The accumulation of insoluble and misfolded proteins is commonly associated with degeneration of neurons in amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) patients. Heat shock proteins (HSPs) play a central role in the regulation of protein homeostasis by facilitating effective folding, trafficking, and degradation of both nascent and aged polypeptides. While it has become increasingly clear that perturbations in in the proteostasis network play a significant role in ALS/FTD, limited emphasis has been placed on investigating the direct causal relationship between the functionality of HSPs and disease pathogenesis. Heterozygous, loss-of-function mutations in the DNAJC7 gene, which encodes for the HSP40 protein DNAJC7 have recently been identified as a cause for rare forms of ALS. The DNAJC7 protein acts as a co-chaperone for HSP70 chaperones, thereby facilitating HSP70-polypeptide interactions and appropriate polypeptide folding. However, little is known about the specific function of DNAJC7 in the central nervous system and motor neurons specifically, the cell type that predominantly degenerates in ALS patients. Our primary hypothesis is that DNAJC7 haploinsufficiency leads to the accumulation of misfolded HSP70 client proteins resulting in the disruption of biological processes critical to the function and survival of vulnerable MNs. Here, we will use mutant DNAJC7 cellular models, patient induced pluripotent stem cell (iPSC)-derived cortical and spinal motor neurons and CRISPR/Cas9 gene-editing, in combination with mass spectrometry (MS)-based quantitative proteomics and RNA-Sequencing to elucidate how ALS/FTD-associated mutations in DNAJC7 contribute towards neuronal dysfunction and degeneration. In Aim 1 we will determine the endogenous interactome of DNAJC7 in mutant and isogenic control human neurons to better understand the functional role that it plays in postmitotic cells. In Aim 2 we will methodically characterize disruptions in protein folding caused by pathogenic DNAJC7 using two independent MS-based proteomics approaches. These experiments will provide an unbiased proteome-wide interrogation of fundamental aspects of proteostasis in ALS-DNAJC7 mutant and isogenic control neurons. In Aim 3, which is based on our preliminary finding that DNAJC7 interacts with the ALS-casual RNA-metabolism proteins FUS and MATR3, we will investigate how DNAJC7 mutations affect RNA processing in patient neurons. Taken together, our proposed aims will shed light upon the cellular mechanisms that are compromised by DNAJC7 haploinsufficiency in distinct human cortical and spinal neurons. Our findings will impact the field by contributing towards the understanding of HSP-dependent proteostasis mechanisms in human neurons as well as to how rare ALS genetic mutations lead to neuron dysfunction and loss. Identifying novel proteins that are susceptible to misfolding in human neurons might highlight cellular pathways critical for ALS/FTD pathophysiology.
|Effective start/end date||1/18/23 → 12/31/24|
- National Institute of Neurological Disorders and Stroke (1R21NS131713-01)
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