Research ArticleNeuroscience Open Access | 10.1172/jci.insight.179525

Ruth D. Azaria,1 Adele B. Correia,1 Kylie J. Schache,1 Manuela Zapata,2 Koralege C. Pathmasiri,2 Varshasnata Mohanty,2 Dharma T. Nannapaneni,3 Brandon L. Ashfeld,3,4 Paul Helquist,3 Olaf Wiest,3 Kenji Ohgane,5 Qingqing Li,6 Ross A. Fredenburg,7 Brian S.J. Blagg,3,4 Stephanie M. Cologna,2 Mark L. Schultz,8 and Andrew P. Lieberman1

1Department of Pathology, University of Michigan Medical School, Ann Arbor, Michigan, USA.

2Department of Chemistry, University of Illinois Chicago, Illinois, USA.

3Department of Chemistry & Biochemistry and

4Warren Family Center for Drug Discovery, University of Notre Dame, Notre Dame, Indiana, USA.

5Department of Chemistry, Ochanomizu University, Tokyo, Japan.

6HitGen Inc, Chengdu, China.

7Ara Parseghian Medical Research Fund at Notre Dame University, Notre Dame, Indiana, USA.

8Stead Family Department of Pediatrics, Carver College of Medicine, University of Iowa, Iowa City, Iowa, USA.

Address correspondence to: Andrew P. Lieberman, Department of Pathology, University of Michigan Medical School, 3510 MSRB1, 1150 W. Medical Center Dr., Ann Arbor, Michigan 48109, USA. Phone: 734.647.4624; Email: liebermn@med.umich.edu. Or to: Mark L. Schultz, Department of Pediatrics, University of Iowa, 2150 ML, 25 S. Grand Avenue, Iowa City, Iowa 52246, USA. Phone: 319.335.0557; Email: mark-schultz-1@uiowa.edu.

Authorship note: RDA, ABC, and KLS contributed equally to this work.

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1Department of Pathology, University of Michigan Medical School, Ann Arbor, Michigan, USA.

2Department of Chemistry, University of Illinois Chicago, Illinois, USA.

3Department of Chemistry & Biochemistry and

4Warren Family Center for Drug Discovery, University of Notre Dame, Notre Dame, Indiana, USA.

5Department of Chemistry, Ochanomizu University, Tokyo, Japan.

6HitGen Inc, Chengdu, China.

7Ara Parseghian Medical Research Fund at Notre Dame University, Notre Dame, Indiana, USA.

8Stead Family Department of Pediatrics, Carver College of Medicine, University of Iowa, Iowa City, Iowa, USA.

Address correspondence to: Andrew P. Lieberman, Department of Pathology, University of Michigan Medical School, 3510 MSRB1, 1150 W. Medical Center Dr., Ann Arbor, Michigan 48109, USA. Phone: 734.647.4624; Email: liebermn@med.umich.edu. Or to: Mark L. Schultz, Department of Pediatrics, University of Iowa, 2150 ML, 25 S. Grand Avenue, Iowa City, Iowa 52246, USA. Phone: 319.335.0557; Email: mark-schultz-1@uiowa.edu.

Authorship note: RDA, ABC, and KLS contributed equally to this work.

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1Department of Pathology, University of Michigan Medical School, Ann Arbor, Michigan, USA.

2Department of Chemistry, University of Illinois Chicago, Illinois, USA.

3Department of Chemistry & Biochemistry and

4Warren Family Center for Drug Discovery, University of Notre Dame, Notre Dame, Indiana, USA.

5Department of Chemistry, Ochanomizu University, Tokyo, Japan.

6HitGen Inc, Chengdu, China.

7Ara Parseghian Medical Research Fund at Notre Dame University, Notre Dame, Indiana, USA.

8Stead Family Department of Pediatrics, Carver College of Medicine, University of Iowa, Iowa City, Iowa, USA.

Address correspondence to: Andrew P. Lieberman, Department of Pathology, University of Michigan Medical School, 3510 MSRB1, 1150 W. Medical Center Dr., Ann Arbor, Michigan 48109, USA. Phone: 734.647.4624; Email: liebermn@med.umich.edu. Or to: Mark L. Schultz, Department of Pediatrics, University of Iowa, 2150 ML, 25 S. Grand Avenue, Iowa City, Iowa 52246, USA. Phone: 319.335.0557; Email: mark-schultz-1@uiowa.edu.

Authorship note: RDA, ABC, and KLS contributed equally to this work.

Find articles by Schache, K. in: JCI | PubMed | Google Scholar

1Department of Pathology, University of Michigan Medical School, Ann Arbor, Michigan, USA.

2Department of Chemistry, University of Illinois Chicago, Illinois, USA.

