Senescent and disease-associated microglia are modifiable features of aged brain white matter.

Brain white matter tracts undergo structural and functional changes linked to late-life cognitive decline, but the cellular and molecular contributions to their selective vulnerability are not well defined. In naturally aged mice, we demonstrate that senescent and disease-associated microglia (DAM) phenotypes converge in hippocampus-adjacent white matter. Through gold-standard gene expression and immunolabeling combined with high-dimensional spatial mapping, we identified microglial cell fates in aged white matter characterized by aberrant morphology, microenvironment reorganization, and expression of senescence and DAM markers, including galectin 3 (GAL3/Lgals3), B-cell lymphoma 2 (Bcl2), and cyclin dependent kinase inhibitors, including Cdkn2a/p16ink4a. Pharmacogenetic or pharmacological targeting of p16ink4a or BCL2 reduced white matter GAL3+ DAM abundance and rejuvenated microglial fimbria organization. Our results demonstrate dynamic changes in microglial identity in aged white matter that can be reverted by senotherapeutic intervention to promote homeostatic maintenance in the aged brain.

Accelerated aging of the brain transcriptome by the common chemotherapeutic doxorubicin.

Cancer is one of the most common age-related diseases, and over one-third of cancer patients will receive chemotherapy. One frequently reported side effect of chemotherapeutic agents like doxorubicin (Doxo) is impaired cognitive function, commonly known as "chemotherapy-induced cognitive impairment (CICI)", which may mimic accelerated brain aging. The biological mechanisms underlying the adverse effects of Doxo on the brain are unclear but could involve mitochondrial dysfunction. Here, we characterized brain (hippocampal) transcriptome and cognitive/behavioral changes in young mice treated with Doxo +/- the mitochondrial therapeutic MitoQ. We found that Doxo altered transcriptome/biological processes related to synaptic transmission and neurotransmitter function, neuronal health and behavior, and that these gene expression changes were: 1) similar to key differences observed in transcriptome data on brain aging; and 2) associated with related, aging-like behavioral differences, such as decreased exploration time and impaired novel object recognition test (NOR, an index of learning/memory) performance. Interestingly, MitoQ partially prevented Doxo-induced transcriptome changes in the brain, but it had no effect on behavior or cognitive function. Collectively, our findings are consistent with the idea that chemotherapeutic agents could induce neuronal/gene expression and behavioral changes similar to those that occur during brain aging. In this context, mitochondrial therapeutics may have potential as treatments for CICI at the biological level, but their effects on behavior/cognitive function require further investigation.

Tumor Necrosis Factor Alpha-Mediated Inflammation and Remodeling of the Extracellular Matrix Underlies Aortic Stiffening Induced by the Common Chemotherapeutic Agent Doxorubicin.

[Figure: see text].

Unmasking Transcriptional Heterogeneity in Senescent Cells.

Cellular senescence is a state of irreversibly arrested proliferation, often induced by genotoxic stress [1]. Senescent cells participate in a variety of physiological and pathological conditions, including tumor suppression [2], embryonic development [3, 4], tissue repair [5-8], and organismal aging [9]. The senescence program is variably characterized by several non-exclusive markers, including constitutive DNA damage response (DDR) signaling, senescence-associated ß-galactosidase (SA-ßgal) activity, increased expression of the cyclin-dependent kinase (CDK) inhibitors p16INK4A (CDKN2A) and p21CIP1 (CDKN1A), increased secretion of many bio-active factors (the senescence-associated secretory phenotype, or SASP), and reduced expression of the nuclear lamina protein LaminB1 (LMNB1) [1]. Many senescence-associated markers result from altered transcription, but the senescent phenotype is variable, and methods for clearly identifying senescent cells are lacking [10]. Here, we characterize the heterogeneity of the senescence program using numerous whole-transcriptome datasets generated by us or publicly available. We identify transcriptome signatures associated with specific senescence-inducing stresses or senescent cell types and identify and validate genes that are commonly differentially regulated. We also show that the senescent phenotype is dynamic, changing at varying intervals after senescence induction. Identifying novel transcriptome signatures to detect any type of senescent cell or to discriminate among diverse senescence programs is an attractive strategy for determining the diverse biological roles of senescent cells and developing specific drug targets.

