Search

CRISPR-mediated genome editing in human iPSC-derived neurons is established as a model system to investigate how the lysosomal pathway is adapted to the unique demands of neurons. JIP3 and JIP4 proteins promote lysosome axonal transport in these neurons with putative relevance for Alzheimer’s disease.

NPC intracellular cholesterol transporter 1 (NPC1) is a multipass, transmembrane glycoprotein mostly recognized for its key role in facilitating cholesterol efflux. Mutations in the NPC1gene result in Niemann-Pick disease, type C (NPC), a fatal, lysosomal storage disease. Due to the progressively expanding implications of NPC1-related disorders, we investigated endogenous NPC1 protein–protein interactions in the mouse cortex and human-derived iPSCs neuronal models of the disease through coimmunoprecipitation-coupled with LC-MS based proteomics. The current study investigated protein–protein interactions specific to the wild-type and the most prevalent NPC1 mutation (NPC1I1061T) while filtering out any protein interactor identified in the Npc1–/–mouse model. Additionally, the results were matched across the two species to map the parallel interactome of wild-type and mutant NPC1I1061T. Most of the identified wild-type NPC1 interactors were related to cytoskeleton organization, synaptic vesicle activity, and translation. We found many putative NPC1 interactors not previously reported, including two SCAR/WAVE complex proteins that regulate ARP 2/3 complex actin nucleation and multiple membrane proteins important for neuronal activity at synapse. Moreover, we identified proteins important in trafficking specific to wild-type and mutant NPC1I1061T. Together, the findings are essential for a comprehensive understanding of NPC1 biological functions in addition to its classical role in sterol efflux.

Using CRISPRi-based depletion in human iPSC-derived neurons we demonstrate that AP-4 adaptor protein complex regulates neuronal lysosome composition and function via regulating sorting of critical lysosomal protein receptors including Sortilin 1 to the organelle. AP-4 also regulates axonal endo-lysosome abundance in these human neurons.