3Department of Chemistry & Biochemistry and

4Warren Family Center for Drug Discovery, University of Notre Dame, Notre Dame, Indiana, USA.

5Department of Chemistry, Ochanomizu University, Tokyo, Japan.

6HitGen Inc, Chengdu, China.

7Ara Parseghian Medical Research Fund at Notre Dame University, Notre Dame, Indiana, USA.

8Stead Family Department of Pediatrics, Carver College of Medicine, University of Iowa, Iowa City, Iowa, USA.

Address correspondence to: Andrew P. Lieberman, Department of Pathology, University of Michigan Medical School, 3510 MSRB1, 1150 W. Medical Center Dr., Ann Arbor, Michigan 48109, USA. Phone: 734.647.4624; Email: liebermn@med.umich.edu. Or to: Mark L. Schultz, Department of Pediatrics, University of Iowa, 2150 ML, 25 S. Grand Avenue, Iowa City, Iowa 52246, USA. Phone: 319.335.0557; Email: mark-schultz-1@uiowa.edu.

Authorship note: RDA, ABC, and KLS contributed equally to this work.

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1Department of Pathology, University of Michigan Medical School, Ann Arbor, Michigan, USA.

2Department of Chemistry, University of Illinois Chicago, Illinois, USA.

3Department of Chemistry & Biochemistry and

4Warren Family Center for Drug Discovery, University of Notre Dame, Notre Dame, Indiana, USA.

5Department of Chemistry, Ochanomizu University, Tokyo, Japan.

6HitGen Inc, Chengdu, China.

7Ara Parseghian Medical Research Fund at Notre Dame University, Notre Dame, Indiana, USA.

8Stead Family Department of Pediatrics, Carver College of Medicine, University of Iowa, Iowa City, Iowa, USA.

Address correspondence to: Andrew P. Lieberman, Department of Pathology, University of Michigan Medical School, 3510 MSRB1, 1150 W. Medical Center Dr., Ann Arbor, Michigan 48109, USA. Phone: 734.647.4624; Email: liebermn@med.umich.edu. Or to: Mark L. Schultz, Department of Pediatrics, University of Iowa, 2150 ML, 25 S. Grand Avenue, Iowa City, Iowa 52246, USA. Phone: 319.335.0557; Email: mark-schultz-1@uiowa.edu.

Authorship note: RDA, ABC, and KLS contributed equally to this work.

Find articles by Pathmasiri, K. in: JCI | PubMed | Google Scholar |

1Department of Pathology, University of Michigan Medical School, Ann Arbor, Michigan, USA.

2Department of Chemistry, University of Illinois Chicago, Illinois, USA.

3Department of Chemistry & Biochemistry and

4Warren Family Center for Drug Discovery, University of Notre Dame, Notre Dame, Indiana, USA.

5Department of Chemistry, Ochanomizu University, Tokyo, Japan.

6HitGen Inc, Chengdu, China.

7Ara Parseghian Medical Research Fund at Notre Dame University, Notre Dame, Indiana, USA.

8Stead Family Department of Pediatrics, Carver College of Medicine, University of Iowa, Iowa City, Iowa, USA.

Address correspondence to: Andrew P. Lieberman, Department of Pathology, University of Michigan Medical School, 3510 MSRB1, 1150 W. Medical Center Dr., Ann Arbor, Michigan 48109, USA. Phone: 734.647.4624; Email: liebermn@med.umich.edu. Or to: Mark L. Schultz, Department of Pediatrics, University of Iowa, 2150 ML, 25 S. Grand Avenue, Iowa City, Iowa 52246, USA. Phone: 319.335.0557; Email: mark-schultz-1@uiowa.edu.

Authorship note: RDA, ABC, and KLS contributed equally to this work.

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1Department of Pathology, University of Michigan Medical School, Ann Arbor, Michigan, USA.

2Department of Chemistry, University of Illinois Chicago, Illinois, USA.

3Department of Chemistry & Biochemistry and

4Warren Family Center for Drug Discovery, University of Notre Dame, Notre Dame, Indiana, USA.

5Department of Chemistry, Ochanomizu University, Tokyo, Japan.

6HitGen Inc, Chengdu, China.

7Ara Parseghian Medical Research Fund at Notre Dame University, Notre Dame, Indiana, USA.

8Stead Family Department of Pediatrics, Carver College of Medicine, University of Iowa, Iowa City, Iowa, USA.

Address correspondence to: Andrew P. Lieberman, Department of Pathology, University of Michigan Medical School, 3510 MSRB1, 1150 W. Medical Center Dr., Ann Arbor, Michigan 48109, USA. Phone: 734.647.4624; Email: liebermn@med.umich.edu. Or to: Mark L. Schultz, Department of Pediatrics, University of Iowa, 2150 ML, 25 S. Grand Avenue, Iowa City, Iowa 52246, USA. Phone: 319.335.0557; Email: mark-schultz-1@uiowa.edu.