Analysis of individual cells identifies cell-to-cell variability following induction of cellular senescence.

Senescent cells play important roles in both physiological and pathological processes, including cancer and aging. In all cases, however, senescent cells comprise only a small fraction of tissues. Senescent phenotypes have been studied largely in relatively homogeneous populations of cultured cells. In vivo, senescent cells are generally identified by a small number of markers, but whether and how these markers vary among individual cells is unknown. We therefore utilized a combination of single-cell isolation and a nanofluidic PCR platform to determine the contributions of individual cells to the overall gene expression profile of senescent human fibroblast populations. Individual senescent cells were surprisingly heterogeneous in their gene expression signatures. This cell-to-cell variability resulted in a loss of correlation among the expression of several senescence-associated genes. Many genes encoding senescence-associated secretory phenotype (SASP) factors, a major contributor to the effects of senescent cells in vivo, showed marked variability with a subset of highly induced genes accounting for the increases observed at the population level. Inflammatory genes in clustered genomic loci showed a greater correlation with senescence compared to nonclustered loci, suggesting that these genes are coregulated by genomic location. Together, these data offer new insights into how genes are regulated in senescent cells and suggest that single markers are inadequate to identify senescent cells in vivo.

Cellular Senescence Promotes Adverse Effects of Chemotherapy and Cancer Relapse.

Cellular senescence suppresses cancer by irreversibly arresting cell proliferation. Senescent cells acquire a proinflammatory senescence-associated secretory phenotype. Many genotoxic chemotherapies target proliferating cells nonspecifically, often with adverse reactions. In accord with prior work, we show that several chemotherapeutic drugs induce senescence of primary murine and human cells. Using a transgenic mouse that permits tracking and eliminating senescent cells, we show that therapy-induced senescent (TIS) cells persist and contribute to local and systemic inflammation. Eliminating TIS cells reduced several short- and long-term effects of the drugs, including bone marrow suppression, cardiac dysfunction, cancer recurrence, and physical activity and strength. Consistent with our findings in mice, the risk of chemotherapy-induced fatigue was significantly greater in humans with increased expression of a senescence marker in T cells prior to chemotherapy. These findings suggest that senescent cells can cause certain chemotherapy side effects, providing a new target to reduce the toxicity of anticancer treatments. SIGNIFICANCE: Many genotoxic chemotherapies have debilitating side effects and also induce cellular senescence in normal tissues. The senescent cells remain chronically present where they can promote local and systemic inflammation that causes or exacerbates many side effects of the chemotherapy. Cancer Discov; 7(2); 165-76. ©2016 AACR.This article is highlighted in the In This Issue feature, p. 115.

Vitamin D Promotes Protein Homeostasis and Longevity via the Stress Response Pathway Genes skn-1, ire-1, and xbp-1.

Vitamin D has multiple roles, including the regulation of bone and calcium homeostasis. Deficiency of 25-hydroxyvitamin D, the major circulating form of vitamin D, is associated with an increased risk of age-related chronic diseases, including Alzheimer's disease, Parkinson's disease, cognitive impairment, and cancer. In this study, we utilized Caenorhabditis elegans to examine the mechanism by which vitamin D influences aging. We found that vitamin-D3-induced lifespan extension requires the stress response pathway genes skn-1, ire-1, and xbp-1. Vitamin D3 (D3) induced expression of SKN-1 target genes but not canonical targets of XBP-1. D3 suppressed an important molecular pathology of aging, that of widespread protein insolubility, and prevented toxicity caused by human ß-amyloid. Our observation that D3 improves protein homeostasis and slows aging highlights the importance of maintaining appropriate vitamin D serum levels and may explain why such a wide variety of human age-related diseases are associated with vitamin D deficiency.

Genomic Analysis Reveals Disruption of Striatal Neuronal Development and Therapeutic Targets in Human Huntington's Disease Neural Stem Cells.

