Abstract
ABSTRACTNiemann-Pick disease type C1 (NPC1) is a rare autosomal recessive lysosomal storage disease primarily caused by mutations in NPC1. NPC1 is characterized by abnormal accumulation of unesterified cholesterol and glycolipids in late endosomes and lysosomes. Common signs include neonatal jaundice, hepatosplenomegaly, cerebellar ataxia, seizures and cognitive decline. Both mouse and feline models of NPC1 mimic the disease progression in humans and have been used in preclinical studies of 2-hydroxypropyl-β-cyclodextrin (2HPβCD; VTS-270), a drug that appeared to slow neurological progression in a Phase 1/2 clinical trial. However, there remains a need to identify additional therapeutic agents. High-throughput drug screens have been useful in identifying potential therapeutic compounds; however, current preclinical testing is time and labor intensive. Thus, development of a high-capacity in vivo platform suitable for screening candidate drugs/compounds would be valuable for compound optimization and prioritizing subsequent in vivo testing. Here, we generated and characterize two zebrafish npc1-null mutants using CRISPR/Cas9-mediated gene targeting. The npc1 mutants model both the early liver and later neurological disease phenotypes of NPC1. LysoTracker staining of npc1 mutant larvae was notable for intense staining of lateral line neuromasts, thus providing a robust in vivo screen for lysosomal storage. As a proof of principle, we were able to show that treatment of the npc1 mutant larvae with 2HPβCD significantly reduced neuromast LysoTracker staining. These data demonstrate the potential value of using this zebrafish NPC1 model for efficient and rapid in vivo optimization and screening of potential therapeutic compounds.This article has an associated First Person interview with the first author of the paper.
Highlights
Niemann-Pick disease type C (NPC) is a rare autosomal recessive disease caused by the accumulation of cholesterol and glycolipids in late endosomes/lysosomes
Two independent sites located within exon 2 and exon 7 were selected as the single guide RNA targeting sites to increase the chance of generating mutations that would disrupt the Npc1 protein near the N-terminus and give rise to a non-functional npc1 allele
To gain further insight into Niemann-Pick disease type C1 (NPC1) pathology and to obtain an in vivo NPC1 model system that would be amenable to high-capacity screening of potential therapeutic compounds, we utilized CRISPR/ Cas9-mediated gene targeting to mutate zebrafish npc1
Summary
Niemann-Pick disease type C (NPC) is a rare autosomal recessive disease caused by the accumulation of cholesterol and glycolipids in late endosomes/lysosomes. The onset of neurological manifestations of NPC is variable and ranges from early-infantile to adolescent/adult onset (Mengel et al, 2017). Mutation of both alleles of either NPC1 or NPC2 can cause NPC. Mutations in NPC1 are reported in 95% of NPC patients, with mutations of NPC2 accounting for the remaining cases (Vanier, 2010) Both NPC1 and NPC2 are lysosomal proteins that facilitate intracellular cholesterol trafficking (Carstea et al, 1997; Naureckiene et al, 2000; Vanier and Millat, 2004)
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