Authorship note: RDA, ABC, and KLS contributed equally to this work.

Find articles by Nannapaneni, D. in: JCI | PubMed | Google Scholar

1Department of Pathology, University of Michigan Medical School, Ann Arbor, Michigan, USA.

2Department of Chemistry, University of Illinois Chicago, Illinois, USA.

3Department of Chemistry & Biochemistry and

4Warren Family Center for Drug Discovery, University of Notre Dame, Notre Dame, Indiana, USA.

5Department of Chemistry, Ochanomizu University, Tokyo, Japan.

6HitGen Inc, Chengdu, China.

7Ara Parseghian Medical Research Fund at Notre Dame University, Notre Dame, Indiana, USA.

8Stead Family Department of Pediatrics, Carver College of Medicine, University of Iowa, Iowa City, Iowa, USA.

Address correspondence to: Andrew P. Lieberman, Department of Pathology, University of Michigan Medical School, 3510 MSRB1, 1150 W. Medical Center Dr., Ann Arbor, Michigan 48109, USA. Phone: 734.647.4624; Email: liebermn@med.umich.edu. Or to: Mark L. Schultz, Department of Pediatrics, University of Iowa, 2150 ML, 25 S. Grand Avenue, Iowa City, Iowa 52246, USA. Phone: 319.335.0557; Email: mark-schultz-1@uiowa.edu.

Authorship note: RDA, ABC, and KLS contributed equally to this work.

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1Department of Pathology, University of Michigan Medical School, Ann Arbor, Michigan, USA.

2Department of Chemistry, University of Illinois Chicago, Illinois, USA.

3Department of Chemistry & Biochemistry and

4Warren Family Center for Drug Discovery, University of Notre Dame, Notre Dame, Indiana, USA.

5Department of Chemistry, Ochanomizu University, Tokyo, Japan.

6HitGen Inc, Chengdu, China.

7Ara Parseghian Medical Research Fund at Notre Dame University, Notre Dame, Indiana, USA.

8Stead Family Department of Pediatrics, Carver College of Medicine, University of Iowa, Iowa City, Iowa, USA.

Address correspondence to: Andrew P. Lieberman, Department of Pathology, University of Michigan Medical School, 3510 MSRB1, 1150 W. Medical Center Dr., Ann Arbor, Michigan 48109, USA. Phone: 734.647.4624; Email: liebermn@med.umich.edu. Or to: Mark L. Schultz, Department of Pediatrics, University of Iowa, 2150 ML, 25 S. Grand Avenue, Iowa City, Iowa 52246, USA. Phone: 319.335.0557; Email: mark-schultz-1@uiowa.edu.

Authorship note: RDA, ABC, and KLS contributed equally to this work.

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1Department of Pathology, University of Michigan Medical School, Ann Arbor, Michigan, USA.

2Department of Chemistry, University of Illinois Chicago, Illinois, USA.

3Department of Chemistry & Biochemistry and

4Warren Family Center for Drug Discovery, University of Notre Dame, Notre Dame, Indiana, USA.

5Department of Chemistry, Ochanomizu University, Tokyo, Japan.

6HitGen Inc, Chengdu, China.

7Ara Parseghian Medical Research Fund at Notre Dame University, Notre Dame, Indiana, USA.

8Stead Family Department of Pediatrics, Carver College of Medicine, University of Iowa, Iowa City, Iowa, USA.

Address correspondence to: Andrew P. Lieberman, Department of Pathology, University of Michigan Medical School, 3510 MSRB1, 1150 W. Medical Center Dr., Ann Arbor, Michigan 48109, USA. Phone: 734.647.4624; Email: liebermn@med.umich.edu. Or to: Mark L. Schultz, Department of Pediatrics, University of Iowa, 2150 ML, 25 S. Grand Avenue, Iowa City, Iowa 52246, USA. Phone: 319.335.0557; Email: mark-schultz-1@uiowa.edu.

Authorship note: RDA, ABC, and KLS contributed equally to this work.

Find articles by Wiest, O. in: JCI | PubMed | Google Scholar

1Department of Pathology, University of Michigan Medical School, Ann Arbor, Michigan, USA.

2Department of Chemistry, University of Illinois Chicago, Illinois, USA.

3Department of Chemistry & Biochemistry and

4Warren Family Center for Drug Discovery, University of Notre Dame, Notre Dame, Indiana, USA.

5Department of Chemistry, Ochanomizu University, Tokyo, Japan.

6HitGen Inc, Chengdu, China.