We utilized induced pluripotent stem cells (iPSCs) derived from Huntington's disease (HD) patients as a human model of HD and determined that the disease phenotypes only manifest in the differentiated neural stem cell (NSC) stage, not in iPSCs. To understand the molecular basis for the CAG repeat expansion-dependent disease phenotypes in NSCs, we performed transcriptomic analysis of HD iPSCs and HD NSCs compared to isogenic controls. Differential gene expression and pathway analysis pointed to transforming growth factor ß (TGF-ß) and netrin-1 as the top dysregulated pathways. Using data-driven gene coexpression network analysis, we identified seven distinct coexpression modules and focused on two that were correlated with changes in gene expression due to the CAG expansion. Our HD NSC model revealed the dysregulation of genes involved in neuronal development and the formation of the dorsal striatum. The striatal and neuronal networks disrupted could be modulated to correct HD phenotypes and provide therapeutic targets.

Pleiotropic age-dependent effects of mitochondrial dysfunction on epidermal stem cells.

Tissue homeostasis declines with age partly because stem/progenitor cells fail to self-renew or differentiate. Because mitochondrial damage can accelerate aging, we tested the hypothesis that mitochondrial dysfunction impairs stem cell renewal or function. We developed a mouse model, Tg(KRT14-cre/Esr1) (20Efu/J) × Sod2 (tm1Smel) , that generates mitochondrial oxidative stress in keratin 14-expressing epidermal stem/progenitor cells in a temporally controlled manner owing to deletion of Sod2, a nuclear gene that encodes the mitochondrial antioxidant enzyme superoxide dismutase 2 (Sod2). Epidermal Sod2 loss induced cellular senescence, which irreversibly arrested proliferation in a fraction of keratinocytes. Surprisingly, in young mice, Sod2 deficiency accelerated wound closure, increasing epidermal differentiation and reepithelialization, despite the reduced proliferation. In contrast, at older ages, Sod2 deficiency delayed wound closure and reduced epidermal thickness, accompanied by epidermal stem cell exhaustion. In young mice, Sod2 deficiency accelerated epidermal thinning in response to the tumor promoter 12-O-tetradecanoylphorbol-13-acetate, phenocopying the reduced regeneration of older Sod2-deficient skin. Our results show a surprising beneficial effect of mitochondrial dysfunction at young ages, provide a potential mechanism for the decline in epidermal regeneration at older ages, and identify a previously unidentified age-dependent role for mitochondria in skin quality and wound closure.

Selective binding of nuclear alpha-synuclein to the PGC1alpha promoter under conditions of oxidative stress may contribute to losses in mitochondrial function: implications for Parkinson's disease.

Alpha-synuclein has been reported to be present in the nucleus and levels enhanced by oxidative stress. Herein, we sought to investigate the mechanistic role of nuclear alpha-synuclein. We found that alpha-synuclein nuclear localization coincided with enhanced chromatin binding both in an in vitro and a corresponding in vivo brain oxidative stress model previously characterized by our laboratory as well as in PD brain tissues. Genome-wide chromatin immunoprecipitation (ChIP)-on-chip analysis of alpha-synuclein:promoter binding in response to oxidative stress in vitro revealed that binding occurs at several promoters belonging to a range of functional categories including transcriptional regulation. Interestingly, given the important role of mitochondrial dysfunction in PD, this included binding to the promoter for the master mitochondrial transcription activator, PGC1alpha in vitro, in vivo, and in human brain tissue with age and PD. To test the possible mechanistic impact of alpha-synuclein PGC1alpha promotor binding, we assessed PGC1alpha promoter activity, mRNA, and protein levels and expression of candidate PGC1alpha target genes in our in vitro model. All were found to be reduced in conjunction with increased levels of aberrant mitochondrial morphology and impaired mitochondrial function. Exogenous PGC1alpha expression was found to attenuate alpha-synuclein-mediated mitochondrial dysfunction and subsequent neurotoxicity in vitro. Our data suggest that nuclear alpha-synuclein localization under conditions of oxidative stress may impact on mitochondrial function in part via the protein's capacity to act as a transcriptional modulator of PGC1alpha. This represents a novel role for alpha-synuclein as it relates to mitochondrial dysfunction in PD.

cep-1/p53-dependent dysplastic pathology of the aging C. elegans gonad.

