Generation and characterization of human iPSC-derived NPC1 (I1061T/I10161T) i(3)Neurons as a model for NPC1 disease.

Niemann-Pick disease, type C is an autosomal recessive, fatal, neurodegenerative disorder caused by pathological variants in NPC1 or NPC2. Dysfunction of either NPC1 or NPC2 results in impaired intracellular cholesterol transport and subsequent storage of unesterified cholesterol in endolysosomal compartments. Earlier cell-based studies utilized patient fibroblasts to study this disease; however, neuronal cells allow for investigation of the neurodegenerative aspect of NPC1. Expression of neurogenin in induced pluripotent stem cells leads to the generation of i(3)Neurons (integrated, isogenic, and inducible), allowing for rapid, synchronized growth of homogenous neurons. In this study, we report the development and characterization of a human iPSC-derived NPC1 (I1061T/I1061T) i(3)Neuronal model system. NPC1 (I1061T) is a missense variant resulting in a misfolded protein targeted for proteasomal degradation in the ER. NPC1 (I1061T/I1061T) i(3)Neurons phenocopied the cellular pathological features of NPC1 disease including endolysosomal cholesterol accumulation, lysosomal morphological changes, and response to the proteostasis modulator, mo56HC. The NPC1 phenotype was alleviated by 2-hydroxypropyl-β-cyclodextrin treatment, a drug demonstrating efficacy both in vitro and in vivo. This NPC1 (I1061T/I1061T) i(3)Neuronal cell line can facilitate future high-throughput drug and genomic screens, particularly those aimed at identifying proteostasis regulators that improve the expression/stability of the mutant NPC1 protein.