, it’s pretty great for us so-called “experts” — skimming over Dominick’s archives, I definitely found myself reminded of a few things I’d forgotten (and then forgotten that I’d forgotten).

So if you’re at all interested in theories of aging, cellular senescence, telomere biology, or just well-researched and well-written science blogging, click on over to Ageing Research and check it out. Belated welcome to the biogerontology blogosphere, Dominick!

 

Tobacco-induced senescence limited to the lung

Smoking, which is bad for you, causes premature senescence in lung fibroblasts. Since senescent cells secrete factors that encourage nearby cancers to grow, this induction of senescence could represent a major contribution to carcinogenesis by tobacco.

One open question in the field is whether tobacco (or, by extension, other toxins) have a more systemic effect on senescence throughout the body — especially because of the broad-spectrum secretory phenotype of senescent cells, some have speculated that the senescent cells formed initially at the site of carcinogen exposure could “act at a distance” and drive senescence elsewhere in the body.

Good news (against an otherwise bleak background) is provided by Müller et al., who find that skin fibroblasts from smokers show no signs of premature senescence — hence the tobacco toxins are not inducing senescence throughout the body. The authors used skin from the upper torso, which is usually protected from smoke by clothing — facial skin is exposed to sunlight, potentially confounding the results, and more to the point is directly exposed to smoke; an observation (one should say “confirmation of the obvious”) that smoking contributed to skin wrinkling via premature senescence would merely underscore what is already known.

 

AGEs and the ECM

Advanced glycation endproducts (AGEs) are the result of nonenzymatic condensation reactions between sugars and proteins. AGEs accumulate in multiple tissues over the course of aging, and they have been implicated in a variety of age-related diseases. The data connecting them to aging isn’t terribly strong, in part because it’s hard to prevent them from forming and thereby observe aging and disease progression in their absence. Most of the evidence that they’re involved is circumstantial, derived from observations of diseases where they accumulate prematurely (e.g., diabetes, where the “sugar spikes” that result from impaired glucose homeostasis cause increased AGE formation).

A mechanistic understanding of the effect of AGE on tissues and cells, then, would go a long way toward boosting the argument that these compounds are causative in aging, rather than merely a harmless epiphenomenon. To that end, Molinari et al. studied the effect of AGEs on gene expression in fibroblasts (mesenchymal cells that provide support and structure to tissues throughout the body, especially the skin):

Effect of advanced glycation endproducts on gene expression profiles of human dermal fibroblasts

The Maillard reaction and its end products, AGE-s (Advanced Glycation End products) are rightly considered as one of the important mechanisms of post-translational tissue modifications with aging. We studied the effect of two AGE-products prepared by the glycation of lysozyme and of BSA, on the expression profile of a large number of genes potentially involved in the above mentioned effects of AGE-s. The two AGE-products were added to human skin fibroblasts and gene expression profiles investigated using microarrays. Among the large number of genes monitored the expression of 16 genes was modified by each AGE-preparations, half of them only by both of them. Out of these 16 genes, 12 were more strongly affected, again not all the same for both preparations. Both of them upregulated MMP and serpin-expression and downregulated some of the collagen-chain coding genes, as well as the cadherin- and fibronectin genes. The BSA-AGE preparation downregulated 10 of the 12 genes strongly affected, only the serpin-1 and MMP-9 genes were upregulated. The lysozyme-AGE preparation upregulated selectively the genes coding for acid phosphatase (ACP), integrin chain α5 (ITGA5) and thrombospondin (THBS) which were unaffected by the BSA-AGE preparation. It was shown previously that the lysozyme-AGE strongly increased the rate of proliferation and also cell death, much more than the BSA-AGE preparation. These differences between these two AGE-preparations tested suggest the possibility of different receptor-mediated transmission pathways activated by these two preparations. Most of the gene-expression modifications are in agreement with biological effects of Maillard products, especially interference with normal tissue structure and increased tissue destruction.

The authors exposed fibroblasts to two types of AGE-modified (AGE-ylated?) proteins, which had overlapping but non-identical effects on gene expression. The common features of the response to the two proteins are most intriguing, however: increased transcription of matrix metalloproteases (MMPs), which break down the extracellular matrix (ECM), and decreased transcription of ECM components like collagen and fibronectin. Taken together, these effects would result in a net weakening of the ECM, which in turn would have profoundly negative effects on organ function, ranging from skin wrinkling to cardiomyopathy.

On another note: increased MMPs and ECM breakdown are hallmarks of fibroblast senescence, which is usually associated with DNA damage or telomere shortening — could AGEs be stimulating premature senescence, either by damaging DNA or via some other pathway?