The C. elegans germline and somatic gonad are actively developing until the animal reaches adulthood, and then continue to undergo striking changes as the animal ages. Reported changes include a depletion of available sperm, a decrease in oocyte quality up till mid-life, a reduction in germline nuclei, a decrease in fertility, and an accumulation of DNA in the midbody of aging C. elegans. Here, we have focused on the aging gonad in old animals, and show in detail that the aging gonad undergoes a massive uterine growth composed of endoreduplicating oocytes, yolk, and expanses of chromatin. We use a novel series of imaging techniques in combination with histological methodology for reconstructing aged worms in 3-dimensions, and show in old animals growing masses swelling inside the uterus to occupy most of the diameter of the worm. We link this accelerated growth to the cep-1/p53 tumor suppressor. Because cep-1 is required for DNA damage induced apoptosis, and daf-2 limits longevity, these results suggest a role for age-related DNA damage in dysplastic uterine growths, which in some respects resemble premalignant changes that can occur in aging mammals.

Massage therapy attenuates inflammatory signaling after exercise-induced muscle damage.

Massage therapy is commonly used during physical rehabilitation of skeletal muscle to ameliorate pain and promote recovery from injury. Although there is evidence that massage may relieve pain in injured muscle, how massage affects cellular function remains unknown. To assess the effects of massage, we administered either massage therapy or no treatment to separate quadriceps of 11 young male participants after exercise-induced muscle damage. Muscle biopsies were acquired from the quadriceps (vastus lateralis) at baseline, immediately after 10 min of massage treatment, and after a 2.5-hour period of recovery. We found that massage activated the mechanotransduction signaling pathways focal adhesion kinase (FAK) and extracellular signal-regulated kinase 1/2 (ERK1/2), potentiated mitochondrial biogenesis signaling [nuclear peroxisome proliferator-activated receptor γ coactivator 1α (PGC-1α)], and mitigated the rise in nuclear factor κB (NFκB) (p65) nuclear accumulation caused by exercise-induced muscle trauma. Moreover, despite having no effect on muscle metabolites (glycogen, lactate), massage attenuated the production of the inflammatory cytokines tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6) and reduced heat shock protein 27 (HSP27) phosphorylation, thereby mitigating cellular stress resulting from myofiber injury. In summary, when administered to skeletal muscle that has been acutely damaged through exercise, massage therapy appears to be clinically beneficial by reducing inflammation and promoting mitochondrial biogenesis.

Mitochondrial oxidative stress caused by Sod2 deficiency promotes cellular senescence and aging phenotypes in the skin.

Cellular senescence arrests the proliferation of mammalian cells at risk for neoplastic transformation, and is also associated with aging. However, the factors that cause cellular senescence during aging are unclear. Excessive reactive oxygen species (ROS) have been shown to cause cellular senescence in culture, and accumulated molecular damage due to mitochondrial ROS has long been thought to drive aging phenotypesin vivo. Here, we test the hypothesis that mitochondrial oxidative stress can promote cellular senescence in vivo and contribute to aging phenotypes in vivo, specifically in the skin. We show that the number of senescent cells, as well as impaired mitochondrial (complex II) activity increase in naturally aged mouse skin. Using a mouse model of genetic Sod2 deficiency, we show that failure to express this important mitochondrial anti-oxidant enzyme also impairs mitochondrial complex II activity, causes nuclear DNA damage, and induces cellular senescence but not apoptosis in the epidermis. Sod2 deficiency also reduced the number of cells and thickness of the epidermis, while increasing terminal differentiation. Our results support the idea that mitochondrial oxidative stress and cellular senescence contribute to aging skin phenotypes in vivo.

Human embryonic stem cells express elevated levels of multiple pro-apoptotic BCL-2 family members.

