| Guarente lab | Amon lab | Tsai lab |
SIR2 and related genes (sirtuins) are NAD-dependent deacylases that link metabolism, epigenetics and aging in a variety of species. Sirtuins are also involved in the longevity conferred by dietary or calorie restriction (CR). The mammalian sirtuins SIRT1-7 also protect against many diseases of aging, including the major neurodegenerative diseases. New data underscore the importance of NAD+ metabolism in aging. NAD+ levels decline in mice and humans with aging, leading to inactivation of sirtuins. Mouse studies demonstrate that declining NAD+ thereby limits health span and life span. Moreover, NAD+ can be replenished by NAD+ precursors in the diet to restore health benefits in aging animals. Our lab has studied the roles of sirtuins and NAD+ in aging in a variety of organisms, including yeast, C. elegans, and mice. We have also examined how expression of sirtuins, especially the SIR2 ortholog SIRT1, can protect against diseases in mouse models, including diabetes, kidney disease, cancer, and ALS. Also we have probed how SIRT1 might influence functions important in restraining aging, such as circadian rhythm and adult stem cell maintenance. For examine, Intestinal stem cells (ISCs) decrease in number and function in old mice, and NAD+ precursors can restore the number and function of ISCs in these animals. In addition, NAD+ precursors have now been shown to boost NAD+ levels in humans sustainably and may bring health benefits. Finally, we are very interested in the aging of the human brain and musculature. In this regard, we have initiated a study of human brain aging based on computational analysis of the transcriptomes of human tissue samples. We have also initiated an analysis of a candidate gene in human muscle aging, dysferlin. This latter study includes an assessment of >100 polymorphisms in the dysferlin gene that change amino acids in the protein may predispose to muscular dystrophy in homozygous people, and we hypothesize may lead to muscle loss with aging in heterozygous people.
Aneuploidy is defined as a chromosome composition that is not a multiple of the haploid complement. The effects of chromosome gains and losses on human health are severe. Aneuploidy is the leading cause of miscarriages and mental retardation and a hallmark of cancer. Our studies and that of others indicate that aneuploidy is a cause of aging. Imbalanced karyotypes lead to senescence in cultured cells and premature aging in mice (Baker et al., 2004; Sheltzer et al., 2017). The cellular consequences of aneuploidy provide an explanation for how the condition causes aging. Aneuploidy elicits many phenotypes characteristic of aging cells such as proteotoxic stress (Torres et al., 2007; Tang et al., 2011; Oromendia et al., 2012; Santaguida et al., 2015), genomic instability (Sheltzer et al., 2011; Santaguida et al., 2017), and metabolic alterations (Torres et al., 2007; Williams et al., 2008). The goal of our studies is to understand how aneuploidy brings about these aging phenotypes.
Aneuploidy - a cause of aging in mammals:
Based on the observations that aneuploidy causes senescence we investigated the karyotypic landscape during normal human aging. We used single cell sequencing to obtain a genome-wide, high-resolution assessment of chromosome copy number alterations in human tissues of individuals ranging in age from 40 – 70. We discovered large copy number variations (CNVs) in 8-9% of cells across tissues indicating that large CNVs are rare within and across individuals (Knouse et al., 2014; Knouse et al., 2016). These observations raised a key question. If aneuploidy is not prevalent in aged tissues why do syndromes that cause high levels of aneuploidy lead to progeria? To address this question we conducted a survey of the karyotypic landscape of mouse models of chromosome instability (CIN) that lead to progeria (Pfau et al., 2016). This analysis led to a remarkable finding. Non-regenerating adult tissues are highly aneuploid in mice with CIN, but regenerative tissues are largely euploid. This observation leads to two important conclusions. First, it indicates that, in vivo, mechanisms exist that select against aneuploid cells. Second, it provides an explanation for why chromosome instability leads to premature aging. The fact that aneuploid cells are eliminated from tissues necessitates increased proliferation of stem cells to replenish the tissue. This in turn leads to stem cell exhaustion and premature aging. In addition, as CIN animals age, aneuploid cells accumulate in tissues, further contributing to the decline in tissue function.
We have begun to study the mechanisms underlying the elimination of aneuploid cells in tissues and why the process becomes less efficient with age. Recently, we found that cells with highly abnormal karyotypes elicit an immune response in vitro (Santaguida et al., 2017). Primary cells with highly abnormal karyotypes involving numerical and structural chromosome abnormalities arrest in G1, senesce and produce pro-inflammatory signals, causing them to be recognized by Natural Killer cells in vitro. Based on these observations we hypothesize that aneuploid cells are generated during cell division that occurs as part of tissue regeneration but are cleared by the immune system. We hypothesize that as we age immune function declines leading to the accumulation of aneuploid cells and hence a decline in tissue function. We are currently testing this idea.
