Heidi Scrable

Fat tissue, aging, and cellular senescence.

Fat tissue, frequently the largest organ in humans, is at the nexus of mechanisms involved in longevity and age‐related metabolic dysfunction. Fat distribution and function change dramatically throughout life. Obesity is associated with accelerated onset of diseases common in old age, while fat ablation and certain mutations affecting fat increase life span. Fat cells turn over throughout the life span. Fat cell progenitors, preadipocytes, are abundant, closely related to macrophages, and dysdifferentiate in old age, switching into a pro‐inflammatory, tissue‐remodeling, senescent‐like state. Other mesenchymal progenitors also can acquire a pro‐inflammatory, adipocyte‐like phenotype with aging. We propose a hypothetical model in which cellular stress and preadipocyte overutilization with aging induce cellular senescence, leading to impaired adipogenesis, failure to sequester lipotoxic fatty acids, inflammatory cytokine and chemokine generation, and innate and adaptive immune response activation. These pro‐inflammatory processes may amplify each other and have systemic consequences. This model is consistent with recent concepts about cellular senescence as a stress‐responsive, adaptive phenotype that develops through multiple stages, including major metabolic and secretory readjustments, which can spread from cell to cell and can occur at any point during life. Senescence could be an alternative cell fate that develops in response to injury or metabolic dysfunction and might occur in nondividing as well as dividing cells. Consistent with this, a senescent‐like state can develop in preadipocytes and fat cells from young obese individuals. Senescent, pro‐inflammatory cells in fat could have profound clinical consequences because of the large size of the fat organ and its central metabolic role.

Phosphorylation Regulates SIRT1 Function

Background SIR2 is an NAD+-dependent deacetylase [1]–[3] implicated in the regulation of lifespan in species as diverse as yeast [4], worms [5], and flies [6]. We previously reported that the level of SIRT1, the mammalian homologue of SIR2 [7], [8], is coupled to the level of mitotic activity in cells both in vitro and in vivo [9]. Cells from long-lived mice maintained SIRT1 levels of young mice in tissues that undergo continuous cell replacement by proliferating stem cells. Changes in SIRT1 protein level were not associated with changes in mRNA level, suggesting that SIRT1 could be regulated post-transcriptionally. However, other than a recent report on sumoylation [10] and identification of SIRT1 as a nuclear phospho-protein by mass spectrometry [11], post-translational modifications of this important protein have not been reported. Methodology/Principal Findings We identified 13 residues in SIRT1 that are phosphorylated in vivo using mass spectrometry. Dephosphorylation by phosphatases in vitro resulted in decreased NAD+-dependent deacetylase activity. We identified cyclinB/Cdk1 as a cell cycle-dependent kinase that forms a complex with and phosphorylates SIRT1. Mutation of two residues phosphorylated by Cyclin B/Cdk1 (threonine 530 and serine 540) disturbs normal cell cycle progression and fails to rescue proliferation defects in SIRT1-deficient cells [12], [13]. Conclusions/Significance Pharmacological manipulation of SIRT1 activity is currently being tested as a means of extending lifespan in mammals. Treatment of obese mice with resveratrol, a pharmacological activator of SIRT1, modestly but significantly improved longevity and, perhaps more importantly, offered some protection against the development of type 2 diabetes mellitus and metabolic syndrome [14]–[16]. Understanding the endogenous mechanisms that regulate the level and activity of SIRT1, therefore, has obvious relevance to human health and disease. Our results identify phosphorylation by cell cycle dependent kinases as a major mechanism controlling the level and function of this sirtuin and complement recent reports of factors that inhibit [17], [18] and activate [19] SIRT1 by protein-protein interactions.

Progressive loss of SIRT1 with cell cycle withdrawal

Sir2 is an NAD+‐dependent deacetylase that regulates lifespan in yeast, worms and flies. The mammalian orthologs of Sir2 include SIRT1 in humans and mice. In this study, we analyzed the level of SIRT1 in human lung fibroblasts (IMR90) and mouse embryonic fibroblasts (MEFs) from mice with normal, accelerated, and delayed aging. SIRT1 protein, but not mRNA, decreased significantly with serial cell passage in both human and murine cells. Mouse SIRT1 decreased rapidly in prematurely senescent (p44 Tg) MEFs, remained high in MEFs with delayed senescence (Igf‐1r–/–), and was inversely correlated with senescence‐activated β‐galactosidase (SA‐βGal) activity. Reacquisition of mitotic capability following spontaneous immortalization of serially passaged wild‐type MEFs restored the level of SIRT1 to that of early passage, highly proliferative MEFs. In mouse and human fibroblasts, we found a significant positive correlation between the levels of SIRT1 and proliferating cell nuclear antigen (PCNA), a DNA processing factor expressed during S‐phase. In the animal, we found that SIRT1 decreased with age in tissues in which mitotic activity also declines, such as the thymus and testis, but not in tissues such as the brain in which there is little change in mitotic activity throughout life. Again, the decreases in SIRT1 were highly correlated with decreases in PCNA. Finally, loss of SIRT1 with age was accelerated in mice with accelerated aging but was not observed in long‐lived growth hormone‐receptor knockout mice. Thus, as mitotic activity ceases in mouse and human cells in the normal environment of the animal or in the culture dish, there is a concomitant decline in the level of SIRT1.

