# Investigating mechanisms of NPC1 proteostasis in human neurons

> **NIH NIH F31** · UNIVERSITY OF MICHIGAN AT ANN ARBOR · 2024 · $41,925

## Abstract

Abstract
Niemann-Pick disease type C is a childhood-onset, autosomal recessive, lysosomal storage disease
characterized by the accumulation of unesterified cholesterol. Clinical phenotypes are heterogeneous, but
typically include progressive neurodegeneration, seizures, and early death. Niemann-Pick C is commonly (~95%
of cases) caused by loss-of-function mutations in the NPC1 gene, which encodes a transmembrane glycoprotein
required for exporting cholesterol from late endosomes and lysosomes. Most commonly, Niemann-Pick C
patients have an isoleucine to threonine missense mutation at position 1061 (I1061T NPC1). In fibroblasts from
these patients, we showed that I1061T NPC1 misfolds in the endoplasmic reticulum (ER) and is rapidly degraded
by ER-associated degradation (ERAD) and by ER-autophagy (ER-phagy). Importantly, escape from these
pathways by genetic or pharmacologic manipulations enables I1061T NPC1 to reach the lysosome, where it is
still functional. As such, NPC1 protein homeostasis (proteostasis) pathways have emerged as prime targets for
therapeutic intervention. While modulators of these pathways have shown success in patient fibroblasts, thus far
they failed to improve neurological phenotypes when tested in vivo. Critically, the extent to which pathways
identified in fibroblasts also function in neurons, a critical disease target cell, is unknown. The overall objective
of this application is to determine the effectors mediating NPC1 folding, trafficking, and degradation in neurons.
Our central hypothesis is that cholesterol acts as a pharmacological chaperone to promote folding of NPC1 in
the ER, and that folding and trafficking of newly synthesized NPC1 to the lysosome are regulated by neuron-
specific pathways. To address this hypothesis, we will use inducible human stem cell-derived neurons (iNeurons)
with WT or I1061T NPC1 to measure NPC1 trafficking, function, and half-life after manipulation of cellular
cholesterol or use of therapeutic cholesterol analogs (Aim 1). We will also use iNeurons expressing fluorescently
tagged WT or I1061T NPC1 to find proteostasis regulators via a targeted CRISPR interference (CRISPRi) screen
of proteostasis-related genes (Aim 2). The rationale for this work is that characterizing the machinery that
regulates NPC1 folding and trafficking will deepen our mechanistic understanding of the NPC1 protein and
identify new targets for development of Niemann-Pick C disease therapeutics. This research plan will not only
deepen our understanding of neuronal NPC1 proteostasis but will provide learning opportunities that will further
my development during my doctoral training. Using the University of Michigan’s vast resources for scientific
investigation, I will gain new skills in live cell and high-content imaging, CRISPR-based techniques, and in written
and oral scientific communication. This new training will position me well for success as a post-doctoral trainee
and long-term as an independent investigator...

## Key facts

- **NIH application ID:** 10998266
- **Project number:** 1F31NS135634-01A1
- **Recipient organization:** UNIVERSITY OF MICHIGAN AT ANN ARBOR
- **Principal Investigator:** Ruth D. Azaria
- **Activity code:** F31 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2024
- **Award amount:** $41,925
- **Award type:** 1
- **Project period:** 2024-07-01 → 2026-06-30

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10998266

## Citation

> US National Institutes of Health, RePORTER application 10998266, Investigating mechanisms of NPC1 proteostasis in human neurons (1F31NS135634-01A1). Retrieved via AI Analytics 2026-05-25 from https://api.ai-analytics.org/grant/nih/10998266. Licensed CC0.

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