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

阿尔茨海默病和相关的痴呆症表现出与年龄相关的发病，错误折叠的蛋白质聚集体，如β-淀粉样蛋白和tau缠结，氧化DNA损伤增加，最终导致神经元细胞死亡。由DNA修复基因突变引起的孟德尔类早老性疾病表现出类似阿尔茨海默氏症的早发性神经变性，这表明基因组完整性受损可以加速衰老并直接导致神经元损失。DNA损伤可以修复，但可能导致称为体细胞突变的基因组的永久性变化，导致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的假设。在阿尔茨海默病的大脑中，对这一假设的直接支持仍然是难以捉摸的，因为标准的dna测序实验不足以全面表征有丝分裂后人类神经元中发生的体细胞突变。这样的实验力量不足，因为有丝分裂后神经元中发生的突变只存在于发生突变的单个细胞中，因此在分析从数百万个脑细胞中分离出来的DNA时，无法将其与背景噪声区分开来。

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.