Retrotransposable Element Expression in Neural Stem Cell Senescence and Aging

神经退行性疾病和痴呆症，包括阿尔茨海默病，创造了显著的经济 它们造成了沉重的负担，并造成了相当大的人类痛苦。老龄化是发展的主要风险因素 这些条件。随着年龄的增长，神经干细胞(NSC)功能的下降是一个主要因素 促进了这些条件的发展。然而，导致NSC功能的机制 人们对衰退知之甚少。我们实验室和其他实验室最近的研究表明，染色质经历了 随年龄增长的整体重塑，异色区开放，等色区相对关闭 地区。高度异染色区含有大量的逆转座子元件(RTE)。我们 先前已经表明，RTE的表达也随着年龄的增长而增加，并在活跃的转座中达到顶峰 事件。体细胞转座可导致插入突变和基因组重排产生基因组 不稳定并引发细胞衰老。这就引出了我们的假设：年龄相关的变化 染色质结构导致RTES的抑制，导致DNA损伤和基因组不稳定， 最终引发细胞衰老和神经干细胞功能下降。为了检验这一假设，我们将 进行一系列实验，以确定随年龄增加的RTE表达在衰老中的作用 NSC功能。在特定的目标1中，我们将首先研究衰老和老年人的染色质结构。 NSCs，并确定对转录组的影响，特别是对rtes表达的影响。为了获得 加强和补充全基因组数据，我们将使用高通量Illumina测序 结合三种方法确定染色质结构和功能的变化：转座酶的测定 可及染色质(ATAC-SEQ)，使用抑制和抑制抗体的染色质免疫沉淀 激活染色质组蛋白标记(ChIP-seq)和rna-seq以定义转录组，包括rte 表情。特异性目标2将研究RTE的表达与DNA损伤的关系 神经干细胞蓄积功能下降。我们将确定DNA损伤积累的动力学和 随着时间的推移，神经干细胞中的衰老。放松RTE管制对NSC功能和衰老的影响将是 使用设计用于过度表达长散布的核元素L1的报告构造来确定。 最后，我们将确定RTE在体内神经干细胞功能下降中的作用。 RTE抑制对神经干细胞在嗅球再生神经元能力的影响将被研究。 通过对年轻和老年小鼠暴露于逆转录酶抑制剂3TC的嗅觉辨别试验， 这抑制了RTE转位。为了确定RTE抑制是否延长了NSC的健康寿命，神经源性 神经干细胞的能力将在3TC处理的小鼠中进行研究。把实验的结果综合起来 本提案中概述的内容将使我们对导致NSC功能下降的机制有新的了解 这可能导致衰老，并可能最终导致可延长人类健康寿命的治疗干预措施。

Neurodegenerative conditions and dementias, including Alzheimer's disease, create a significant economic burden and are responsible for considerable human suffering. Aging is the primary risk factor for development of these conditions. The decline in neural stem cell (NSC) function that occurs with age is a major factor contributing to the development of these conditions. However, the mechanisms resulting in NSC functional decline are poorly understood. Recent work from our laboratory and others reveals that chromatin undergoes global remodeling with age, with an opening of heterochromatic regions and a relative closing of euchromatic regions. The highly heterochromatic regions contain large numbers of retrotransposable elements (RTEs). We have previously shown that RTE expression also increases with age and culminates in active transposition events. Somatic transposition can lead to insertional mutagenesis and genome rearrangements creating genome instability and triggering cellular senescence. This leads us to our hypothesis: Age-associated changes in chromatin structure lead to de-repression of RTEs, resulting in DNA damage and genome instability, ultimately triggering cellular senescence and a decline in NSC function. To test this hypothesis we will perform a set of experiments designed to determine the role of increased RTE expression with age in loss of NSC function. In Specific Aim 1 we will begin by investigating the chromatin architecture in senescent and aged NSCs, and determine the effects on the transcriptome, in particular on the expression of RTEs. To obtain reinforcing and complementary whole genome data, we will use high-throughput Illumina sequencing in conjunction with three methods to define changes in chromatin structure and function: assay for transposase accessible chromatin (ATAC-seq), chromatin immunoprecipitation using antibodies against repressive and activating chromatin histone marks (ChIP-seq), and RNA-seq to define the transcriptome, including RTE expression. Specific Aim 2 will investigate the relationship between RTE expression and DNA damage accumulation in NSC functional decline. We will determine the dynamics of DNA damage accumulation and senescence in NSCs over time. The effects of RTE deregulation on NSC function and senescence will be determined using a reporter construct designed to over express the long interspersed nuclear element L1. Finally, we will determine the role of RTE expression in NSC functional decline in vivo in Specific Aim 3. The effect of RTE de-repression on the ability of NSCs to regenerate neurons in the olfactory bulb will be investigated through olfactory discrimination tests in young and aged mice exposed to the reverse transcriptase inhibitor 3TC, which represses RTE transposition. To determine if RTE repression extends NSC healthspan, the neurogenic capabilities of NSCs will be investigated in 3TC treated mice. Taken together the results of the experiments outlined in this proposal will give us new insight into the mechanisms that result in NSC functional decline with age and may ultimately lead to therapeutic interventions that can extend human healthspan.