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表现为与年龄相关的发病，错误折叠的蛋白质聚集体，如β-淀粉样蛋白和tau缠结，增加氧化dna损伤，最终导致神经细胞死亡。由DNA修复基因突变引起的孟德尔程序性疾病表现出类似阿尔茨海默氏症的早发性神经退行性变，这表明基因组完整性受损会加速衰老，直接导致神经元丢失。DNA损伤是可以修复的，但可能会导致称为体细胞突变的基因组永久变化，这导致了一种假设，即体细胞突变负担增加可能在年龄相关的神经退行性疾病中很常见。这一假说在阿尔茨海默病的大脑中仍然难以直接支持，因为标准的DNA测序实验不足以全面描述发生在有丝分裂后人类神经元中的体细胞突变。这样的实验动力不足，因为在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一无二的，因此在分析从数百万个脑细胞中分离出的DNA时，它与背景噪音无法区分。 我们最近开发了在单细胞分辨率下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来表明人类神经元的永久性体细胞突变伴随着衰老，特定的突变签名指定了在人脑中产生DNA损伤的离散路径。患有遗传性早发性神经退行性疾病的捐赠者，特别是Cockayne综合征和着色性干皮病的捐赠者，以及晚发型散发性阿尔茨海默病捐赠者的神经元，突变数量和特征不同，这表明神经变性与特定的体细胞突变模式有关。我们没有发现已知的阿尔茨海默病基因(如APOE、PSEN1、PSEN2或APP)中存在突变热点的证据，相反，我们发现阿尔茨海默病神经元中的体细胞突变代表着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，以前所未有的详细程度研究神经变性过程中的体细胞突变。我们将关注晚期阿尔茨海默病捐赠者和晚期帕金森氏病捐赠者的神经元，帕金森氏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、错误折叠的蛋白质聚集体和神经细胞死亡。我们将重点关注那些具有这些疾病的已知病理特征的神经元，例如β-淀粉样蛋白和tau聚集体，以检验这些神经元经历更多的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、神经退行性变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.