Development of a neuronal model of Niemann-Pick type C1 disease using human induced pluripotent stem cells

Authors

• AV Prabhu
• ME Ward
• FD Porter

Abstract

Niemann-Pick Type C1 disease (NPC) is a lethal, neurodegenerative disorder with typical onset during childhood. It is caused by mutations in the NPC1 gene, which is responsible for cholesterol efflux from the lysosome. In the absence of functional NPC1 protein, endocytosed cholesterol accumulates in the lysosome. Many in vitro studies of NPC have been performed with peripheral cells. However, NPC is fundamentally a neurodegenerative disease. Thus, there is a need for a useful cellular model of NPC that can examine processes contributing specifically to neuronal loss, with the ability to identify therapeutics that are effective in neurons. We have generated a neuronal model of NPC1 disease using human induced pluripotent stem cells (iPSCs) that harbor a neurogenin 2 transgene which can be turned on in the presence of the compound doxycyline. Expression of neurogenin 2 results in the rapid and reproducible differentiation of iPSCs to glutamatergic cortical neurons. Unlike many commonly used immortalized cell lines, an isogenic iPSC line has the advantage of a normal, stable karyotype, and using CRISPR/Cas-9 technology, we have successfully knocked-out NPC1 in the iPSC line. In both the wild-type and NPC1 null cells, neuron-like morphology is clear just 7 days post-differentiation with doxycycline. At this time, significant accumulation of unesterified cholesterol is evident in the perinuclear region of NPC1 null cells using markers of cholesterol – filipin and perfringolysin O. We are currently investigating the major changes in the lipidome or proteome of neurons due to the absence of functional NPC1. We also aim to characterize the effect of NPC1 knockout on the biogenesis, transport and distribution of lysosomes in the neuron. Any lysosomal defects identified could provide insights into the cellular pathology that contributes to neuronal dysfunction in NPC1 disease. Furthermore, significant phenotypes in the NPC1 model cell line, such as the changes in the accumulation and distribution of cholesterol could also be used for primary screens. In general, high-throughput screens using neurons are disadvantaged by high costs, low neuronal yields, heterogeneous neuronal populations, and complex differentiation processes. With our NPC model, we are able to avoid these issues and will perform two critical high-throughput screens. The first is a drug repurposing screen using a collection of 2,816 clinically approved drugs, and the second uses existing CRISPR-interference libraries to identify gene modifiers of NPC1. These unbiased screens could reveal new clues to NPC pathogenesis, and help identify viable therapies that impede neurodegeneration in NPC.

Scientific Focus Area: Cell Biology

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