Modeling Niemann-Pick disease type C1 in zebrafish: a robust platform for in vivo screening of candidate therapeutic compounds

ABSTRACT Niemann-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.

Niemann-Pick disease, type C1 (NPC1) is a rare, often fatal, disease caused by mutations in the NPC1 gene, which is essential for transporting cholesterol across vesicles called lysosomes within the cell. Mutations in the NPC1 gene result in the accumulation of cholesterol and lipids in these lysosomes, and further cause liver defects and neurological defects in the brain. Currently there is no FDA-approved drug available for the treatment of NPC1. Therefore, speeding up the drug-screening process for potential NPC1 treatments is very important. We generated a zebrafish genetic NPC1 model that shows some of the same disease features found in NPC1 patients. We have shown that our zebrafish NPC1 model can be used as a new platform to boost the screening of candidate drugs for treating NPC1.
What are the potential implications of these results for your field of research?
Previously, most of the drug screening and development of potential NPC1 treatments has been performed using an in vitro cell culture model, followed by testing a few candidates in mammalian models. However, it takes a long time to conduct drug tests in mammals, and drugs that show efficacy in cell culture may not behave the same way in mammals. Therefore, an intermediate model for testing candidate drugs identified by in vitro screening before moving on to mammalian model testing is necessary to reduce the false-positive rate. We have shown that the lateral line neuromasts of our npc1-null mutant zebrafish larvae provide a great in vivo system for screening candidate drugs for treating NPC1. A proof-of-principle experiment with 2HPβCD treatment and staining live npc1-null mutant zebrafish larvae with LysoTracker showed that reduction of LysoTracker intensity can be easily captured by fluorescence microscopy, and serves as a readout for drug treatment. The results suggest that our zebrafish NPC1 model can be used as an in vivo model to bridge the gap between in vitro and mammalian models for NPC1 drug screening.
"The advantages of using zebrafish as the model system include larger clutch size and the transparency of embryos and larvae." What are the main advantages and drawbacks of the model system you have used as it relates to the disease you are investigating?
The advantages of using zebrafish as the model system include larger clutch size and the transparency of embryos and larvae. We can easily obtain 100 npc1 homozygous mutants for drug screening in a single clutch from the intercross of npc1 heterozygous mutants, while only getting one or two npc1 mutants per litter for mice. The transparency of embryos and larvae allows us to examine the phenotype of internal structures in zebrafish npc1-null mutant larvae when they are still alive. Moreover, we took the advantage of neuromasts, which are unique mechanosensory organs located on the skin of zebrafish but not in mammals, as the target for the drug treatment. The accessibility of neuromasts to the external environment makes it easier for drugs to be delivered into cells. Analyzing the effect of drug treatment is also easier due to the high concentration of LysoTracker-positive spots in npc1 mutant neuromasts. The drawback of using zebrafish is the generation time of about two to four months, making it even slightly slower than the generation time of mice.

Wei-Chia Tseng
Wei-Chia Tseng's contact details: Section on Molecular Dysmorphology, Division of Translational Research, Eunice Kennedy Shriver National Institute of Child Health and Human Development, National Institutes of Health, Department of Health and Human Services, Bethesda, MD 20892, USA. E-mail: wei-chia.tseng@nih.gov What has surprised you the most while conducting your research?
Our npc1 mutants exhibited very intense LysoTracker staining in neuromasts as well as in olfactory placodes. This phenotype is unique and useful for sorting the live npc1 mutant larvae from their wild-type and heterozygous mutant siblings.
Describe what you think is the most significant challenge impacting your research at this time and how will this be addressed over the next 10 years?
The most significant challenge of studying Niemann-Pick disease, type C1 is that the disease varies widely in both symptoms and the age at onset between individuals. Some patients develop severe, often fatal, visceral manifestations such as the liver defect very early in their life, while other patients develop variable neurological defects at ages ranging from early childhood to adulthood. The cause of the variability is still not clear. Over the next three to five years, we will understand more about the mechanism of disease progression as new pathways or proteins involved in the process are identified. This will help us to develop and test potential therapeutic methods for a variety of NPC1 patients over the next decade.
What's next for you?
I will continue to work on screening candidate drugs for treating NPC1 using our zebrafish model. At the same time, exploring the pathological mechanism of the disease in the zebrafish model is also important as it will allow me to find potential targets for therapeutic intervention. My goal is to identify drugs that reduce NPC1 pathological features significantly in our zebrafish model and test those drugs in mammalian NPC1 models.