ABSTRACT 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 spinal motor neurons (MNS) 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, which is based on our preliminary finding that DNAJC7 interacts with the ALS-causal RNA metabolism protein MATR3, we will determine if pathogenic mutations in DNAJC7 impede MATR3 folding. Next, we will investigate the functional consequences of disruptions in DNAJC7-substrate interface by measuring how DNAJC7 mutations affect MATR3-associated RNA processing in patient MNs. In Aim 2, which is based on our preliminary finding that DNAJC7 impedes the HSF1 response, we will define the mechanistic role DNAJC7 plays in the HSF1- mediated stress response in patient derived mutant and isogenic control MNs. We will directly measure DNAJC7- dependent loss of HSF1 transcriptional activity upon stress, determine if DNAJC7 is involved in the dissociation of the inhibitory HSP70-HSF1 complex, and methodically characterize disruptions in protein solubility caused by pathogenic DNAJC7 using MS-based proteomics approaches. Taken together, our proposed aims will shed light upon the cellular mechanisms that are compromised by DNAJC7 haploinsufficiency. 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.

Project Narrative The purpose of our proposed study is to elucidate how mutations in the heat shock protein DNAJC7 cause ALS/FTD, an untreatable and devastating neurodegenerative disease. We will use induced pluripotent stem cell- derived neurons in combination with quantitative mass spectrometry-based proteomics to elucidate the role of DNAJC7 in human neurons and determine how its dysfunction leads to their dysfunction.