Isolation and Proteomic Analysis of Mouse Sperm Detergent-Resistant Membrane Fractions. Evidence for Dissociation of Lipid Rafts During Capacitation

Abstract Mammalian sperm acquire fertilization capacity after residing in the female tract during a process known as capacitation. The present study examined whether cholesterol efflux during capacitation alters the biophysical properties of the sperm plasma membrane by potentially reducing the extent of lipid raft domains as analyzed by the isolation of detergent-resistant membrane fractions using sucrose gradients. In addition, this work investigated whether dissociation of the detergent-resistant membrane fraction during capacitation alters resident sperm raft proteins. Mouse sperm proteins associated with such fractions were studied by silver staining, tandem mass spectrometry, and Western blot analysis. Caveolin 1 was identified in sperm lipid rafts in multimeric states, including a high-molecular-weight oligomer that is sensitive to degradation under reducing conditions at high pH. Capacitation resulted in reduction of the light buoyant-density, detergent-resistant membrane fraction and decreased the array of proteins isolated within this fraction, including loss of the high-molecular-weight caveolin 1 oligomers. Proteomic analysis of sperm proteins isolated in the light buoyant-density fraction identified several proteins, including hexokinase 1, testis serine proteases 1 and 2, TEX101, hyaluronidase (PH20, SPAM1), facilitated glucose transporter 3, lactate dehydrogenase A, carbonic anhydrase IV, IZUMO, pantophysin, basigin, and cysteine-rich inhibitory secretory protein 1. Capacitation also resulted in a significant reduction of sperm labeling by the fluorescent lipid-analog DiIC16, indicating that capacitation alters the liquid-ordered domains in the sperm plasma membrane. The observations that capacitation alters the protein composition of the detergent-resistant membrane fractions is consistent with the hypothesis that cholesterol efflux during capacitation dissociates lipid raft constituents, initiating signaling events that lead to sperm capacitation.

An aging pathway controls the TrkA to p75NTR receptor switch and amyloid β‐peptide generation

Aging of the brain is characterized by marked changes in the expression levels of the neurotrophin receptors, TrkA and p75NTR. An expression pattern in which TrkA predominates in younger animals switches to one in which p75NTR predominates in older animals. This TrkA‐to‐p75NTR switch is accompanied by activation of the second messenger ceramide, stabilization of β‐site amyloid precursor protein‐cleaving enzyme‐1 (BACE1), and increased production of amyloid β‐peptide (Aβ). Here, we show that the insulin‐like growth factor‐1 receptor (IGF1‐R), the common regulator of lifespan and age‐related events in many different organisms, is responsible for the TrkA‐to‐p75NTR switch in both human neuroblastoma cell lines and primary neurons from mouse brain. The signaling pathway that controls the level of TrkA and p75NTR downstream of the IGF1‐R requires IRS2, PIP3/Akt, and is under the control of PTEN and p44, the short isoform of p53. We also show that hyperactivation of IGF1‐R signaling in p44 transgenic animals, which show an accelerated form of aging, is characterized by early TrkA‐to‐p75NTR switch and increased production of Aβ in the brain.

Regenerative capacity of neural precursors in the adult mammalian brain is under the control of p53.

The question of whether or not stem cell loss drives aging in the brain has not been fully resolved. Here, we used mice over-expressing the short isoform of p53 (DeltaNp53 or p44) as a model of aging to gain insight into the cellular mechanisms underlying age-related functional deficits in the brain. By BrdU labeling, we observed an accelerated decline in the number of subventricular zone proliferating cells with age in p44Tg mice compared to mice with normal p53 expression. A 2-3-fold reduction in the number of slowly dividing stem cells was evident in the subventricular zone of 9-12-month-old p44Tg mice, but not in younger p44Tg mice or in normal mice. Consequently, the supply of new olfactory bulb neurons was also reduced. The number and size of neurospheres generated from subventricular zone cells from p44Tg mice was significantly reduced, and cells derived from these neurospheres had limited self-renewal and amplification capacities. At the cellular level, p44 lengthened the cell cycle and affected cell cycle reentry properties, evident by an increased proportion of cells in G0. At the functional level, p44 expression resulted in impaired olfactory discrimination in 15-16-month-old mice. This phenotype is driven by constitutive activation of p53 and constitutive expression of p21(Cip1/waf1) in neural stem cells. Our results demonstrate that p53 plays a crucial role in the maintenance of the regenerative capacity of the brain by regulating the proliferation of stem and progenitor cells.