 

A clinical view of Hutchinson-Gilford progeria

Sometimes, biogerontologists get so enraptured by the molecular and cell-biological details of our studies that we forget what might be called the “human element” — the experience of an individual who is going through the process of aging. This process can be associated with significant discomfort and indignity, so it is all the more tragic when it occurs too early, as in the case of progeroid syndromes such as Hutchinson-Gilford progeria syndrome (HGPS).

I’ve generally focused on the molecular underpinnings of the disease, especially on recent findings that mutations in the lamin A gene are responsible for HGPS, possibly by causing accelerated cellular senescence or interfering with the cell cycle. What I haven’t done is talk much about what it’s like to have the condition.

Therefore, I read with interest this article by Merideth et a great many al., in which the authors report a detailed study of the clinical progression of this rare and devastating genetic disease:

Phenotype and course of Hutchinson-Gilford progeria syndrome

BACKGROUND: Hutchinson-Gilford progeria syndrome is a rare, sporadic, autosomal dominant syndrome that involves premature aging, generally leading to death at approximately 13 years of age due to myocardial infarction or stroke. The genetic basis of most cases of this syndrome is a change from glycine GGC to glycine GGT in codon 608 of the lamin A (LMNA) gene, which activates a cryptic splice donor site to produce abnormal lamin A; this disrupts the nuclear membrane and alters transcription. METHODS: We enrolled 15 children between 1 and 17 years of age, representing nearly half of the world’s known patients with Hutchinson-Gilford progeria syndrome, in a comprehensive clinical protocol between February 2005 and May 2006. RESULTS: Clinical investigations confirmed sclerotic skin, joint contractures, bone abnormalities, alopecia, and growth impairment in all 15 patients; cardiovascular and central nervous system sequelae were also documented. Previously unrecognized findings included prolonged prothrombin times, elevated platelet counts and serum phosphorus levels, measured reductions in joint range of motion, low-frequency conductive hearing loss, and functional oral deficits. Growth impairment was not related to inadequate nutrition, insulin unresponsiveness, or growth hormone deficiency. Growth hormone treatment in a few patients increased height growth by 10% and weight growth by 50%. Cardiovascular studies revealed diminishing vascular function with age, including elevated blood pressure, reduced vascular compliance, decreased ankle-brachial indexes, and adventitial thickening. CONCLUSIONS: Establishing the detailed phenotype of Hutchinson-Gilford progeria syndrome is important because advances in understanding this syndrome may offer insight into normal aging. Abnormal lamin A (progerin) appears to accumulate with aging in normal cells.

The situation sounds grim, and it is. Happily, the news is not all bad: as a result of the identification of the disease gene and a brilliant insight into the biochemistry of the underlying pathology, HGPS patients are currently showing promising responses to treatment with farnesyltransferase inhibitors. These compounds, originally designed as antitumor drugs, have demonstrated beneficial effects on some (but not all, as pointed out in an article under discussion at Longevity Meme) of the symptoms of HGPS.

 

Histone H1 in aging immune cells

From Happel et al., a detailed characterization of histone H1 profiles in peripheral blood lymphocytes over the course of aging. The authors demonstrate significant differences in post-translational modifications of several histone H1 isoforms, and conclude that these changes may be implicated in age-related chromatin remodeling:

H1 histone subtype constitution and phosphorylation state of the ageing cell system of human peripheral blood lymphocytes

Until a few years ago, the H1 histones were exclusively considered to be the architectural proteins of chromatin involved in chromatin condensation. However there is now increasing data to support the hypothesis that the H1 subtypes are involved in genomic integrity and that they may have unexpected functional roles in various biological processes such as in differentiation and DNA repair, apoptosis and lifespan. Moreover, the H1 histones are phosphorylated to a great extent. Recent work has implicated phosphorylation of H1 in the regulation of chromatin remodeling. In light of the fact that chromatin reorganization and heterochromatin formation has been shown to take place during ageing and senescence, in the present investigation, we have analyzed the changes that take place in the somatic H1 linker histone subtype profile and their phosphorylation states in human peripheral blood lymphocytes as a function of donor age. Results from this work show that there is a significant age-related dephosphorylation of H1.4 and H1.5 and an increase in the heterochromatin protein HP1α as a function of donor age. These results indicate that dephosphorylation of H1 histones may be related to an increase in senescence-associated heterochromatin formation during the in vivo ageing of human peripheral blood lymphocytes.

The specific situation in immune cells appears to be quite different from the case in fibroblasts, where histone H1 is completely lost at senescence. In both cases, however, the change in histone H1 profile is tightly associated with the form of chromatin modeling termed Senescence-Associated Heterochromatic Foci (SAHFs).

