Impact of DNA double-strand breaks on 3D genome organization and genome stability in Alzheimer’s disease

项目摘要/摘要 神经元中DNA双链断裂(DSB)的积累是阿尔茨海默病的早期特征 (Ad)。DSB的增加也与衰老有关，这是AD的最大风险因素。广告也是一个复杂的问题 涉及所有主要脑神经胶质细胞类型的疾病。因此，迫切需要了解分子 DSB诱导神经元和神经胶质细胞改变的机制。基因组的结构稳定性是 最重要的是维持一个有功能的基因组，最近，基因组的3D组织已经出现 作为基因组功能的主要调节者。我的总体假设是3D基因组重组和结构 DSB介导的基因组不稳定性是AD发病和脑老化的主要驱动因素。我的目标是 确定神经元内的DSB如何和在多大程度上影响基因组组织和神经胶质细胞 反应，目标是确定可以作为预防疾病新疗法的分子途径 或者阻止神经退行性变的进程。我将使用概括症状前期的小鼠模型 DSB在神经元和人类IPSC模型中的积累以确定3D基因组破坏的程度 由DSB及其潜在的分子机制引起。我还将确定神经细胞的后果 DSB介导的染色体频率测定对基因组结构稳定性的影响 人类阿尔茨海默病神经元的易位。有趣的是，正常的神经元活动会在即刻早期引起DSB 基因启动子。此外，有报道称阿尔茨海默病患者存在神经元过度活动。我会测试IEG是不是经常出现的地方 神经细胞培养中诱发的神经元过度活动后的染色体易位。 先前的研究表明，小胶质细胞在响应于 神经退行性变。为了理解3D基因组组织在小胶质细胞转变中的作用，我将使用 单细胞Hi-C，以测量介导反应性小胶质细胞状态的独特染色质相互作用。 随后，使用单细胞Hi-C整合分析的转录因子预测将在IPSC中进行测试 衍生的小胶质细胞-神经元共培养，以研究它们调节反应性小胶质细胞反应的潜力。这 方法将扩展到研究少突胶质细胞在独立期的反应。 我将与我的导师蔡立辉博士、我的导师委员会、布鲁斯·扬克纳博士和马诺利斯博士一起工作 Kellis，我的技术支持和咨询委员会，Frederick Alt博士和Peter Fraser博士开展我的 建议的培训计划。我会从Tsai实验室获得iPSC技术和差异化的经验来调制 这种疾病与小胶质细胞对神经元DSB的反应有关，并与Manolis Kellis博士合作实施 转录因子预测的计算管道。弥补我在生物学方面的训练中的差距 对于AD和大脑老化，我将旁听相关课程，并接受Bruce Yankner博士的额外指导。

PROJECT SUMMARY/ABSTRACT The accumulation of DNA Double-Strand Breaks (DSBs) in neurons is an early hallmark of Alzheimer’s disease (AD). Increased DSBs are also associated with aging, which is the largest risk factor for AD. AD is also a complex disease involving all major brain glial cell types. Thus, there is a critical need to understand the molecular mechanisms of DSB induced changes in both neurons and glia. The structural stability of the genome is paramount in maintaining a functional genome, and recently, the 3D organization of the genome has emerged as a major regulator of genome function. My overall hypothesis is that 3D genome reorganization and structural genome instability mediated by DSBs are principle drivers of AD pathogenesis and brain aging. My objective is to determine how and to what extent DSBs within the neurons impact genome organization and the glial response, with the goal of identifying molecular pathways that can be targeted as novel therapies for preventing or halting the progression of neurodegeneration. I will use mouse models that recapitulate the pre-symptomatic accumulation of DSBs in neurons and human iPSC models to determine the degree of the 3D genome disruption caused due to DSBs and the underlying molecular mechanisms. I will also identify the consequences of neuronal DSBs on the structural stability of the genome by measuring the frequency of DSB mediated chromosomal translocations in human AD neurons. Interestingly, normal neuronal activity causes DSBs at immediate early gene promoters. Also, neuronal hyperactivity has been reported in AD. I will test if IEGs are locations of frequent chromosomal translocations after neuronal hyperactivity induced in neuronal culture. Previous studies have shown that microglia transitions to a reactive state in response to neurodegeneration. To understand the role of the 3D genome organization in this microglia transition, I will use single cell Hi-C to measure the unique chromatin interactions that mediate the reactive microglia state. Subsequently, transcription factor predictions using integrative analysis of single cell Hi-C will be tested in iPSC derived microglia-neuron co-cultures for their potential to modulate the reactive microglia response. This approach will be extended to study oligodendrocyte response in the independent phase. I will work with my mentor Dr. Li-Huei Tsai, my mentorship committee, Dr. Bruce Yankner and Dr. Manolis Kellis, my technical support and advisory committee, Dr. Frederick Alt and Dr. Peter Fraser to carry out my proposed training plan. I will gain experience in iPSC technology and differentiation from the Tsai lab to modulate the disease associated microglia response to neuronal DSBs and work with Dr. Manolis Kellis to implement the computational pipelines for the transcription factor predictions. To bridge the gap in my training in the biology of AD and brain aging, I will audit relevant courses and receive additional mentoring from Dr. Bruce Yankner.