The role of CSPalpha in Adult onset neuronal Lipofuscinosis pathogenesis

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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.