Single Cell Transcriptomic Profiling of Multiple System Atrophy Brain

多系统萎缩脑的单细胞转录组分析

• 批准号: 10799995
• 负责人: Un Jung Kang
• 金额: $ 59.33万
• 依托单位: NEW YORK UNIVERSITY SCHOOL OF MEDICINE
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-05-04 至 2024-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10799995
• 关键词: Affect; Age; Astrocytes; Atlases; Autopsy; Brain; Brain region; Cell Nucleus; Cell physiology; Cells; Cerebellar Diseases; Cerebellum; Classification; Clinical; Complex; Corpus striatum structure; Cytoplasmic Inclusion; Data; Data Set; Dysautonomias; Event; Exhibits; Experimental Models; Fluorescent in Situ Hybridization; Functional disorder; Gene Expression; Gene Expression Profile; Genes; Genetic Transcription; Human; Immunohistochemistry; In Situ Hybridization; Individual; Informatics; Mediating; Messenger RNA; Microglia; Modeling; Molecular; Molecular Profiling; Morphology; Multiple System Atrophy; Myelin; Nerve Degeneration; Neurodegenerative Disorders; Neurons; Oligodendroglia; Olivopontocerebellar Atrophies; Parkinsonian Disorders; Pathologic; Pathologic Processes; Pathology; Pathway interactions; Patients; Phenotype; Physiology; Property; Sampling; Secondary to; Site; Stress; Striatonigral Degeneration; System; Techniques; Testing; Tissues; Up-Regulation; alpha synuclein; brain tissue; cell type; cerebral atrophy; differential expression; human tissue; insight; neuron loss; nonhuman primate; overexpression; proteostasis; putamen; response; single nucleus RNA-sequencing; single-cell RNA sequencing; synuclein; therapeutic target; transcriptome; transcriptomics

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.

项目总结/摘要 多系统萎缩（MSA）是一种罕见的进行性神经退行性疾病，其特征在于选择性地 α-突触核蛋白在少突胶质细胞内的胶质细胞质内含物（GCI）中的积累。临床上，MSA 患者表现为帕金森综合征、小脑功能障碍和自主神经功能障碍的各种组合。MSA是 根据植物学的优势进行细分，这与主要的 神经变性：MSA-P用于帕金森综合征和纹状体黑质变性或MSA-C用于小脑特征 和橄榄体脑桥小脑萎缩，尽管大多数病例涉及两个系统。α-synuclein的成因 在少突胶质细胞内的积累和MSA中少突胶质细胞生理学的后果是 大部分未知。同样，少突胶质细胞功能障碍如何导致神经元死亡仍然不清楚。在这 我们将使用单核RNA测序（snRNA-seq）来产生单细胞的转录组 从MSA患者的死后脑组织中提取细胞，以描绘细胞类型特异性转录变化 与MSA有关。我们将探测所有患者的纹状体、小脑和皮质组织集。 我们的样本集包含MSA-C和MSA-P病例。这使我们能够捕捉到 发生在每种MSA亚型的主要病理部位、MSA-P的纹状体和小脑的变化 对于MSA-C，沿着次要部位，以及受影响最小的大脑区域（皮质）。在目标1中，我们 收集额外的MSA病例并从所有组织组中生成snRNA-seq图谱。这些数据将 整合和聚类，以确定主要的细胞类型和亚型， 差异表达的基因将用于鉴定调控网络并推断功能变化。在Aim中 2、我们将用免疫组织化学和多重测序技术验证snRNA-seq在组织中鉴定的变化， 使用RNAscope进行荧光原位杂交，可以评估细胞是否携带 失调的基因表达模式具有功能障碍的证据。最初，我们专注于少突胶质细胞， 将确定携带GCI的细胞是否表现出失调的转录组，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完整性。在目标3中，我们将测试是否强制 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谱 使用非人灵长类MSA模型从MSA组织中分离。完成后，该提案将产生一个 纹状体、小脑和小脑中几乎所有细胞类型的MSA依赖性转录变化图谱 皮质，确定基因表达的关键改变和受影响的途径，确定α-突触核蛋白是否 GCI细胞中α-突触核蛋白mRNA的表达增加， 概括了与MSA相关联的小区状态。