The Role of Damage Response Pathways in Stem Cell Aging

If one accepts the premise that stem cell depletion contributes to aging, inhibition of checkpoints that regulate cellular damage by shunting such cells into pathways ending in apoptosis or senescence may preserve stem

Fasl Induced Calcium

Fig. 1.4. Homeostatic balance between stem cell replication and carcinogenesis. The balance between malignancy and stem cell frequency is tightly regulated by cell cycle checkpoints and DNA damage sensors. This pathway demonstrates the upregulation of p21 in response to critically short telomeres resulting in the cell elimination in order to prevent tumorigenesis. However, if p21 is unable to respond, the outcome is an increase in lifespan and an increase in stem cell frequency

Fig. 1.4. Homeostatic balance between stem cell replication and carcinogenesis. The balance between malignancy and stem cell frequency is tightly regulated by cell cycle checkpoints and DNA damage sensors. This pathway demonstrates the upregulation of p21 in response to critically short telomeres resulting in the cell elimination in order to prevent tumorigenesis. However, if p21 is unable to respond, the outcome is an increase in lifespan and an increase in stem cell frequency cell numbers at the risk of increased tumorigenesis. For example, p21, encoded by Cdknla, a cell cycle inhibitor and downstream target of p53, is upregulated in response to short telomeres, consistent with it being important in averting catastrophic genomic rearrangements via processes that eliminate cells (74, 77). In the context of telomeres and aging, a recent study examined the combined effects of Terc and Cdknla deletion. The absence of p21 in fourth generation (G4) Terc~'~ mice, with critically short telom-eres, prolonged their lifespan and improved stem cell function in the hematopoietic system and intestinal epithelium (78). Surprisingly, it did so without an acceleration of tumorigenesis. The fact that p21 deletion did not extend lifespan to that found in G4 Terc~'+ mice, demonstrates that both p21-dependent and p21-independent mechanisms limit longevity in the context of telomere dysfunction (78).

Fifty years ago, Denham Harman posited and championed what is now called the free radical theory of aging (79). Long-lived cells such as stem cells run the risk of accumulating debilitating damage from a lifetime of normal metabolism and exposure to toxins and cellular stressors (Fig. 1.5). However, until recently, the mechanisms by which reactive oxygen species (ROS) impinge on and are responded to by stem cells has been murky.

Fig. 1.5. Common sources of stress in aging hematopoietic stem cells that can accumulate, resulting in cellular damage

Elucidation of the role in stem cells of the ATM (Ataxia Telangiectasia Mutated) damage-response pathway has begun to clear up the picture. To interrelate telomere regulation with cellular damage responses, it should be noted that one of the cardinal roles of ATM is in telomere regulation (80). In Atm~'~ mice, not only do telomeres dysfunction but cellular oxidant levels rise and it was found that the hematopoietic stem cell population was diminished resulting in aplasia and severe anemia in mice only 6 months old (81, 82). Treatment of Atm~'~ mice with the hydrogen peroxide scavenger N-acetyl-cysteine rescued the stem cell aplasia and restored self-renewal via mechanisms independent from the role of ATM in maintaining telomere stability (81). In a recently published extension of these studies, Ito and colleagues showed that oxidative stress in stem cells, but not progenitor cells, activated the p38 MAPK pathway, a relatively general damage control pathway responding to diverse cellular insults (83). This pathway was con-stitutively activated in Atm~'~ mice and was associated with heightened stem cell proliferation. Inhibition of the p38 MAPK pathway reversed stem cell aplasia by returning the bulk of stem cells to a quiescent state. To relate these findings to aging, the authors simulated stem cell aging by serial transplant experiments in wild-type mice. With each transplant not only did the levels of ROS increase in purified stem cells, but p38 MAPK pathway activity increased in concert (83). Thus, stem cells may be depleted during natural aging by an entrained series of events beginning with the accumulation of ROS, subsequent activation of p38, and an increased stem cell cycling but not self-renewal. Since in other cell types increased proliferation is associated with increased cellular ROS (84, 85), a positive feedback loop may similarly be established that enforces the demise of the population. These recent findings are outlined in scenarios depicted in Fig. 1.6.

