Single Cell Transcriptomic Profiling of Multiple System Atrophy Brain

项目摘要/摘要 多系统萎缩(MSA)是一种罕见的进行性神经退行性疾病，其特征是选择性 α-突触核蛋白在少突胶质细胞内的胶质胞质包涵体中的积聚。临床上，MSA 患者出现帕金森症、小脑功能障碍和自主神经障碍的各种组合。MSA是 根据症状学的优势进行细分，与原发部位相关 神经变性：MSA-P用于帕金森氏症和纹状体黑质变性或MSA-C用于小脑特征 和橄榄桥小脑萎缩，尽管大多数病例涉及这两个系统。α-突触核蛋白产生的原因 MSA中少突胶质细胞的蓄积及其对少突胶质细胞生理学的影响 很大程度上是未知的。同样，少突胶质细胞功能障碍如何导致神经元死亡仍不清楚。在这 建议，我们将使用单核RNA测序(SnRNA-seq)来生成单细胞的转录本 从MSA患者的死后脑组织中描绘细胞类型特异性转录变化 与MSA关联。我们将为所有患者探测相同患者的纹状体、小脑和皮质组织集 我们的样本集包括MSA-C和MSA-P病例。这使我们能够捕获 每种MSA亚型、纹状体区和小脑的原发病变部位的变化 对于MSA-C，以及次要部位，以及受影响最小的大脑区域(皮质)。在目标1中，我们将 收集更多的MSA病例，并从所有组织集生成SnRNA-seq图谱。这些数据将是 集成和聚类，以确定主要细胞类型和亚型，从中进行信息分析 差异表达的基因将被用来识别调控网络并推断功能变化。在AIM 2、我们将通过免疫组织化学和多重分析来验证SnRNA-seq在组织中鉴定的变化。 用RNAScope进行荧光原位杂交，可以评估细胞是否具有 失调的基因表达模式有功能障碍的证据。最初专注于少突胶质细胞，我们 将决定携带GCI的细胞是否表现出失调的转录，α-突触核蛋白mRNA是否 在这些细胞中过度表达，以及这是否影响髓鞘的完整性。在目标3中，我们将测试强制 α-突触核蛋白在少突胶质细胞中的过表达足以概括所获得的SnRNA-seq图谱。 使用非人灵长类MSA模型从MSA组织中提取。完成后，这项建议将产生一个 纹状体、小脑和大脑中几乎所有细胞类型依赖于MSA的转录变化图谱 确定基因表达的关键变化和受影响的通路，确定α-突触核蛋白 α-突触核蛋白基因在GCI细胞中的表达增加 概括了与MSA关联的单元状态。

Project Summary/Abstract Multiple system atrophy (MSA) is a rare progressive neurodegenerative disease characterized by selective accumulation of α-synuclein in glial cytoplasmic inclusions (GCIs) within oligodendrocytes. Clinically, MSA patients present with various combinations of parkinsonism, cerebellar dysfunction, and dysautonomia. MSA is subclassified based on predominance of symptomology, which is associated with the primary site of neurodegeneration: MSA-P for parkinsonism and striatonigral degeneration or MSA-C for cerebellar features and olivopontocerebellar atrophy, though most cases involve both systems. The causes of α-synuclein accumulation within oligodendrocytes and the consequences for oligodendrocyte physiology in MSA are largely unknown. Likewise, how oligodendrocyte dysfunction causes neuronal death remains obscure. In this proposal, we will use single nucleus RNA sequencing (snRNA-seq) to generate transcriptomes of single cells from postmortem brain tissue from MSA patients to delineate the cell type specific transcriptional changes associated with MSA. We will probe striatal, cerebellar, and cortical tissue sets from the same patients for all patients of our sample set which contains both MSA-C and MSA-P cases. This allows us to capture the changes that occur in the primary site of pathology for each MSA subtype, striatum for MSA-P and cerebellum for MSA-C, along with the secondary sites, and a minimally affected brain region (cortex). In Aim 1, we will collect additional MSA cases and generate snRNA-seq profiles from all tissue sets. These data will be integrated and clustered to identify major cell types and subtypes from which informatic analysis of differentially-expressed genes will be used to identify regulatory networks and infer change in function. In Aim 2, we will validate the changes identified by snRNA-seq in tissue with immunohistochemistry and multiplexed fluorescence in situ hybridization with RNAscope, allowing the assessment of whether cells bearing dysregulated gene expression patterns have evidence of dysfunction. Initially focusing on oligodendrocytes, we will determine whether cells bearing GCI exhibit dysregulated transcriptomes, whether α-synuclein mRNA is overexpressed in these cells, and whether this affects myelin integrity. In Aim 3, we will test whether forced overexpression of α-synuclein in oligodendrocytes is sufficient to recapitulate the snRNA-seq profiles obtained from MSA tissues using a nonhuman primate MSA model. Upon completion, this proposal will generate an atlas of the MSA-dependent transcriptional changes of nearly all cell types in the striatum, cerebellum, and cortex, identify key alterations in gene expression and the pathways affected, determine whether α-synuclein expression is increased in cells with GCI, and whether de novo expression of α-synuclein mRNA fully recapitulates the cell states associated with MSA.