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Characterizing Senescence in Astrocytes and its Effect on Neurons

  • Author(s): Limbad, Chandani Bhupendrabhai
  • Advisor(s): Campisi, Judith
  • et al.
Abstract

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.

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