7Ara Parseghian Medical Research Fund at Notre Dame University, Notre Dame, Indiana, USA.

8Stead Family Department of Pediatrics, Carver College of Medicine, University of Iowa, Iowa City, Iowa, USA.

Address correspondence to: Andrew P. Lieberman, Department of Pathology, University of Michigan Medical School, 3510 MSRB1, 1150 W. Medical Center Dr., Ann Arbor, Michigan 48109, USA. Phone: 734.647.4624; Email: liebermn@med.umich.edu. Or to: Mark L. Schultz, Department of Pediatrics, University of Iowa, 2150 ML, 25 S. Grand Avenue, Iowa City, Iowa 52246, USA. Phone: 319.335.0557; Email: mark-schultz-1@uiowa.edu.

Authorship note: RDA, ABC, and KLS contributed equally to this work.

Find articles by Ohgane, K. in: JCI | PubMed | Google Scholar

1Department of Pathology, University of Michigan Medical School, Ann Arbor, Michigan, USA.

2Department of Chemistry, University of Illinois Chicago, Illinois, USA.

3Department of Chemistry & Biochemistry and

4Warren Family Center for Drug Discovery, University of Notre Dame, Notre Dame, Indiana, USA.

5Department of Chemistry, Ochanomizu University, Tokyo, Japan.

6HitGen Inc, Chengdu, China.

7Ara Parseghian Medical Research Fund at Notre Dame University, Notre Dame, Indiana, USA.

8Stead Family Department of Pediatrics, Carver College of Medicine, University of Iowa, Iowa City, Iowa, USA.

Address correspondence to: Andrew P. Lieberman, Department of Pathology, University of Michigan Medical School, 3510 MSRB1, 1150 W. Medical Center Dr., Ann Arbor, Michigan 48109, USA. Phone: 734.647.4624; Email: liebermn@med.umich.edu. Or to: Mark L. Schultz, Department of Pediatrics, University of Iowa, 2150 ML, 25 S. Grand Avenue, Iowa City, Iowa 52246, USA. Phone: 319.335.0557; Email: mark-schultz-1@uiowa.edu.

Authorship note: RDA, ABC, and KLS contributed equally to this work.

Find articles by Li, Q. in: JCI | PubMed | Google Scholar

1Department of Pathology, University of Michigan Medical School, Ann Arbor, Michigan, USA.

2Department of Chemistry, University of Illinois Chicago, Illinois, USA.

3Department of Chemistry & Biochemistry and

4Warren Family Center for Drug Discovery, University of Notre Dame, Notre Dame, Indiana, USA.

5Department of Chemistry, Ochanomizu University, Tokyo, Japan.

6HitGen Inc, Chengdu, China.

7Ara Parseghian Medical Research Fund at Notre Dame University, Notre Dame, Indiana, USA.

8Stead Family Department of Pediatrics, Carver College of Medicine, University of Iowa, Iowa City, Iowa, USA.

Address correspondence to: Andrew P. Lieberman, Department of Pathology, University of Michigan Medical School, 3510 MSRB1, 1150 W. Medical Center Dr., Ann Arbor, Michigan 48109, USA. Phone: 734.647.4624; Email: liebermn@med.umich.edu. Or to: Mark L. Schultz, Department of Pediatrics, University of Iowa, 2150 ML, 25 S. Grand Avenue, Iowa City, Iowa 52246, USA. Phone: 319.335.0557; Email: mark-schultz-1@uiowa.edu.

Authorship note: RDA, ABC, and KLS contributed equally to this work.

Find articles by Fredenburg, R. in: JCI | PubMed | Google Scholar

1Department of Pathology, University of Michigan Medical School, Ann Arbor, Michigan, USA.

2Department of Chemistry, University of Illinois Chicago, Illinois, USA.

3Department of Chemistry & Biochemistry and

4Warren Family Center for Drug Discovery, University of Notre Dame, Notre Dame, Indiana, USA.

5Department of Chemistry, Ochanomizu University, Tokyo, Japan.

6HitGen Inc, Chengdu, China.

7Ara Parseghian Medical Research Fund at Notre Dame University, Notre Dame, Indiana, USA.

8Stead Family Department of Pediatrics, Carver College of Medicine, University of Iowa, Iowa City, Iowa, USA.

Address correspondence to: Andrew P. Lieberman, Department of Pathology, University of Michigan Medical School, 3510 MSRB1, 1150 W. Medical Center Dr., Ann Arbor, Michigan 48109, USA. Phone: 734.647.4624; Email: liebermn@med.umich.edu. Or to: Mark L. Schultz, Department of Pediatrics, University of Iowa, 2150 ML, 25 S. Grand Avenue, Iowa City, Iowa 52246, USA. Phone: 319.335.0557; Email: mark-schultz-1@uiowa.edu.