Δ40p53 controls the switch from pluripotency to differentiation by regulating IGF signaling in ESCs

Δ40p53 is a transactivation-deficient isoform of the tumor suppressor p53. We discovered that Δ40p53, in addition to being highly expressed in embryonic stem cells (ESCs), is the major p53 isoform during early stages of embryogenesis in the mouse. By altering the dose of Δ40p53 in ESCs, we identified a critical role for this isoform in maintaining the ESC state. Haploinsufficiency for Δ40p53 causes a loss of pluripotency in ESCs and acquisition of a somatic cell cycle, while increased dosage of Δ40p53 prolongs pluripotency and inhibits progression to a more differentiated state. Δ40p53 controls the switch from pluripotent ESCs to differentiated somatic cells by controlling the activity of full-length p53 at critical targets such as Nanog and the IGF-1 receptor (IGF-1R). The IGF axis plays a central role in the switch between pluripotency and differentiation in ESCs-and Δ40p53, by controlling the level of the IGF-1R, acts as a master regulator of this switch. We propose that this is the primary function of Δ40p53 in cells of the early embryo and stem cells, which are the only normal cells in which this isoform is expressed.

Altered longevity-assurance activity of p53:p44 in the mouse causes memory loss, neurodegeneration and premature death

The longevity‐assurance activity of the tumor suppressor p53 depends on the levels of Δ40p53 (p44), a short and naturally occurring isoform of the p53 gene. As such, increased dosage of p44 in the mouse leads to accelerated aging and short lifespan. Here we show that mice homozygous for a transgene encoding p44 (p44+/+) display cognitive decline and synaptic impairment early in life. The synaptic deficits are attributed to hyperactivation of insulin‐like growth factor 1 receptor (IGF‐1R) signaling and altered metabolism of the microtubule‐binding protein tau. In fact, they were rescued by either Igf1r or Mapt haploinsufficiency. When expressing a human or a ‘humanized’ form of the amyloid precursor protein (APP), p44+/+ animals developed a selective degeneration of memory‐forming and ‐retrieving areas of the brain, and died prematurely. Mechanistically, the neurodegeneration was caused by both paraptosis‐ and autophagy‐like cell deaths. These results indicate that altered longevity‐assurance activity of p53:p44 causes memory loss and neurodegeneration by affecting IGF‐1R signaling. Importantly, Igf1r haploinsufficiency was also able to correct the synaptic deficits of APP695/swe mice, a model of Alzheimer’s disease.

Δ40 Isoform of p53 Controls β-Cell Proliferation and Glucose Homeostasis in Mice

OBJECTIVE Investigating the dynamics of pancreatic β-cell mass is critical for developing strategies to treat both type 1 and type 2 diabetes. p53, a key regulator of the cell cycle and apoptosis, has mostly been a focus of investigation as a tumor suppressor. Although p53 alternative transcripts can modulate p53 activity, their functions are not fully understood. We hypothesized that β-cell proliferation and glucose homeostasis were controlled by Δ40p53, a p53 isoform lacking the transactivation domain of the full-length protein that modulates total p53 activity and regulates organ size and life span in mice. RESEARCH DESIGN AND METHODS We phenotyped metabolic parameters in Δ40p53 transgenic (p44tg) mice and used quantitative RT-PCR, Western blotting, and immunohistochemistry to examine β-cell proliferation. RESULTS Transgenic mice with an ectopic p53 gene encoding Δ40p53 developed hypoinsulinemia and glucose intolerance by 3 months of age, which worsened in older mice and led to overt diabetes and premature death from ∼14 months of age. Consistent with a dramatic decrease in β-cell mass and reduced β-cell proliferation, lower expression of cyclin D2 and pancreatic duodenal homeobox-1, two key regulators of proliferation, was observed, whereas expression of the cell cycle inhibitor p21, a p53 target gene, was increased. CONCLUSIONS These data indicate a significant and novel role for Δ40p53 in β-cell proliferation with implications for the development of age-dependent diabetes.

A genetic program for deletion of foreign DNA from the mammalian genome

Mammalian genomes are in constant jeopardy of invasion by prokaryotic DNA sequences because of their extensive exposure to bacteria; however, mammalian genomes appear to be protected from horizontal transmission of bacterial DNA. Transgenic mice provide a convenient model system for investigating the capacity of mammalian genomes in vivo to retain, silence, and/or reject foreign DNAs. We have previously reported that bacterial genes encoding the Lac repressor (lacI) are subject to sequence-dependent methylation and silencing in the transgenic mouse. In this paper, we report that bacterially derived lacI transgenes, but not their mammalian counterparts, can also be eliminated from the somatic cell DNA of affected animals. This somatic instability is heritable, strain-dependent, and conferred in cis. Our data are consistent with a model of genome surveillance in the mouse which can lead to loss of foreign DNA and which may be analogous to restriction-modification systems that maintain the integrity of the bacterial genome.