Aneuploidy - a cause of replicative senescence in budding yeast:
Budding yeast cells produce a finite number of daughter cells before they die. We recently found that the permanent cell cycle arrest that marks the end of a yeast cell’s life is a direct consequence of massive genome instability caused by deregulation of the G1-S phase transition. We find that age-induced accumulation of Whi5, the functional equivalent of the Retinoblastoma protein in yeast, and defects in G1 cyclin transcription cause genomic instability and aneuploidy that result in cell death. Our studies aimed at determining why the G1 – S phase transition becomes impaired in old cells identified extra chromosomal rDNA circles (ERCs) as the cause of G1 cyclin expression defects in old cells. While ERCs have not been found in senescing mammalian cells, a failure to induce expression of G1/S phase genes and accumulation of cell cycle entry inhibitors are key characteristics of mammalian cell senescence. Our findings suggest that the ultimate cause of age-induced cell cycle defects and cell death – deregulation of the G1/S phase transition that causes genome instability and aneuploidy- is fundamentally conserved across eukaryotes. We are now studying how ERCs affect G1 – S regulation and plan to probe whether the mechanisms we discovered in yeast are conserved in mammals.
Damage to the genetic material of the cells, particular nuclear DNA of long-lived neurons, if not properly repaired can result in genomic instability, a common denominator seen in aging brains and neurodegenerative disorders such as Alzheimer’s disease (AD), amyotrophic lateral sclerosis (ALS), and frontotemporal dementia (FTD). In addition, neuroinflammation has emerged as a key feature of many neurodegenerative diseases. Excitedly, recent findings open up the exciting possibility that therapeutic interventions geared toward enhancing the capacity of cells to self-repair their damaged genomes could stall or potentially reverse the accumulated genotoxic effect of damaged DNA, and perhaps preserve cognitive functions in aging brains and prevent the development of neurodegenerative disorders. Exploring the links between DNA damage and neuroinflammation that could open up novel therapeutic interventions against age-related disorders and neurodegeneration will be the primary objectives of our proposed research.
In recent years, work in our lab has identified the accumulation of DNA damage in the form of DNA double strand breaks (DSBs) as an early pathological event in several mouse models of neurodegenerative diseases. In addition, we identified two enzymes, the NAD+-dependent deacetylase, SIRT1, and the class I histone deacetylase, HDAC1, as essential for the repair of DNA DSBs in neurons. Furthermore, pharmacological activation of SIRT1 can promote genomic stability and confer neuroprotection by preventing neuronal loss in CK-p25 mouse model of AD-like neurodegeneration and P301S FTD/tauopathy mice. However, the precise mechanisms that link DNA damage to the development of neurodegenerative diseases remain poorly understood. A major confounding factor is that the DNA lesions and sources of damage that are most pertinent to neurodegeneration remain unknown.
We recently reported that DSBs are produced by neurons in response to normal stimuli (Madabhushi et al., Cell 2015). We showed that DSBs do not occur randomly across the genome, but rather appear then are repaired at specific loci corresponding to immediate early response genes (IEGs) activated in response to stimuli associated with learning and memory. These DSBs are likely mediated by the type II topoisomerase IIβ (Topo IIβ) as reduced Topo IIβ levels by RNAi attenuates DSB formation and IEG expression (Madabhushi et al., Cell 2015). We are currently investigating 1) whether DSBs at certain neuronal activity-regulated genes could be produced in vivo during the normal process of physiological learning; 2) whether DNA damage accumulates randomly or at specific genomic “hotspots” in neurons of aging mice and those suffering neurodegeneration; 3) how gene expression and genome organization are affected in neurons sustaining DNA damage (i.e., gamma-H2AX positive) and their impacts on neuronal function.
We are also pursuing potential mechanistic links between DNA damage and other pathological features in aging brains and neurodegenerative diseases. We recently showed that gene expression patterns in CK-p25 mice are highly correlated with expression changes in human hippocampal gray matter in AD post-mortem brains (Gjoneska et al., Nature 2015). One of the most salient observation from this comparative analysis is the concurrent increased expression of both DNA damage response and immune response/inflammation genes at the earliest stages of pathology in the CK-p25 mice. While DNA damage has been shown to occur predominantly in neurons, it is likely that microglia or other glial cells capable of triggering or relaying immune signals up-regulate the expression of inflammatory response pathways. To deconvolute which specific cell types are involved in the neuroinflammatory response in mouse models of neurodegeneration and aging brains, we will isolate individual brain cell types via FACs analysis against cell type specific markers. We will profile in cell-type specific manner the dynamics of the transcriptomes (i.e., RNA-seq), epigenomes (i.e., ATAC-seq, ChIP-seq), and proteomes (i.e., mass spectrometry). These analyses will enable us to identify which inflammatory pathways and key regulators among the major brain cell types including neurons, oligodendrocytes, astrocytes, and microglia mediate neuroinflammation in the context of neurodegeneration and aging.