A human lysosomal storage disorder toolkit for decoding proteome landscapes in cortical and dopaminergic-like induced neurons

Lysosomes maintain cellular homeostasis by degrading proteins delivered via endocytosis and autophagy, and by recycling building blocks for organelle biogenesis. Lysosomal Storage Disorders (LSDs) comprise a broad group of diseases affecting diverse lysosomal functions. To facilitate molecular phenotyping across diverse LSD gene classes, we are developing a library of human embryonic stem cells engineered to lack individual LSD genes. Here, we report the generation and initial analysis of a first-generation toolkit of ES cells lacking one of 23 LSD genes, including the majority of genes associated with sphingolipidoses and neuronal ceroid lipofucinoses. Global proteomic analysis of induced cortical-like and midbrain dopaminergic-like neurons coupled with in-depth abundance and correlation profiling across organelles and sub-organelle components revealed potential vulnerabilities that reflect distinct patterns of proteome alterations across both genotypes and neuronal cell types. We characterize alterations in the mitochondrial proteome associated with GBA1 and ASAH1 deficiency, and identify synaptic defects in ASAH1-/- induced neurons that correlate with defects in neuronal firing rates. Moreover, we developed an informatic pipeline for proteome-wide identification of individual protein interactions and protein complexes that may be disrupted as a result of LSD gene deficiency. Finally, we visualized structural alterations of ASAH1-deficient endolysosomes in situ using cryo-electron tomography, revealing swollen organelles that were largely devoid of dense internal membranes characteristic of wild-type cells, but containing numerous intralumenal vesicle compartments. This toolkit and associated proteomic landscapes provide a resource for defining molecular signatures associated with LSD gene dysfunction and organelle vulnerability.