Authorship note: RDA, ABC, and KLS contributed equally to this work.

Find articles by Blagg, B. in: JCI | PubMed | Google Scholar

1Department of Pathology, University of Michigan Medical School, Ann Arbor, Michigan, USA.

2Department of Chemistry, University of Illinois Chicago, Illinois, USA.

3Department of Chemistry & Biochemistry and

4Warren Family Center for Drug Discovery, University of Notre Dame, Notre Dame, Indiana, USA.

5Department of Chemistry, Ochanomizu University, Tokyo, Japan.

6HitGen Inc, Chengdu, China.

7Ara Parseghian Medical Research Fund at Notre Dame University, Notre Dame, Indiana, USA.

8Stead Family Department of Pediatrics, Carver College of Medicine, University of Iowa, Iowa City, Iowa, USA.

Address correspondence to: Andrew P. Lieberman, Department of Pathology, University of Michigan Medical School, 3510 MSRB1, 1150 W. Medical Center Dr., Ann Arbor, Michigan 48109, USA. Phone: 734.647.4624; Email: liebermn@med.umich.edu. Or to: Mark L. Schultz, Department of Pediatrics, University of Iowa, 2150 ML, 25 S. Grand Avenue, Iowa City, Iowa 52246, USA. Phone: 319.335.0557; Email: mark-schultz-1@uiowa.edu.

Authorship note: RDA, ABC, and KLS contributed equally to this work.

Find articles by Cologna, S. in: JCI | PubMed | Google Scholar |

1Department of Pathology, University of Michigan Medical School, Ann Arbor, Michigan, USA.

2Department of Chemistry, University of Illinois Chicago, Illinois, USA.

3Department of Chemistry & Biochemistry and

4Warren Family Center for Drug Discovery, University of Notre Dame, Notre Dame, Indiana, USA.

5Department of Chemistry, Ochanomizu University, Tokyo, Japan.

6HitGen Inc, Chengdu, China.

7Ara Parseghian Medical Research Fund at Notre Dame University, Notre Dame, Indiana, USA.

8Stead Family Department of Pediatrics, Carver College of Medicine, University of Iowa, Iowa City, Iowa, USA.

Address correspondence to: Andrew P. Lieberman, Department of Pathology, University of Michigan Medical School, 3510 MSRB1, 1150 W. Medical Center Dr., Ann Arbor, Michigan 48109, USA. Phone: 734.647.4624; Email: liebermn@med.umich.edu. Or to: Mark L. Schultz, Department of Pediatrics, University of Iowa, 2150 ML, 25 S. Grand Avenue, Iowa City, Iowa 52246, USA. Phone: 319.335.0557; Email: mark-schultz-1@uiowa.edu.

Authorship note: RDA, ABC, and KLS contributed equally to this work.

Find articles by Schultz, M. in: JCI | PubMed | Google Scholar

1Department of Pathology, University of Michigan Medical School, Ann Arbor, Michigan, USA.

2Department of Chemistry, University of Illinois Chicago, Illinois, USA.

3Department of Chemistry & Biochemistry and

4Warren Family Center for Drug Discovery, University of Notre Dame, Notre Dame, Indiana, USA.

5Department of Chemistry, Ochanomizu University, Tokyo, Japan.

6HitGen Inc, Chengdu, China.

7Ara Parseghian Medical Research Fund at Notre Dame University, Notre Dame, Indiana, USA.

8Stead Family Department of Pediatrics, Carver College of Medicine, University of Iowa, Iowa City, Iowa, USA.

Address correspondence to: Andrew P. Lieberman, Department of Pathology, University of Michigan Medical School, 3510 MSRB1, 1150 W. Medical Center Dr., Ann Arbor, Michigan 48109, USA. Phone: 734.647.4624; Email: liebermn@med.umich.edu. Or to: Mark L. Schultz, Department of Pediatrics, University of Iowa, 2150 ML, 25 S. Grand Avenue, Iowa City, Iowa 52246, USA. Phone: 319.335.0557; Email: mark-schultz-1@uiowa.edu.

Authorship note: RDA, ABC, and KLS contributed equally to this work.

Find articles by Lieberman, A. in: JCI | PubMed | Google Scholar

Authorship note: RDA, ABC, and KLS contributed equally to this work.