Recent evidence demonstrates that this may occur not only through the p38 pathway but via the p16 INK4A-retinoblastoma (RB) activation of protein kinase C (86). Evidence supporting this arm of the cellular damage control

P16 And Mapk Pathways

Fig. 1.6. The ATM pathway effects stem cell number. (A) The normal function of the ATM pathway yields a normal stem cell number. Here, ATM detects the levels of ROS. When ROS levels reach a critical amount, p38 MAPK is activated, resulting in stem cell self-renewal and proper maintenance of the stem cell pool. (B) Mice lacking ATM cannot control the levels of ROS, leading to an increase in oxidative stress. This increase causes constitutive activation of p38 MAPK and excessive stem cell proliferation resulting in differentiation rather than self-renewal. This depletion of the stem cell pool manifests in the whole animal as stem cell aplasia. (C) Long-term treatment with antioxidants can deter the constitutive activation of p38 MAPK seen in the ATM knockout mice. With this intervention, the stem cell pool is maintained at normal levels. (D) Pharmacologic inhibition of p38 MAPK is another method of restoring normal stem cell proliferation rates and maintaining the stem cell pool in ATM knockout mice. This is not surprising since p38 inhibitors are often incorporated into aplastic anemia therapies

Fig. 1.6. The ATM pathway effects stem cell number. (A) The normal function of the ATM pathway yields a normal stem cell number. Here, ATM detects the levels of ROS. When ROS levels reach a critical amount, p38 MAPK is activated, resulting in stem cell self-renewal and proper maintenance of the stem cell pool. (B) Mice lacking ATM cannot control the levels of ROS, leading to an increase in oxidative stress. This increase causes constitutive activation of p38 MAPK and excessive stem cell proliferation resulting in differentiation rather than self-renewal. This depletion of the stem cell pool manifests in the whole animal as stem cell aplasia. (C) Long-term treatment with antioxidants can deter the constitutive activation of p38 MAPK seen in the ATM knockout mice. With this intervention, the stem cell pool is maintained at normal levels. (D) Pharmacologic inhibition of p38 MAPK is another method of restoring normal stem cell proliferation rates and maintaining the stem cell pool in ATM knockout mice. This is not surprising since p38 inhibitors are often incorporated into aplastic anemia therapies pathway is outlined below and summarized in Fig. 1.7. Thep16INK4A cyclin-dependent kinase inhibitor has been shown to enforce G1 cell cycle arrest by activating the RB tumor suppressor which subsequently induces irreversible cellular senescence. In a recent series of papers the role the p16 ink4A-rb pathway has been brought to the fore in the biology of stem cell aging, not only in the hematopoietic system but in pancreatic islets and in neural stem cells as well (6, 87, 88). In all three regenerative systems, p16 ink4a rose in stem cells during aging. In the hematopoietic system the increase in p16 inK4A was found only in the most primitive stem cell population, and not in any of their immediate progeny (6). In p16 INK4A-/- mice the size of the hematopoietic stem cell population was similar to that of wild-type animals and stem cells from the two strains had equal abilities to reconstitute irradiated recipients. However, after aging 14-24 months, the

Effects of Age

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Fig. 1.7. The effects of age on the p16 INK4A pathway. The levels of p16INK4A are low in the primitive hematopoietic stem cells of young mice. Thus, there are low activation levels of the RB tumor suppressor leading to few cells in cell cycle arrest (low senescence). In the primitive hematopoietic stem cells of old mice, the levels of p16 INK4A are high, leading to an increase in RB activation and an increase in the number of cells in G1 cell cycle arrest (high senescence). In p16 INK4A knockout mice subjected to serial transplantation the effects of the gene deletion on the stem cell pool were dramatic. Young cells lacking p16 INK4A were unable to activate the RB pathway to decrease cell cycling and maintain levels of stem cell self-renewal. With the stress of serial transplantation, these mice demonstrated premature exhaustion compared to the wild-type young mice. The old mice also lacked p16 INK4A and therefore could not activate the RB tumor suppressor pathway. Accordingly, they demonstrate low levels of G1 cell cycle arrest. Thus, the cells have increased cell cycling compared to their wild-type counterparts and are able to maintain superior regenerative capacity during serial transplantation knockout mice had a significantly larger stem cell population with a larger fraction proliferating and were better able to engraft in a transplant setting. Surprisingly, when subjected to serial transplantation, stem cells from young pl6 INK4A-1- mice showed premature exhaustion compared to age-matched wild-type controls. However, when the same serial transplantation comparison was made using old donor animals, the opposite was true: stem cells from old p16 knockout mice had superior regenerative capacity (6). Thus p16 INK4A has age-specific effects on hematopoietic stem cell repopulation as has been shown in other populations (89-91). Prevention of the age-related increase of p16 INK4A in stem cells clearly had significant advantages in terms of both numbers and repopulating function. However, the p16 INK4A-deficient animals are cancer-prone and have reduced lifespans as a result (92). As pointed out in a commentary to these three papers, the capacity of stem cells for regeneration must be balanced against the probabilities of tumorigenesis (91). While the p16 INK4A-RB pathway may limit stem cell numbers through senescence and apoptosis, it acts as a tumor suppressor by eliminating pre-cancerous cells and thus extending a healthy lifespan.

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