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，创造了重大的经济效益。 它们造成了巨大的人类痛苦。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）功能下降是一个主要因素 有助于这些条件的发展。然而，导致NSC功能的机制 下降的原因知之甚少。我们实验室和其他实验室最近的工作表明，染色质经历了 随着年龄的增长，整体重塑，异染色质区域开放，常染色质区域相对关闭 地区高度异染色质区含有大量的逆转录转座因子（RTEs）。我们 先前的研究表明，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， 不稳定性和触发细胞衰老。这就引出了我们的假设： 染色质结构导致RTEs的去阻遏，导致DNA损伤和基因组不稳定， 最终引发细胞衰老和NSC功能下降。为了验证这个假设，我们将 进行一组实验，旨在确定RTE表达随年龄增长而增加在 国家安全委员会的职能。在具体目标1中，我们将开始研究衰老和老年人的染色质结构。 NSC，并确定对转录组的影响，特别是对RTEs表达的影响。获得 为了加强和补充全基因组数据，我们将使用高通量Illumina测序技术， 结合三种方法确定染色质结构和功能的变化：转座酶测定 可接近染色质（ATAC-seq），使用针对抑制性和非抑制性的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 表情具体目标2将研究RTE表达与DNA损伤之间的关系 NSC功能下降。我们将确定DNA损伤累积的动力学， 神经干细胞随时间的衰老。RTE失调对NSC功能和衰老的影响将在 使用设计为过表达长散布核元件L1的报道构建体测定。 最后，我们将确定RTE表达在特定目标3中体内NSC功能下降中的作用。的 将研究RTE去阻遏对NSC再生嗅球中神经元的能力的影响 通过暴露于逆转录酶抑制剂3 TC的年轻和老年小鼠的嗅觉辨别测试， 抑制RTE转座。为了确定RTE抑制是否延长了NSC的健康寿命， 将在3 TC处理的小鼠中研究NSC的能力。综合实验结果 该提案中概述的这一点将使我们对导致NSC功能下降的机制有新的认识， 并可能最终导致可以延长人类健康寿命的治疗干预。

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.