Two of the greatest challenges in regenerative medicine today remain (1) the ability to culture human embryonic stem cells (hESCs) at a scale sufficient to satisfy clinical demand and (2) the ability to eliminate teratoma-forming cells from preparations of cells with clinically desirable phenotypes. Understanding the pathways governing apoptosis in hESCs may provide a means to address these issues. Limiting apoptosis could aid scaling efforts, whereas triggering selective apoptosis in hESCs could eliminate unwanted teratoma-forming cells. We focus here on the BCL-2 family of proteins, which regulate mitochondrial-dependent apoptosis. We used quantitative PCR to compare the steady-state expression profile of all human BCL-2 family members in hESCs with that of human primary cells from various origins and two cancer lines. Our findings indicate that hESCs express elevated levels of the pro-apoptotic BH3-only BCL-2 family members NOXA, BIK, BIM, BMF and PUMA when compared with differentiated cells and cancer cells. However, compensatory expression of pro-survival BCL-2 family members in hESCs was not observed, suggesting a possible explanation for the elevated rates of apoptosis observed in proliferating hESC cultures, as well as a mechanism that could be exploited to limit hESC-derived neoplasms.

Cellular senescence: a link between cancer and age-related degenerative disease?

Cellular senescence is an established cellular stress response that acts primarily to prevent the proliferation of cells that experience potentially oncogenic stress. In recent years, it has become increasingly apparent that the senescence response is a complex phenotype, which has a variety of cell non-autonomous effects. The senescence-associated secretory phenotype, or SASP, entails the secretion of numerous cytokines, growth factors and proteases. The SASP can have beneficial or detrimental effects, depending on the physiological context. One recently described beneficial effect is to aid tissue repair. Among the detrimental effects, the SASP can disrupt normal tissue structures and function, and, ironically, can promote malignant phenotypes in nearby cells. These detrimental effects in many ways recapitulate the degenerative and hyperplastic pathologies that develop during aging. Because the SASP is largely a response to genomic or epigenomic damage, we suggest it may be a model for a cellular damage response that can propagate damage signals both within and among tissues. We propose that both the degenerative and hyperplastic diseases of aging may be fueled by such damage signals.

The garlic constituent diallyl trisulfide increases the lifespan of C. elegans via skn-1 activation.

Medicinal benefits of Allium vegetables, such as garlic, have been noted throughout recorded history, including protection against cancer and cardiovascular disease. We now demonstrate that garlic constituent diallyl trisulfide (DATS) increases longevity of Caenorhabditis elegans by affecting the skn-1 pathway. Treatment of worms with 5-10 µM DATS increased worm mean lifespan even when treatment is started during young adulthood. To explore the mechanisms involved in the DATS-mediated increase in longevity, we treated daf-2, daf-16, and eat-2 mutants and found that DATS increased the lifespan of daf-2 and daf-16 mutants, but not the eat-2 mutants. Microarray experiments demonstrated that a number of genes regulated by oxidative stress and the skn-1 transcription factor were also changed by DATS treatment. Consistently, DATS treatment leads to the induction of the skn-1 target gene gst-4, and this induction was dependent on skn-1. We also found that the effects of DATS on worm lifespan depend on skn-1 activity in both in the intestine and ASI neurons. Together our data suggest that DATS is able to increase worm lifespan by enhancing the function of the pro-longevity transcription factor skn-1.

Eccentric exercise activates novel transcriptional regulation of hypertrophic signaling pathways not affected by hormone changes.

Unaccustomed eccentric exercise damages skeletal muscle tissue, activating mechanisms of recovery and remodeling that may be influenced by the female sex hormone 17beta-estradiol (E2). Using high density oligonucleotide based microarrays, we screened for differences in mRNA expression caused by E2 and eccentric exercise. After random assignment to 8 days of either placebo (CON) or E2 (EXP), eighteen men performed 150 single-leg eccentric contractions. Muscle biopsies were collected at baseline (BL), following supplementation (PS), +3 hours (3H) and +48 hours (48H) after exercise. Serum E2 concentrations increased significantly with supplementation (P<0.001) but did not affect microarray results. Exercise led to early transcriptional changes in striated muscle activator of Rho signaling (STARS), Rho family GTPase 3 (RND3), mitogen activated protein kinase (MAPK) regulation and the downstream transcription factor FOS. Targeted RT-PCR analysis identified concurrent induction of negative regulators of calcineurin signaling RCAN (P<0.001) and HMOX1 (P = 0.009). Protein contents were elevated for RND3 at 3H (P = 0.02) and FOS at 48H (P<0.05). These findings indicate that early RhoA and NFAT signaling and regulation are altered following exercise for muscle remodeling and repair, but are not affected by E2.