Published August 29, 2024 - More info

Published in Volume 9, Issue 20 on October 22, 2024
JCI Insight. 2024;9(20):e179525. https://doi.org/10.1172/jci.insight.179525.
© 2024 Azaria et al. This work is licensed under the Creative Commons Attribution 4.0 International License. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/. View PDF Abstract

Therapeutics that rescue folding, trafficking, and function of disease-causing missense mutants are sought for a host of human diseases, but efforts to leverage model systems to test emerging strategies have met with limited success. Such is the case for Niemann-Pick type C1 disease, a lysosomal disorder characterized by impaired intracellular cholesterol trafficking, progressive neurodegeneration, and early death. NPC1, a multipass transmembrane glycoprotein, is synthesized in the endoplasmic reticulum and traffics to late endosomes/lysosomes, but this process is often disrupted in disease. We sought to identify small molecules that promote folding and enable lysosomal localization and functional recovery of mutant NPC1. We leveraged a panel of isogenic human induced neurons expressing distinct NPC1 missense mutations. We used this panel to rescreen compounds that were reported previously to correct NPC1 folding and trafficking. We established mo56-hydroxycholesterol (mo56Hc) as a potent pharmacological chaperone for several NPC1 mutants. Furthermore, we generated mice expressing human I1061T NPC1, a common mutation in patients. We demonstrated that this model exhibited disease phenotypes and recapitulated the protein trafficking defects, lipid storage, and response to mo56Hc exhibited by human cells expressing I1061T NPC1. These tools established a paradigm for testing and validation of proteostatic therapeutics as an important step toward the development of disease-modifying therapies.

Introduction

Protein synthesis and folding in the endoplasmic reticulum (ER) is an iterative and highly regulated process, as nascent polypeptides undergo conformational changes and posttranslational modifications in an attempt to reach their functional, or native, conformation (1). Frequently, this process requires assistance from ER-resident chaperones that facilitate protein folding to enable subsequent export from the ER (2). These folding and trafficking processes govern a protein’s homeostasis, or “proteostasis.” However, commonly in human disease, missense mutations impair these processes. The resulting misfolded proteins are subject to degradation by ER quality control mechanisms, leading to the loss of functional proteins that underlies disease pathogenesis (2).

This cascade of events occurs in many individuals with Niemann-Pick type C1 disease, an autosomal recessive lysosomal disorder characterized by the accumulation of unesterified cholesterol in late endosomes and lysosomes (3). Disease course and severity are variable but include progressive neurodegeneration, hepatomegaly, and early death (4). Niemann-Pick type C1 disease is caused by mutations in the NPC1 gene, which encodes a multipass transmembrane glycoprotein required for export of cholesterol from late endosomes and lysosomes (5). The disease hallmark of impaired intracellular lipid transport has many downstream consequences, including dysregulated ER calcium (6), disrupted autophagy (7–14), altered mTOR signaling (15, 16), lysosomal membrane permeabilization (16–18), and mitochondrial abnormalities (13, 19). Together, these result in myelination defects (20, 21) and neuron loss in the central nervous system (22–24).

Many of the more than 250 disease-causing mutations in NPC1 are missense mutations that result in misfolded proteins that are either nonfunctional or degraded before reaching the lysosome (25). The latter is exemplified by the most common mutation in patients of western European ancestry, I1061T NPC1. Human I1061T NPC1 misfolds in the ER and is targeted to ER-associated degradation and ER-targeted macroautophagy (26–28). However, treatment with histone deacetylase inhibitors or ryanodine receptor antagonists corrects I1061T NPC1 folding and trafficking, enabling the protein to bypass ER quality control pathways and reach the lysosome, where it is functional (29, 30).

These observations have spurred interest in developing proteostatic therapeutics for Niemann-Pick type C1 disease, with the goal of rescuing folding and trafficking of the mutant protein to promote functional recovery. Notably, recent work has discovered significant differences in the trafficking of mouse versus human I1061T NPC1 that are attributable to differences in amino acid sequence (31). These distinctions affect the response of the mutant protein to proteostatic therapeutics and diminish the utility of I1061T Npc1–knockin mice for testing these compounds in vivo (32). Moreover, much prior work on proteostatic modulators used patient fibroblasts or other non-neuronal cell types that may have significantly different ER folding environments from primary neurons, a critical target cell in this disease.

To address these limitations, we utilized a panel of isogenic human induced pluripotent stem cell–derived (iPSC-derived) inducible neurons (iNeurons) with distinct NPC1 disease-causing missense mutations. Here, we employed this panel to rescreen many of the small molecules that were reported previously to act as NPC1 proteostatic modulators. We established mo56-hydroxycholesterol (mo56Hc) (33, 34) as a potent pharmacological chaperone for several NPC1 missense mutants. Furthermore, we developed and characterized a mouse model expressing human I1061T NPC1. We demonstrated that this model exhibited critical disease phenotypes and recapitulated the trafficking defects, cholesterol storage, and response to mo56Hc exhibited by human cells expressing I1061T NPC1. These tools established a pipeline for testing and validation of NPC1 proteostatic therapeutics as an important step toward the development of disease-modifying therapies.

