npc2-Deficient Zebrafish Reproduce Neurological and Inflammatory Symptoms of Niemann-Pick Type C Disease

Abstract

Niemann-Pick type C (NPC) disease is an autosomal recessive lysosomal storage disease that is caused by a mutation of the <i>NPC1</i> or <i>NPC2</i> gene, in which un-esterified cholesterol and sphingolipids accumulate mainly in the liver, spleen, and brain. Abnormal lysosomal storage leads to cell damage, neurological problems, and premature death. The time of onset and severity of symptoms of NPC disease are highly variable. The molecular mechanisms that are responsible for NPC disease pathology are far from being understood. The present study generated and characterized a zebrafish mutant that lacks Npc2 protein that may be useful for studies at the organismal, cellular, and molecular levels and both small-scale and high-throughput screens. Using CRISPR/Cas9 technology, we knocked out the zebrafish homolog of <i>NPC2</i>. Five-day-old <i>npc2</i> mutants were morphologically indistinguishable from wildtype larvae. We found that live <i>npc2^-/-</i> larvae exhibited stronger Nile blue staining. The <i>npc2^-/-</i> larvae exhibited low mobility and a high anxiety-related response. These behavioral changes correlated with downregulation of the <i>mcu</i> (mitochondrial calcium uniporter) gene, <i>ppp3ca</i> (calcineurin) gene, and genes that are involved in myelination (<i>mbp</i> and <i>mpz</i>). Histological analysis of adult <i>npc2^-/-</i> zebrafish revealed that pathological changes in the nervous system, kidney, liver, and pancreas correlated with inflammatory responses (i.e., the upregulation of <i>il1</i>, <i>nf</i>κβ, and <i>mpeg</i>; i.e., hallmarks of NPC disease). These findings suggest that the <i>npc2</i> mutant zebrafish may be a model of NPC disease.