A novel post-translational proteomics platform identifies neurite outgrowth impairments in Parkinson's disease GBA-N370S dopamine neurons

Abstract

The causes of Parkinsons disease (PD) likely involve complex interactions between environmental factors and susceptibility genes with variants at the GBA locus encoding the glucocerebrosidase (GCase) enzyme being the strongest common genetic risk factor for PD. To understand GBA-related disease mechanisms, we used a novel multipart-enrichment proteomics and post-translational modification workflow to simultaneously identify peptides with phosphorylation, reversible cysteine-modifications or sialylated N-linked glycosylation, alongside unmodified proteins. We identified large numbers of dysregulated proteins and post-translational modifications (PTMs) in heterozygous GBA-N370S PD patient induced pluripotent stem cells (iPSC)-derived dopamine neurons. Alterations in glycosylation status of lysosomal proteins identified disturbances in the autophagy-lysosomal pathway, concurrent with upstream perturbations in mTOR phosphorylation and activity in GBA-N370S iPSC-dopamine neurons. In addition, the strategy revealed several native and modified proteins encoded by PD-associated genes to be dysregulated in GBA-N370S neurons, enhancing our understanding of the wider role of GBA mutations on the neuronal proteome. Integrated pathway analysis of all datasets revealed impaired neuritogenesis in GBA-N370S PD iPSC-dopamine neurons and identified tau (MAPT) as a key mediator of this process. Using a functional assay, we confirmed neurite outgrowth deficits in GBA-N370S PD neurons and a central role for tau in this process. Furthermore, pharmacological restoration of GCase activity in GBA-N370S PD patient neurons rescued the neurite outgrowth deficit. Overall, this study demonstrates the potential of PTMomics to elucidate novel neurodegeneration-associated pathways and identify phenotypes and potential drug targets in complex disease models.