Results

Identification of an NPC1 proteostasis modulator using human iNeurons.

We sought to identify small molecules that promote trafficking of mutant NPC1 from the ER to late endosome/lysosomes to recover function in cell types that are critical targets of disease. To accomplish this, we leveraged a panel of isogenic iPSC-derived human iNeurons expressing wild-type (WT) or disease-causing NPC1 missense mutations that were established using CRISPR/Cas9 gene editing. Cells expressing R1186H and P1007A NPC1 were generated to complement the recently reported human iNeurons expressing I1061T or R934L NPC1 (31). iPSCs with these mutations exhibited normal karyotypes (Supplemental Figure 1 and Supplemental Tables 1 and 2; supplemental material available online with this article; https://doi.org/10.1172/jci.insight.179525DS1) and pluripotency (Supplemental Figure 2). Following introduction of a human Neurogenin-2 cassette, these cells were rapidly and efficiently differentiated into iNeurons after treatment with doxycycline (Supplemental Figure 3). In aggregate, this collection of isogenic iNeurons encompasses all 3 described classes of NPC1 disease-causing missense mutations, including mutants that are largely retained in the ER (I1061T, R1186H), mutants that are found in both the ER and late endosomes/lysosomes (R934L), and mutants that traffic efficiently to late endosomes/lysosomes but have reduced function (P1007A) (25). Our studies used immature neurons, cultured 5 days after differentiation. Lysotracker staining showed significant lysosomal enlargement in iNeurons expressing I1061T and R1186H NPC1 (Supplemental Figure 3), correlating with limited trafficking of these mutants through the medial Golgi, as shown by endoglycosidase H (Endo H) assay (Figure 1).

Figure 1

mo56Hc increases Endo H resistance of I1061T NPC1 in iNeurons. (A and B) Differentiated WT and I1061T NPC1 iNeurons were treated with vehicle (NT), 0.2 μM vorinostat (Vori.), 0.2 μM CI-994, 0.005 μM onalespib (Onal.), 100 μM 4-phenylbutyric acid (4-PBA), 10 μM quinestrol (Quin.), 10 μM abiraterone (Abir.), 1 μM 25-hydroxycholesterol (25Hc), 1 μM mo56-hydroxycholesterol (mo56Hc), or 400 μM arimoclomol (Ari.) for 48 hours. Lysates were digested with Endo H and analyzed by Western blot (values on right indicate ladder standard weights in kilodaltons). Quantified at right. (C and D) Differentiated I1061T NPC1 iNeurons were treated (C) for 48 hours with the indicated concentrations of mo56Hc or (D) with 1 µM mo56Hc for the indicated times.” Both experiments utilize mo56Hc treatment. Lysates were digested with Endo H and analyzed by Western blot. Quantified at right. All data are mean ± SEM from indicated number of independent experiments. *P ≤ 0.05, ***P ≤ 0.001, ****P ≤ 0.0001 by (A, C, and D) 1-way ANOVA with Tukey’s post hoc test or (B) t test. (A) n = 5 WT, 9 I1061T NPC1 NT, 3 I1061T NPC1 plus each drug; (B) n = 3 I1061T NPC1 NT, 3 I1061T NPC1 Ari.; (C) n = 3 I1061T NPC1 plus mo56Hc at each concentration; (D) n = 3 I1061T NPC1 plus mo56Hc for each treatment duration.

iNeurons were used to examine the efficacy of compounds reported previously to promote the folding and trafficking of I1061T NPC1 in patient-derived fibroblasts as well as structurally similar small molecules. These compounds included the histone deacetylase inhibitors vorinostat (35) and CI-994 (35), the HSP90 inhibitor onalespib (36), and HSP modulator arimoclomol (37). These compounds are thought to act indirectly on I1061T NPC1, potentially through the regulation of molecular chaperones (25, 36–38). Other tested compounds included the chemical chaperone 4-phenylbutyric acid (27), as well as quinestrol (39), abiraterone (39), 25-hydroxycholesterol (25Hc) (33, 39), the previously described mo56Hc (33, 34), and U18666A (40). Although the mechanism of action of these small molecules is less well characterized, some may act as pharmacological chaperones (41) to stabilize folding intermediates (33, 39).

After identifying nontoxic doses using iNeurons (Supplemental Figure 4), we assessed these compounds for their effect on NPC1 trafficking through the medial Golgi by analysis of NPC1 glycan maturation. The simple high-mannose glycans added to NPC1 during synthesis and folding in the ER are sensitive to cleavage by the enzyme Endo H. However, Endo H is not able to cleave the complex glycans formed after NPC1 is processed in the medial and trans-Golgi. Thus, the sensitivity of glycans to Endo H cleavage indicates NPC1 trafficking relative to the Golgi.