Limb immobilization induces a coordinate down-regulation of mitochondrial and other metabolic pathways in men and women.

Advancements in animal models and cell culture techniques have been invaluable in the elucidation of the molecular mechanisms that regulate muscle atrophy. However, few studies have examined muscle atrophy in humans using modern experimental techniques. The purpose of this study was to examine changes in global gene transcription during immobilization-induced muscle atrophy in humans and then explore the effects of the most prominent transcriptional alterations on protein expression and function. Healthy men and women (N = 24) were subjected to two weeks of unilateral limb immobilization, with muscle biopsies obtained before, after 48 hours (48 H) and 14 days (14 D) of immobilization. Muscle cross sectional area (approximately 5%) and strength (10-20%) were significantly reduced in men and women (approximately 5% and 10-20%, respectively) after 14 D of immobilization. Micro-array analyses of total RNA extracted from biopsy samples at 48 H and 14 D uncovered 575 and 3,128 probes, respectively, which were significantly altered during immobilization. As a group, genes involved in mitochondrial bioenergetics and carbohydrate metabolism were predominant features at both 48 H and 14 D, with genes involved in protein synthesis and degradation significantly down-regulated and up-regulated, respectively, at 14 D of muscle atrophy. There was also a significant decrease in the protein content of mitochondrial cytochrome c oxidase, and the enzyme activity of cytochrome c oxidase and citrate synthase after 14 D of immobilization. Furthermore, protein ubiquitination was significantly increased at 48 H but not 14 D of immobilization. These results suggest that transcriptional and post-transcriptional suppression of mitochondrial processes is sustained throughout 14 D of immobilization, while protein ubiquitination plays an early but transient role in muscle atrophy following short-term immobilization in humans.

Increase in mitochondrial biogenesis, oxidative stress, and glycolysis in murine lymphomas.

Lymphomas adapt to their environment by undergoing a complex series of biochemical changes that are currently not well understood. To better define these changes, we examined the gene expression and gene ontology profiles of thymic lymphomas from a commonly used model of carcinogenesis, the p53(-/-) mouse. These tumors show a highly significant upregulation of mitochondrial biogenesis, mitochondrial protein translation, mtDNA copy number, reactive oxygen species, antioxidant defenses, proton transport, ATP synthesis, hypoxia response, and glycolysis, indicating a fundamental change in the bioenergetic profile of the transformed T cell. Our results suggest that T cell tumorigenesis involves a simultaneous upregulation of mitochondrial biogenesis, mitochondrial respiration, and glycolytic activity. These processes would allow cells to adapt to the stressful tumor environment by facilitating energy production and thereby promote tumor growth. Understanding these adaptations is likely to result in improved therapeutic strategies for this tumor type.

Pharmacogenetic analysis of lithium-induced delayed aging in Caenorhabditis elegans.

Lithium (Li(+)) has been used to treat mood affect disorders, including bipolar, for decades. This drug is neuroprotective and has several identified molecular targets. However, it has a narrow therapeutic range and the one or more underlying mechanisms of its therapeutic action are not understood. Here we describe a pharmacogenetic study of Li(+) in the nematode Caenorhabditis elegans. Exposure to Li(+) at clinically relevant concentrations throughout adulthood increases survival during normal aging (up to 46% median increase). Longevity is extended via a novel mechanism with altered expression of genes encoding nucleosome-associated functions. Li(+) treatment results in reduced expression of the worm ortholog of LSD-1 (T08D10.2), a histone demethylase; knockdown by RNA interference of T08D10.2 is sufficient to extend longevity ( approximately 25% median increase), suggesting Li(+) regulates survival by modulating histone methylation and chromatin structure.