The role of CSPalpha in Adult onset neuronal Lipofuscinosis pathogenesis

常染色体显性遗传性成人起病神经性蜡样脂褐素沉着症(ANCL)是一种进展迅速的致死性神经退行性痴呆，目前尚无治疗方法。我们小组和其他人同时报道，DNAJC5基因突变是常染色体显性遗传性ANCL最常见的原因。我们还发现，DNAJC5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存储物质(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。高通量和无假说的“组学”技术取得了前所未有的进步，可以为ANCL生成非常详细的分子图谱。我们假设CSPα在内切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DNAJC5突变的四个大脑区域以细胞特异性的方式对差异表达的基因和与DNAJC5突变共表达的基因进行批量RNA序列、单核RNAseq和靶向蛋白质组学。我们的初步数据表明，小胶质细胞中的DNAJC5转录水平高于神经元。我们将在带有DNAJC5突变的人IPSC来源的神经元和小胶质细胞中进行Bulk-RNAseq和蛋白质组学研究，并通过CRISPR/Cas9产生的等基因对照来确定DNAJC5对细胞自主特异性通路的影响。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DNAJC5突变携带者的大脑中定义主要脑细胞类型的DEG和特定细胞类型的转录状态。(目标1)。为了确定DNAJC5突变对人IPSC来源的神经元和小胶质细胞ANCL病理的细胞自主影响(AIM 2)，我们将在IPSC来源的神经元和小胶质细胞中使用遗传学(CRISPR/Cas9)和药理学方法来确定DNAJC5突变如何影响吞噬能力、细胞活力、溶酶体功能以及致病HMWA和AFSM的积累。我们将利用人类IPSC来源的神经元和小胶质细胞的蛋白质组学来测试DNAJC5突变对分泌组以及易聚集和促炎蛋白水平的影响。我们将协调跨大脑区域和IPSC衍生细胞的RNA-seq和蛋白质组数据，以确定DNAJC5相关的神经退化路径。

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.