As anticipated, WT NPC1 was Endo H resistant, reflective of efficient trafficking through the Golgi to late endosomes/lysosomes (Figure 1A). In contrast, I1061T NPC1 exhibited Endo H sensitivity (Figure 1A), consistent with protein misfolding within the ER and impaired trafficking, as shown previously (31). Of the small molecule proteostatic modulators tested, only the modified hydroxycholesterol mo56Hc significantly increased the fraction of Endo H–resistant I1061T NPC1, suggesting improved trafficking (Figure 1, A and B, and Supplemental Figure 5). Similar effects were seen in human fibroblasts expressing I1061T NPC1 (Supplemental Figure 6B). Moreover, only mo56Hc significantly reduced lysosomal abundance and area as assessed by lysosomal-associated membrane protein 1 (LAMP1) staining in iNeurons expressing I1061T NPC1 (Supplemental Figure 6A), indicating an improvement in lysosomal pathology. The effects of mo56Hc on I1061T NPC1 Endo H resistance were both dose and time dependent (Figure 1, C and D). Similarly, vehicle-treated R934L NPC1 exhibited both Endo H–sensitive and –resistant species (Figure 2A). Of the modulators tested, mo56Hc significantly increased the fraction of Endo H–resistant R934L NPC1, demonstrating effects on multiple NPC1 trafficking mutants (Figure 2, A and B). Validating this conclusion, mo56Hc also significantly increased the Endo H resistance of R1186H NPC1 (Figure 2C). In contrast, relatively minor effects were seen on the fraction of Endo H–resistant P1007A NPC1, a mutant that traffics efficiently to late endosome/lysosomes but is functionally impaired (Figure 2D). Together, these data suggest that mo56Hc robustly corrects the trafficking of ER-retained NPC1 mutants in iNeurons and fibroblasts.

Figure 2

mo56Hc increases Endo H resistance of NPC1 trafficking mutants in iNeurons. (A and B) Differentiated WT and R934L NPC1 iNeurons were treated with vehicle (NT), 0.2 μM vorinostat (Vori.), 0.2 μM CI-994, 0.005 μM onalespib (Onal.), 100 μM 4-phenylbutyric acid (4-PBA), 10 μM quinestrol (Quin.), 10 μM abiraterone (Abir.), 1 μM 25-hydroxycholesterol (25Hc), 1 μM mo56-hydroxycholesterol (mo56Hc), or 400 μM arimoclomol (Ari.) for 48 hours. Lysates were digested with Endo H and analyzed by Western blot. Quantified at right. (C and D) Differentiated R1186H and P1007A iNeurons were treated with vehicle (NT), 1 μM 25Hc, or 1 μM mo56Hc for 48 hours. Lysates were digested with Endo H and subjected to Western blot. Quantified at right. All data are mean ± SEM from indicated number of independent experiments. **P ≤ 0.01, ***P ≤ 0.001, ****P ≤ 0.0001 by (A, C, and D) 1-way ANOVA with Tukey’s post hoc test or (B) t test. (A) n = 3 WT, 7 R934L NPC1 NT, 3 R934L NPC1 plus each drug; (B) n = 3 R934L NPC1 NT, 3 R934L NPC1 Ari.; (C) n = 3 R1186H NPC1 NT, 3 R1186H NPC1 plus each drug; (D) n = 3 P1007A NPC1 NT, 5 P1007A NPC1 plus each drug.

To glean insights into this compound’s mechanism of action, direct effects of mo56Hc and 25Hc, a structurally similar but inactive oxysterol, were examined on purified NPC1 protein using a thermal stability assay. NPC1 unfolds in 2 distinct thermal transitions as seen in this assay (Figure 3). The 2 tested compounds had distinct effects on NPC1 thermal stability. 25Hc shifted the melting temperature of the later thermal transition by 3°C to the right, while mo56Hc shifted both the melting temperature and slope of the early thermal transition; both effects are known to indicate stabilization of a protein (42). Combined treatment with 25Hc and mo56Hc stabilized both thermal transitions, suggesting that these structurally related molecules do not cross-react between their respective binding sites on the NPC1 protein. Notably, mo56Hc stabilized NPC1 at concentrations well below 50 μM, displaying an EC50 of approximately 3 μM in the thermal stability assay (Supplemental Figure 8). Microscale thermophoresis showed mo56Hc binds NPC1 with an EC50 of 177 nM (Supplemental Figure 8). These data are compatible with activity in cellular assays in the high nanomole to low micromole concentration range, as shown in Figure 1. These analyses support a model in which mo56Hc stabilizes NPC1 in a conformation that favors trafficking.

Figure 3

m56Hc increases thermal stability of NPC1 protein. Purified WT