 

A bridge between sirtuins and Werner’s

Because every longevity-control gene eventually is eventually shown to interact with every other longevity-control gene, it is perhaps not surprising that SIRT1 deacetylates WRN, the protein whose gene is mutated in the devastating human progeria Werner’s Syndrome. Both the helicase and exonuclease activities of the WRN protein are more active in the deacetylated state; thus, the longevity-assurance gene (SIRT1) is responsible for boosting the activity of the major player in the cellular response to DNA damage (WRN), which is the way we’d expect it to work.

Two other recent reports describing progress on WRN reveal that the protein plays a significant role in DNA metabolism under normal growth and after DNA damage: WRN is required both for replication fork progression after genotoxic stress as well as suppressing the spontaneous formation of telomeric DNA circles. These latter structures (which remind me of the extrachromosomal ribosomal DNA circles from the early years of the sirtuin field) are associated with both telomere shortening and cellular senescence.

 

Natural products that fight molecular aging

Skin wrinkling, one of the most conspicuous signs of aging, is the result of breakdown of structural molecules such as collagen and elastin. In our lab and others, we’re accumulating evidence that proteases secreted by senescent fibroblasts are to blame.

Therefore, my ears always prick up when I hear reports like a recent one from Kim and Chung, who describe a plant alkaloid (berberine) that can reduce the production of matrix metalloproteases by dermal fibroblasts in response to UV irradiation. Is the compound decreasing senescence in response to DNA damage — possibly allowing mutated cells to escape cell cycle arrest — or is it instead diminishing the senescence-associated secretion of degradative enzymes? If the latter is true, the protein targeted by berberine will be an excellent target for pharmaceutical development.

Also on a plant-related note, another group has identified a botanical compound that slows the production of advanced glycation endproducts (AGEs); we’ve seen similar results from corn silk extracts. It does make one wonder whether an anti-aging pharmacopoeia is just sitting there in nature, waiting to be discovered.

 

Stress, APP, and Alzheimer’s disease

File under “Stress is bad for you, Reason #6508.” Chronic stress increases the expression of the amyloid precursor protein (APP); the abnormal processing of this protein is a key event in the development of Alzheimer’s disease (AD).

Of course that’s not the whole story: Numerous factors contribute to the pathologenesis of AD, including inflammatory factors secreted by glial cells; this is likely exacerbated by glial senescence, which accumulates with age.

 

Elastin degradation in cardiovascular aging

If you removed every living cell from a human body and looked at the result, you’d still see something recognizably human: bones, of course, and the keratins that make up our skin and hair…and, forming a fine lacework throughout the entire body, the extracellular matrix (ECM). The ECM, which is a particularly prominent feature of connective tissue, consists primarily of large protein complexes that provide structural support (e.g., collagen) as well as elasticity (e.g., the appropriately named elastin).

Elastin is involved in one of the most visible consequences of aging: Over time, elastin is broken down (possibly by proteases secreted by senescent cells), and the skin becomes less resistant to mechanical force. We fight the good fight but eventually gravity wins, and we get wrinkles, wattles, and various other sorts of unmentionable sags. This is specific to later life in part because elastin is only produced during early development and childhood: What you have when you’re an adult is basically all you’ll ever have.

Elastin also has important roles inside the body, most significantly in providing the vasculature and heart with resilience and load-bearing capacity. Indeed, as reported by Pezet et al., mice that are haploinsufficient for elastin display several vascular anomalies and signs of premature cardiac aging. These animals have high blood pressure, narrow and rigid arteries, and cardiac hypertrophy even as young adults. The mice have normal lifespans, but the strain used in these studies all die of a stereotyped set of tumors at an early-to-medium age (for a mouse), so total longevity may be uninformative here.

In light of such findings, it has been suggested (as in this review by Robert et al.) that the age-related breakdown of elastin may place an upper bound on the maximum natural lifespan of the human cardiovascular system (and therefore of any human dependent on such a system).

Solution. “More elastin” sounds obvious, though one would have to be very careful: even though elastin provides elasticity, too much of it might make the arteries and heart overly rigid and unable to perform functionally necessary deformations (think about trying to blow up two nested balloons). Furthermore, excessive deposition of ECM protein in general could result in fibrosis. I would propose a two-fold approach: attack the sources of elastin degradation — calcium deposition, sun damage, and proteases secreted by senescent cells — and in the meantime, figure out how to synthesize more elastin exactly (and only) when it’s needed, so that tissue homeostasis can be preserved without untoward consequences.