The role of CSPalpha in Adult onset neuronal Lipofuscinosis pathogenesis

CSPα 在成人神经元脂褐质沉着症发病机制中的作用

• 批准号: 10592180
• 负责人: Bruno A. Benitez
• 金额: $ 49.05万
• 依托单位: BETH ISRAEL DEACONESS MEDICAL CENTER
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-09-20 至 2024-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10592180
• 关键词: Adult; Affect; Amyloid beta-Protein; Animal Model; Area; Atlases; Autophagocytosis; Brain; Brain Pathology; Brain region; CRISPR/Cas technology; Cell Line; Cell Nucleus; Cell Survival; Cell model; Cells; Data; Disease; Exhibits; Family; Fibroblasts; Functional disorder; Generations; Genes; Genetic; Genetic Transcription; Human; Impairment; Induced pluripotent stem cell derived neurons; Knock-out; Knowledge; Lead; Lysosomes; Maps; Microglia; Molecular; Molecular Chaperones; Molecular Weight; Mus; Mutation; Nerve Degeneration; Neurodegenerative Disorders; Neuronal Ceroid-Lipofuscinosis; Neurons; Occipital lobe; Parietal Lobe; Pathogenesis; Pathogenicity; Pathology; Pathway interactions; Patients; Phagocytes; Pharmacology; Phenocopy; Physiological; Play; Predisposition; Protein Secretion; Proteins; Proteome; Proteomics; Reporting; Resolution; Role; Small Nuclear RNA; Synapses; Technology; Temporal Lobe; Testing; Therapeutic; Time; Transcript; alpha synuclein; brain cell; cell type; cysteine string protein; differential expression; early onset; fly; frontal lobe; gene network; induced pluripotent stem cell; member; misfolded protein; mouse model; mutant; neurodegenerative dementia; overexpression; palmitoylation; protein TDP-43; resilience; screening; spatiotemporal; synaptic function; tau Proteins; tau-1; transcriptome; transcriptome sequencing

Autosomal dominant adult-onset neuronal ceroid lipofuscinosis (ANCL) is a rapidly progressing fatal neurodegenerative dementia with no treatment currently available. Our group and others simultaneously reported that mutations in the DNAJC5 gene are the most common cause of autosomal dominant ANCL. We also showed that DNAJC5 mutations reduce lysosomal function and autophagic flux while causing the accumulation of autofluorescent storage material (AFSM) and high molecular weight aggregates (HMWA) in patient-derived fibroblasts. However, the mechanisms leading to neurodegeneration in ANCL are not known. Furthermore, there are no studies done in relevant patient-derived brain cellular models. Unprecedented advances in high-throughput and hypothesis-free “omics” technologies can generate highly detailed molecular atlas for ANCL. We hypothesize that CSPα plays a role in the endo-lysosomal pathway and that CSPα mutations lead to neuronal and microglial dysfunction and neurodegeneration. To test this hypothesis, we plan to perform bulk-RNA Seq, single nucleus RNAseq and targeted proteomics in four brain regions with and without DNAJC5 mutations on differentially expressed genes and genes co-expressed with DNAJC5 mutants in a cell-specific manner. Our preliminary data demonstrate that DNAJC5 transcript levels are higher in microglial cells than neurons. We will perform bulk-RNAseq and proteomics in human iPSC-derived neurons and microglia with DNAJC5 mutations and an isogenic control generated by CRISPR/Cas9 to define the impact of DNAJC5 on cell autonomous-specific pathways. We will apply snRNA-seq to multiple brain areas affected differently by ANCL pathology, thus tracing pseudotemporal trajectories of pathology progression, defining DEG in major brain cell types and cell-type-specific transcriptional states for the first time in brains of carriers of DNAJC5 mutations. (Aim 1). To determine the cell-autonomous effect of DNAJC5 mutations on ANCL pathology in human iPSC-derived neurons and microglia (Aim 2), we will use genetic (CRISPR/Cas9) and pharmacologic approaches in iPSC-derived neurons and microglia to determine how DNAJC5 mutants impact the phagocytic capacity, cell viability, lysosomal function, and the accumulation of pathogenic HMWA and AFSM. We will use proteomics in human iPSC-derived neurons and microglia to test the effect of DNAJC5 mutations on the secretome and the levels of prone-to-aggregate and proinflammatory proteins. We will harmonize RNA-seq and proteomic data across the brain regions and iPSC-derived cells to determine DNAJC5-associated neurodegenerative pathways.

常染色体显性遗传成人型神经元蜡样质脂褐质沉积症（ANCL）是一种进展迅速的致命性神经退行性痴呆，目前尚无治疗方法。我们的研究组和其他研究组同时报道了DNAJC 5基因突变是常染色体显性ANCL最常见的病因。我们还表明，DNAJC 5突变降低了溶酶体功能和自噬通量，同时导致患者来源的成纤维细胞中自体荧光储存物质（AFSM）和高分子量聚集体（HMWA）的积累。然而，导致ANCL神经变性的机制尚不清楚。 此外，还没有在相关的患者来源的脑细胞模型中进行研究。在高通量和无假设的“组学”技术方面取得了前所未有的进展，可以生成高度详细的ANCL分子图谱。我们假设CSPα在内-溶酶体途径中发挥作用，CSPα突变导致神经元和小胶质细胞功能障碍和神经变性。为了验证这一假设，我们计划在有和没有DNAJC 5突变的四个大脑区域中以细胞特异性方式对差异表达基因和与DNAJC 5突变体共表达的基因进行批量RNA Seq，单核RNAseq和靶向蛋白质组学。我们的初步数据表明，DNAJC 5转录水平在小胶质细胞比神经元更高。我们将在具有DNAJC 5突变的人类iPSC衍生的神经元和小胶质细胞中进行批量RNAseq和蛋白质组学研究，并通过CRISPR/Cas9产生等基因对照，以确定DNAJC 5对细胞特异性通路的影响。我们将snRNA-seq应用于受ANCL病理影响不同的多个脑区，从而追踪病理进展的伪时间轨迹，首次在DNAJC 5突变携带者的大脑中定义主要脑细胞类型和细胞类型特异性转录状态中的DEG。 (Aim 1）。为了确定DNAJC 5突变对人iPSC衍生的神经元和小胶质细胞中ANCL病理学的细胞自主作用（Aim 2），我们将在iPSC衍生的神经元和小胶质细胞中使用遗传（CRISPR/Cas9）和药理学方法来确定DNAJC 5突变体如何影响吞噬能力、细胞活力、溶酶体功能以及致病性HMWA和AFSM的积累。我们将在人类iPSC衍生的神经元和小胶质细胞中使用蛋白质组学来测试DNAJC 5突变对分泌组以及易聚集蛋白和促炎蛋白水平的影响。我们将协调整个大脑区域和iPSC衍生细胞的RNA-seq和蛋白质组学数据，以确定DNAJC 5相关的神经退行性通路。