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Open Access Publications from the University of California

Scholarly Works (6 results)

  • Thesis
  • Peer Reviewed
UC Berkeley Electronic Theses and Dissertations (2017)

Cellular senescence, characterized by a permanent cell cycle arrest and an inflammatory phenotype called the senescence-associated secretory phenotype (SASP), has been described during aging and various age-related diseases. Neurodegenerative diseases are a major part of age-related diseases. Cognitive decline has been shown to happen in diseases such as Alzheimer’s (AD) and in cancer patients after chemotherapy or radiotherapy treatments. Even though there is a link between inflammation and neurodegenerative diseases, there is no in-depth analysis of senescence in the brain. Among various cell types in the brain, astrocytes represent the most abundant population. Astrocytes have proliferative capacity and are essential for neuron survival.

In this study, I first induced senescence in cultured human primary astrocytes using X- irradiation, and determined that astrocytes exhibit various senescence markers including p16, SASP, and downregulation of LMNB1 and HMGB1. Interestingly, as it was particular to senescent (SEN) astrocytes, we detected a downregulation of a specific family of genes, coding for glutamate and potassium transporters, both at the RNA and protein levels. Further we performed unbiased RNA-seq study on non-senescent (NS) and SEN astrocytes to determine the pathways specifically affected in astrocytes. RNA-seq data showed that almost 50% of the genes were upregulated and 50% of the genes were downregulated in SEN astrocytes compared to NS astrocytes. RNA-seq data also confirmed the downregulation of glutamate and potassium transporters upon senescence. These genes are essential for normal astrocyte function in order to maintain homeostasis of neurotransmitter glutamate, potassium ion and water transport, and the strong decrease in their expression in senescent astrocytes suggest a key role of senescence in various brain pathologies.

Further, I performed co-culture assays of neurons and astrocytes in the presence, or the absence, of glutamate to determine whether SEN astrocytes trigger glutamate toxicity and lead to neuronal death. The results showed that SEN astrocytes indeed produced neuronal death in the presence of glutamate.

Then, I analyzed senescence in ten different regions of the mouse brain. For this study, I used six different age groups of C57BL/6 wild type (WT) mice, starting from 2 months to 22 months. Using real-time PCR, I determined variations in the expression of p16, SASP factors, and LMNB1 across the different regions of the mouse brains. The expression of p16 and several SASP factors was upregulated in most of the brain regions during aging.

I also investigated the presence of senescent cells after X-irradiation in vivo in the mouse brain, and detected an upregulation of p16 and SASP expression in brain samples after irradiation. These data suggest that senescence induced in astrocytes may fuel an inflammatory microenvironment in the brain, which would lead to various brain pathologies, and may also lead to brain cancer recurrence after radiotherapy. To test for a potential role of senescence during neurodegenerative diseases, I used the J20 Alzheimer’s disease (AD) mouse model. Real-time PCR results showed an upregulation of p16 and SASP factors in the hippocampus and cortex tissues of the J20 mice compared to wild-type (WT) controls. In addition, the hippocampus from J20 mice showed a downregulation of glutamate and potassium transporters in comparison with hippocampus from age-matched WT mice.

Together this project represents a comprehensive study of the senescent phenotype in astrocytes, either grown in culture or in a mouse brain. Overall these results show that senescence can affect the normal function of astrocytes and cause neuronal cell death. Various mouse models, including natural aging, radiation therapy and AD, are showing the presence of senescent cells and suggest a deleterious role of these cells. Therefore, these findings may open up novel ways to develop treatments for brain diseases.

