The age at onset of motor symptoms in Huntington’s disease (HD) is driven byHTTCAG repeat length but modified by other genes. In this study, we used exome sequencing of 683 patients with HD with extremes of onset or phenotype relative to CAG length to identify rare variants associated with clinical effect. We discovered damaging coding variants in candidate modifier genes identified in previous genome-wide association studies associated with altered HD onset or severity. Variants in FAN1 clustered in its DNA-binding and nuclease domains and were associated predominantly with earlier-onset HD. Nuclease activities of purified variants in vitro correlated with residual age at motor onset of HD. Mutating endogenous FAN1 to a nuclease-inactive form in an induced pluripotent stem cell model of HD led to rates of CAG expansion similar to those observed with completeFAN1knockout. Together, these data implicate FAN1 nuclease activity in slowing somatic repeat expansion and hence onset of HD.

Variants at the GBA locus, encoding glucocerebrosidase, are the strongest common genetic risk factor for Parkinson's disease (PD). To understand GBA-related disease mechanisms, we use a multi-part-enrichment proteomics and post-translational modification (PTM) workflow, identifying large numbers of dysregulated proteins and PTMs in heterozygous GBA-N370S PD patient induced pluripotent stem cell (iPSC) dopamine neurons. Alterations in glycosylation status show disturbances in the autophagy-lysosomal pathway, which concur with upstream perturbations in mammalian target of rapamycin (mTOR) activation in GBA-PD neurons. Several native and modified proteins encoded by PD-associated genes are dysregulated in GBA-PD neurons. Integrated pathway analysis reveals impaired neuritogenesis in GBA-PD neurons and identify tau as a key pathway mediator. Functional assays confirm neurite outgrowth deficits and identify impaired mitochondrial movement in GBA-PD neurons. Furthermore, pharmacological rescue of glucocerebrosidase activity in GBA-PD neurons improves the neurite outgrowth deficit. Overall, this study demonstrates the potential of PTMomics to elucidate neurodegeneration-associated pathways and potential drug targets in complex disease models.

Parkinson’s disease (PD) remains highly challenging to model in vitro, particularly when seeking robust functional phenotypes for target discovery. Patient-derived induced pluripotent stem cell dopaminergic neurons (iPSC-DANs) offer a promising platform to address this challenge. Because calcium dyshomeostasis is well-described in PD pathophysiology, it offers a platform for phenotypic screening. We have previously shown that GBA-N370S iPSC-DANs display impaired intracellular calcium release. Here, we used this disease-relevant phenotype to undertake a 4,108-compound phenotypic screen in GBA-N370S ( GBA-NS ) iPSC-DANs. This screen identified fatty acid synthase (FASN) as the most enriched protein target. The ability of compounds to rescue calcium release correlated with biochemical potency against FASN across multiple chemical series. We confirmed that FASN knockdown increased intracellular calcium release, supporting an on-target effect. Untargeted lipidomics demonstrated broad remodelling of the neuronal lipidome after FASN inhibition, confirming target engagement. FASN inhibition also increased active β-glucocerebrosidase (GCase), reduced extracellular α-synuclein release and ER stress, and altered RASGRF1 expression. Mechanistically, FASN inhibition activated sterol regulatory element-binding protein 1 (SREBP1), and SREBP1 overexpression alone was sufficient to increase intracellular calcium release, identifying a FASN-SREBP1-calcium homeostasis axis in human neurons. Finally, the FASN inhibitor BI 99179 achieved brain exposure and reduced brain fatty acid levels while inducing an SRE-associated transcriptional response in a Parkinson’s mouse model, providing evidence of in vivo target engagement. These findings establish patient iPSC-neuron functional screening as a tractable discovery platform to identify targets which can be taken in vivo and nominate the FASN-SREBP1 axis as a candidate therapeutic pathway in PD. One Sentence Summary: We applied a disease-relevant phenotypic screen in patient neurons to uncover a lipid-calcium regulatory axis that rescues multiple pathogenic pathways in Parkinson’s.