Single-cell analysis of DNA damage, somatic mutation, and gene expression in human Alzheimer’s disease brain

项目摘要 阿尔茨海默病（AD）显示出与年龄相关的疾病发作，但年龄相关的疾病发病的机制 疾病风险的影响是未知的。DNA损伤是正常衰老的标志， 可能的致病机制。我们最近创新了新的方法来研究单个 从死后人脑中提取的神经元利用这些新技术，我们发现正常的神经元 在出生时每个基因组含有至少几十个体细胞单核苷酸变异（SNV）。体细胞SNV 在出生后的有丝分裂后神经元中线性增加，在老年时达到数千人的水平。体细胞SNV 可以通过碱基对取代的模式进行分析，类似于癌症突变，其中不同的 突变“特征”标记暴露于特定诱变剂或特定DNA缺陷的肿瘤 损伤修复途径。对我们数据的分析揭示了至少两个神经元突变特征， 在来自不同来源的神经元中：一个与衰老有关，另一个与衰老中的氧化损伤有关 尤其是在神经退行性疾病中。神经元体细胞SNV的基因组分布分析显示， 在转录区域和活性增强子元件中的富集，表明体细胞突变 直接影响基因表达网络。这些初步数据表明了一种探索 AD中神经元功能障碍的机制可能是已知风险因素（如蛋白质）的下游 错误折叠 在我们最近发表的工作中，我们发现在海马神经元的兴奋性神经元中，体细胞SNV增加， 晚期AD患者。这些发现引发了几个额外的问题。首先，AD处于什么阶段 进展增加的体细胞突变是否开始？回答这个问题。我们将使用新的和改进的 scWGS方案对AD过程中的体细胞突变模式进行全面分析， 包括SNV和其它类型的变体，例如短插入缺失和结构变体。第二，身体 突变导致基因表达失调？我们将单核RNA测序应用于AD， 控制大脑来回答这个问题最后，DNA修复蛋白的活性如何影响 体细胞突变的代？我们将应用定量方法进行免疫荧光染色， 各种DNA修复蛋白和其他DNA损伤的标志物， 突变，以确定人类大脑突变的根本原因。 因此，这项提议旨在了解体细胞突变何时发生，这些突变的结果是什么。 突变是什么，以及最初是什么引起的。

Project Summary Alzheimer’s disease (AD) displays an age-related disease onset, but the mechanisms by which age influences disease risk are unknown. DNA damage is a hallmark of normal aging and has been implicated as a possible pathogenic mechanism in AD. We recently innovated new methods to study the genome of single neurons from the postmortem human brain. Using these novel technologies, we showed that normal neurons contain at least dozens of somatic single nucleotide variants (SNVs) per genome at birth. Somatic SNVs increase linearly in postmitotic neurons after birth, reaching levels in the thousands in old age. Somatic SNVs can be analyzed by patterns of base-pair substitution, analogous to cancer mutations, where distinct mutational “signatures” mark tumors exposed to specific mutagens or those with deficiencies in specific DNA damage repair pathways. This analysis of our data revealed at least two neuronal mutational signatures arising in neurons from distinct sources: one related to aging generally, another related to oxidative damage in aging but especially in neurodegeneration. Analysis of the genomic distribution of neuronal somatic SNVs revealed an enrichment in transcribed regions and in active enhancer elements, suggesting that somatic mutations directly impact gene expression networks. These preliminary data suggest an approach to exploring mechanisms of neuronal dysfunction in AD that may be downstream of known risk factors such as protein misfolding. In our recently published work, we showed that somatic SNVs are increased in excitatory neurons of late-stage AD patients. These findings prompted several additional questions. First, at what stage of AD progression does increased somatic mutation begin? To answer this question. we will use a new and improved scWGS protocol to perform a comprehensive analysis of patterns of somatic mutation over the course of AD, including SNV and other types of variants, such as short indels and structural variants. Second, does somatic mutation result in dysregulation in gene expression? We will apply single-nucleus RNA-sequencing to AD and control brains to answer this question. Finally, how does the activity of DNA repair proteins impact the generation of somatic mutations? We will apply a quantitative approach to immunofluorescence staining for various DNA repair proteins and other marks of DNA damage on brian donors with known levels of somatic mutation to identify the root causes of mutation in the human brain. Thus, this proposal aims to understand when somatic mutations occur, what the result of those mutations are, and what caused them in the first place.