    Cover page: Characterizing Senescence in Astrocytes and its Effect on Neurons
    • Article
    • Peer Reviewed
    Biological Systems & Engineering (2022)
    Cellular senescence is a driver of many age-related pathologies. There is an active search for pharmaceuticals termed senolytics that can mitigate or remove senescent cells in vivo by targeting genes that promote the survival of senescent cells. We utilized single-cell RNA sequencing to identify CRYAB as a robust senescence-induced gene and potential target for senolysis. Using chemical inhibitor screening for CRYAB disruption, we identified 25-hydroxycholesterol (25HC), an endogenous metabolite of cholesterol biosynthesis, as a potent senolytic. We then validated 25HC as a senolytic in mouse and human cells in culture and in vivo in mouse skeletal muscle. Thus, 25HC represents a potential class of senolytics, which may be useful in combating diseases or physiologies in which cellular senescence is a key driver.
      Cover page: Senolysis induced by 25-hydroxycholesterol targets CRYAB in multiple cell types
      • Article
      • Peer Reviewed
      LBL Publications (2020)
      Neurodegeneration is a major age-related pathology. Cognitive decline is characteristic of patients with Alzheimer's and related dementias and cancer patients after chemo- or radio-therapies. A recently emerged driver of these and other age-related pathologies is cellular senescence, a cell fate that entails a permanent cell cycle arrest and pro-inflammatory senescence-associated secretory phenotype (SASP). Although there is a link between inflammation and neurodegenerative diseases, there are many open questions regarding how cellular senescence affects neurodegenerative pathologies. Among the various cell types in the brain, astrocytes are the most abundant. Astrocytes have proliferative capacity and are essential for neuron survival. Here, we investigated the phenotype of primary human astrocytes made senescent by X-irradiation, and identified genes encoding glutamate and potassium transporters as specifically downregulated upon senescence. This down regulation led to neuronal cell death in co-culture assays. Unbiased RNA sequencing of transcripts expressed by non-senescent and senescent astrocytes confirmed that glutamate homeostasis pathway declines upon senescence. Our results suggest a key role for cellular senescence, particularly in astrocytes, in excitotoxicity, which may lead to neurodegeneration including Alzheimer's disease and related dementias.
        Cover page: Astrocyte senescence promotes glutamate toxicity in cortical neurons
        • Article
        • Peer Reviewed
        UC Berkeley Previously Published Works (2022)
        Aging is accompanied by a loss of muscle mass and function, termed sarcopenia, which causes numerous morbidities and economic burdens in human populations. Mechanisms implicated in age-related sarcopenia or frailty include inflammation, muscle stem cell depletion, mitochondrial dysfunction, and loss of motor neurons, but whether there are key drivers of sarcopenia are not yet known. To gain deeper insights into age-related muscle loss, we performed transcriptome profiling on lower limb muscle biopsies from 72 young, elderly, and frail human subjects using bulk RNA-seq (N = 72) and single-nuclei RNA-seq (N = 17). This combined approach revealed changes in gene expression that occur with age and frailty in multiple cell types comprising mature skeletal muscle. Notably, we found increased expression of the genes MYH8 and PDK4, and decreased expression of the gene IGFN1, in aged muscle. We validated several key genes changes in fixed human muscle tissue using digital spatial profiling. We also identified a small population of nuclei that express CDKN1A, present only in aged samples, consistent with p21cip1-driven senescence in this subpopulation. Overall, our findings identify unique cellular subpopulations in aged and sarcopenic skeletal muscle, which will facilitate the development of new therapeutic strategies to combat age-related frailty.
          Cover page: Single nuclei profiling identifies cell specific markers of skeletal muscle aging, frailty, and senescence.
          • Article
          • Peer Reviewed
          Biological Systems & Engineering (2018)
          Exposure to the herbicide paraquat (PQ) is associated with an increased risk of idiopathic Parkinson's disease (PD). Therapies based on PQ's presumed mechanisms of action have not, however, yielded effective disease therapies. Cellular senescence is an anticancer mechanism that arrests proliferation of replication-competent cells and results in a pro-inflammatory senescence-associated secretory phenotype (SASP) capable of damaging neighboring tissues. Here, we demonstrate that senescent cell markers are preferentially present within astrocytes in PD brain tissues. Additionally, PQ was found to induce astrocytic senescence and an SASP in vitro and in vivo, and senescent cell depletion in the latter protects against PQ-induced neuropathology. Our data suggest that exposure to certain environmental toxins promotes accumulation of senescent cells in the aging brain, which can contribute to dopaminergic neurodegeneration. Therapies that target senescent cells may constitute a strategy for treatment of sporadic PD, for which environmental exposure is a major risk factor.
            • Article
            • Peer Reviewed
            LBL Publications (2019)
            Cellular senescence irreversibly arrests cell proliferation, accompanied by a multi-component senescence-associated secretory phenotype (SASP) that participates in several age-related diseases. Using stable isotope labeling with amino acids (SILACs) and cultured cells, we identify 343 SASP proteins that senescent human fibroblasts secrete at 2-fold or higher levels compared with quiescent cell counterparts. Bioinformatic analysis reveals that 44 of these proteins participate in hemostasis, a process not previously linked with cellular senescence. We validated the expression of some of these SASP factors in cultured cells and in vivo. Mice treated with the chemotherapeutic agent doxorubicin, which induces widespread cellular senescence in vivo, show increased blood clotting. Conversely, selective removal of senescent cells using transgenic p16-3MR mice showed that clearing senescent cells attenuates the increased clotting caused by doxorubicin. Our study provides an in-depth, unbiased analysis of the SASP and unveils a function for cellular senescence in hemostasis.
              Cover page: SILAC Analysis Reveals Increased Secretion of Hemostasis-Related Factors by Senescent Cells
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