Single-nuclei multiomic analysis of DLB progression

项目总结 路易体痴呆症(DLB)与其患病率相比，历史上一直调查不足 联核病的研究主要集中在PD和PDD。然而，越来越多的证据表明，DLB是一种独特的 与年龄相关的神经退行性痴呆。和帕金森病一样，各种类型的细胞包括神经元、小胶质细胞、 星形胶质细胞、少突胶质细胞、内皮细胞和外周淋巴细胞可能与DLB有关 发病机制。为了了解DLB发病机制的复杂性，更全面的方法 在疾病发展过程中研究不同类型的细胞和多个脑区是必要的。 我们的实验室在死后脑组织的单核分析方面投入了大量的努力，使用的是最近发布的 铬单细胞多组体ATAC PLUS基因表达平台(10X基因组学)成功 建立了所有关键技术和数据分析管道。为了跟踪疾病的发展，使用RNA- 和atac-seq数据在同一个细胞中，我们开发了一种新的策略：“相关假病机” (CPP)“轨迹分析。我们还建立了“基因峰值”分析，使我们能够分析人际关系。 在单个细胞中基因表达和染色质可及性之间的关系。我们的技术进步和 创新的数据分析技能将满足这一FOA的要求，即“识别ADRD后细胞变化的项目”。 在疾病发展过程中的死亡脑组织。为实现现行《全面和平协议》设定的目标，我们将继续 目标如下： 目的1.弥漫性脑白质瘤的神经病理分期及对照尸检标本的验证。 来自HBTRC的尸检脑组织将通过H&E和α进一步验证神经病理分期- SYN免疫组织化学染色按路易体疾病统一分期系统(USSLB)进行。至 排除偶发性路易体病，对照组织也将进行α-SYN病理学研究。 目的2.从每个样本中分离细胞核，进行SnRNA-seq和SnATAC-seq分析。 来自三组的4个脑区：1)对照组；2)阶段II(边缘或脑干占优势)/III(边缘和 脑干)；3)和IV期(新皮质)，将接受单核多体分析。 目的3.拓扑域映射的空间转录分析。 不同脑区微区基因的差异表达及其与α-SYN的关系 病理将使用空间条形码基因表达谱阵列进行研究。

PROJECT SUMMARY Dementia with Lewy body (DLB) has been historically under-investigated relative to its prevalence as most studies of synucleinopathies focus on PD and PDD. Accumulating evidence indicates, however, DLB is a distinct age-associated neurodegenerative dementia. Like PD, various types of cells including neurons, microglia, astrocytes, oligodendrocytes, endothelial cells, and peripheral lymphocytes might contribute to DLB pathogenesis. To understand complexities of DLB pathogenesis, more comprehensive approaches to investigating different cell types and multiple brain regions over the course of disease progression are necessary. Our lab has put extensive effort into single-nuclei analysis of postmortem brain tissues using a recently released Chromium Single-Cell Multiome ATAC plus Gene Expression platform (10X Genomics) and successfully established all key techniques and a data analysis pipeline. To track the progression of disease using both RNA- and ATAC-seq data in the same cell, we have developed a novel strategy: “correlated pseudo-pathogenesis (cPP)” trajectory analysis. We also established “gene-peak” analysis allowing us to analyze relationships between gene expression and chromatin accessibility in a single cell. Our technical advancements and innovative data analysis skills will satisfy this FOA requesting “projects to identify cellular changes in ADRD post- mortem brain tissue across disease progression.” To achieve the goal set by the current FOA, we will pursue the following aims: Aim 1. Neuropathological staging of DLB and validation of control postmortem samples. Postmortem brain tissues from the HBTRC will be further validated for neuropathological staging by H&E and α- SYN immunohistochemical staining according to Unified Staging System for Lewy Body Disorders (USSLB). To eliminate incidental Lewy body disease (ILBD), control tissues will also be investigated for α-SYN pathology. Aim 2. Isolate nuclei from each sample and perform snRNA-seq and snATAC-seq analysis. 4 brain regions from three groups: 1) control; 2) stage II (limbic or brainstem predominant)/III (both limbic and brainstem); 3) and stage IV (neocortical), will be subject to single-nuclei multiomic analysis. Aim 3. Spatial transcriptomic analysis for topological domain mapping. Differential gene expression in the specific microdomains of each brain region and relationship to α-SYN pathology will be investigated using a spatially barcoded gene expression profiling array.