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Lysosomal cholesterol accumulation contributes to the movement phenotypes associated with NUS1 haploinsufficiency

Abstract

Purpose

Variants in NUS1 are associated with a congenital disorder of glycosylation, developmental and epileptic encephalopathies, and are possible contributors to Parkinson disease pathogenesis. How the diverse functions of the NUS1-encoded Nogo B receptor (NgBR) relate to these different phenotypes is largely unknown. We present three patients with de novo heterozygous variants in NUS1 that cause a complex movement disorder, define pathogenic mechanisms in cells and zebrafish, and identify possible therapy.

Methods

Comprehensive functional studies were performed using patient fibroblasts, and a zebrafish model mimicking NUS1 haploinsufficiency.

Results

We show that de novo NUS1 variants reduce NgBR and Niemann–Pick type C2 (NPC2) protein amount, impair dolichol biosynthesis, and cause lysosomal cholesterol accumulation. Reducing nus1 expression 50% in zebrafish embryos causes abnormal swim behaviors, cholesterol accumulation in the nervous system, and impaired turnover of lysosomal membrane proteins. Reduction of cholesterol buildup with 2-hydroxypropyl-ß-cyclodextrin significantly alleviates lysosomal proteolysis and motility defects.

Conclusion

Our results demonstrate that these NUS1 variants cause multiple lysosomal phenotypes in cells. We show that the movement deficits associated with nus1 reduction in zebrafish arise in part from defective efflux of cholesterol from lysosomes, suggesting that treatments targeting cholesterol accumulation could be therapeutic.

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Fig. 1: De novo NUS1 variants cause a spectrum of lysosomal defects in patient cells.
Fig. 2: Reduction in nus1 expression alters swim behavior of zebrafish embryos.
Fig. 3: nus1 morphants exhibit impaired lysosomal function and cholesterol accumulation.
Fig. 4: Treatment with ßCD reduces cholesterol accumulation and restores lysosomal function.
Fig. 5: Lowering cholesterol accumulation improves swim behaviors in nus1 morphant embryos.

Web Resources

Provean (protein variation effect analyser), http://provean.jcvi.org/index.php. PolyPhen2, http://genetics.bwh.harvard.edu/pph2/. MutationTaster, http://mutationtaster.org/. dbSNP, https://www.ncbi.nlm.nih.gov/snp/. gnomAD, http://gnomad.broadinstitute.org/. Mutalyzer, https://mutalyzer.nl/batch-jobs. ClinVar, https://www.ncbi.nlm.nih.gov/clinvar/.

Data and code availability

All variants described in this study have been deposited in ClinVar; accession numbers are VCV000981036, VCV000981034, VCV000981035 (Submission ID: SUB8124960; Organization ID: 1019).

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Acknowledgements

We acknowledge the patients and their families for their willingness to participate in this study. This work was supported by the Greenwood Genetic Center and grants from the National Institutes of Health (5R01-GM086524-11 to R.S. and H.F-S; AI108819 to M.B.C). We acknowledge the support of the Hazel and Bill Allin Aquaculture Facility housed at the Greenwood Genetic Center and thank the facility staff for their excellent animal care.

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Authors

Contributions

Conceptualization: R.S., H.F.-S., M.J.L. Data curation: S.Y., T.W., E.F.M., M.B.C., R.J.L., H.F.-S., R.S. Formal analysis: S.Y., T.W., R.J.L., H.F.-S., R.S. Funding acquisition: R.S., H.F.-S., M.B.C. Investigation: S.Y., T.W., K.W., R.J.L., E.F.M., H.F.-S. Methodology: S.Y., T.W., M.B.C., H.F.-S., R.S. Project administration: R.S., H.F.-S., M.J.L. Resources: C.S., K.M., P.G., N.R., D.C., M.J.L. Supervision: R.S., H.F.-S. Validation: T.W., H.F.-S. Visualization: S.Y., T.W., H.F.-S. Writing—original draft: R.S., H.F.-S., M.J.L., R.J.L. Writing—review & editing: R.S., H.F.-S.

Corresponding author

Correspondence to Richard Steet.

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Ethics declaration

Informed consents were signed by the parents of the proband and other patients prior to participation in the research. All procedures were employed after being reviewed and approved by the Institutional Review Board, and compliant with practices, at the Greenwood Genetic Center (GGC). Handling and euthanasia of fish complied with policies of the GGC, as approved by the GGC’s Institutional Animal Care and Use Committee (permit #A2019-01-003-A1).

Competing interests

The authors declare no competing interests.

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Yu, SH., Wang, T., Wiggins, K. et al. Lysosomal cholesterol accumulation contributes to the movement phenotypes associated with NUS1 haploinsufficiency. Genet Med 23, 1305–1314 (2021). https://doi.org/10.1038/s41436-021-01137-6

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