Spatial single-cell analysis of somatic mutation in human brain during aging and neurodegeneration

阿尔茨海默病和相关痴呆显示出年龄相关的发病、错误折叠的蛋白质聚集体（如β-淀粉样蛋白和tau缠结）、增加的氧化性DNA损伤以及最终的神经元细胞死亡。由DNA修复基因突变引起的孟德尔早老性疾病显示出类似阿尔茨海默氏症的早发性神经变性，这表明受损的基因组完整性可以加速衰老并直接导致神经元损失。DNA损伤可以被修复，但可能导致基因组的永久性变化，称为体细胞突变，从而导致这样的假设，即体细胞突变负担增加可能在与年龄相关的神经退行性疾病中很常见。在阿尔茨海默病的大脑中，对这一假设的直接支持仍然难以捉摸，因为标准的DNA测序实验无法全面表征有丝分裂后人类神经元中发生的体细胞突变。这样的实验是不够有力的，因为有丝分裂后神经元中出现的突变只对发生突变的单个细胞是独特的，因此当分析从数百万个脑细胞中分离出来的DNA时，它将无法与背景噪音区分开来。 我们最近开发了以单细胞分辨率研究人类神经元体细胞突变的方法，包括单细胞全基因组测序（scWGS）方法。我们使用scWGS来显示永久性体细胞突变伴随着人类神经元的衰老，特定的突变特征提名了在人脑中产生DNA损伤的离散途径。来自遗传性早发性神经退行性疾病（特别是Cockayne综合征和着色性干皮病）供体的神经元和迟发性散发性阿尔茨海默病供体的神经元的突变计数和特征不同，表明神经退行性疾病与特定的体细胞突变模式相关。我们没有发现已知阿尔茨海默氏症基因（如APOE，PSEN 1，PSEN 2或APP）突变热点的证据，而是发现阿尔茨海默氏症神经元中的体细胞突变代表了对每个细胞基因组的随机攻击。 这项新创新者奖提案的目标是开发和应用新的scWGS方法，以前所未有的细节研究神经变性过程中的体细胞突变。我们将重点关注来自晚期阿尔茨海默病供体和晚期帕金森病供体的神经元，帕金森病是另一种与年龄相关的神经退行性疾病，其特征是DNA损伤增加，错误折叠蛋白质聚集和神经元细胞死亡。我们将专注于这些疾病的已知病理标志的神经元，例如，β-淀粉样蛋白和tau聚集体，以测试这些神经元经历增加的DNA损伤从而增加体细胞突变的假设。这项工作将在单细胞基因组学、衰老、神经变性和人类发育领域产生广泛影响。

Alzheimer’s disease and related dementias display an age-related onset, misfolded protein aggregates such as β-amyloid and tau tangles, increased oxidative DNA damage, and ultimately neuron cell death. Mendelian progeroid diseases caused by mutations in DNA repair genes show early-onset neurodegeneration resembling Alzheimer’s, suggesting that compromised genomic integrity can accelerate aging and directly cause neuron loss. DNA damage can be repaired but may result in permanent changes to the genome called somatic mutations, leading to the hypothesis that increased somatic mutation burden may be common across age-related neurodegenerative disorders. Direct support for this hypothesis has remained elusive in the Alzheimer’s disease brain because standard DNA-sequencing experiments are underpowered to characterize somatic mutations that occur in postmitotic human neurons comprehensively. Such experiments are underpowered because a mutation arising in a postmitotic neuron would be unique to only the single cell in which it occurred and thus it would be indistinguishable from background noise when analyzing DNA isolated from millions of brain cells. We recently developed methods to study somatic mutations in human neurons at single-cell resolution, including methods for single-cell, whole genome sequencing (scWGS). We used scWGS to show that permanent somatic mutations accompany aging in human neurons, with specific mutation signatures nominating discrete pathways generating DNA damage in the human brain. Mutation counts and signatures differ in neurons from donors with genetic early-onset neurodegenerative diseases, specifically Cockayne syndrome and Xeroderma Pigmentosum, and in the late-onset sporadic Alzheimer’s donors, suggesting neurodegeneration is associated with specific patterns of somatic mutation. We found no evidence for mutational hotspots in known Alzheimer’s genes such as APOE, PSEN1, PSEN2, or APP, instead finding that somatic mutations in Alzheimer’s neurons represent a stochastic assault on the genome in each cell. The goal of this New Innovator Award proposal is to develop and apply new scWGS methods to study somatic mutations during neurodegeneration in unprecedented detail. We will focus on neurons from late-stage Alzheimer’s disease donors and from donors with late-stage Parkinson’s disease, another age-associated neurodegenerative disorder characterized by increased DNA damage, aggregates of misfolded protein, and neuron cell death. We will focus on those neurons with known pathological hallmarks of these diseases, for example, β-amyloid and tau aggregates, to test the hypothesis that these neurons experience increased DNA damage and thus increased somatic mutations. This work will have broad impacts in the fields of single-cell genomics, aging, neurodegeneration, and human development.