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 NPC1 or NPC2 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 NPC2. Five-day-old npc2 mutants were morphologically indistinguishable from wildtype larvae. We found that live npc2–/– larvae exhibited stronger Nile blue staining. The npc2–/– larvae exhibited low mobility and a high anxiety-related response. These behavioral changes correlated with downregulation of the mcu (mitochondrial calcium uniporter) gene, ppp3ca (calcineurin) gene, and genes that are involved in myelination (mbp and mpz). Histological analysis of adult npc2–/– zebrafish revealed that pathological changes in the nervous system, kidney, liver, and pancreas correlated with inflammatory responses (i.e., the upregulation of il1, nfκβ, and mpeg; i.e., hallmarks of NPC disease). These findings suggest that the npc2 mutant zebrafish may be a model of NPC disease.

Severe neurometabolic phenotype innpc1-/-zebrafish with a C-terminal mutation

“…Both our npc1 mutant models carried mutations in exon 22 and died either before or at the beginning of the juvenile stage. This is remarkably different to previous zebrafish NPC mutant models ( npc1 and npc2 mutant zebrafish had almost the same phenotype), which carried mutations in the first exons and, although they had a reduced lifespan dying between 2-9 months, they survived until at least the start of adult stage (Lin et al, 2018; Tseng et al, 2018; Tseng et al, 2021; Wiweger et al, 2021). Similarly to previous mutant NPC zebrafish models, our mutant NPC zebrafish showed growth retardation resulting in a statistically significant reduced body length.…”

“…Additionally, by 2 wpf, we found vacuolated lesions in digestive organs (liver and intestine), as well as in the renal tubules and brain, these being compatible with the accumulation of lipids. Despite similar lesions were also found in previous NPC zebrafish mutants, they were identified at later stages (Lin et al, 2018; Tseng et al, 2018; Wiweger et al, 2021). Therefore, whereas previous NPC zebrafish models appear to undergo a late onset and slowly progressive form of the NPC disease, our npc1 mutant zebrafish shows a more severe phenotype with an infantile or juvenile onset, resulting in lethality prior to adult stage.…”

“…In addition, they showed statistically significant impaired motor function which started at 2 wpf. Locomotion was previous reported to be significant reduced in a npc2 zebrafish model from 5dpf (Wiweger et al, 2021). Balance defect and trembling was observed but not measured in latter stages of previous npc1 and npc2 zebrafish models (Lin et al, 2018; Tseng et al, 2018; Tseng et al, 2021).…”

“…Zebrafish has an NPC1 gene with a 70% of homology to the orthologous human gene. NPC zebrafish models had been created previously by using morpholinos (Schwend et al, 2011; Louwette et al, 2013) and, more recently, by CRISPR/Cas9 mutagenesis of npc1 (Lin et al, 2018; Tseng et al, 2018) and npc2 genes (Tseng et al, 2021; Wiweger et al, 2021). Since there is high variability of mutation type and location leading to human NPC disease, model organisms of this disorder with mutations in different domains are necessary to help elucidate molecular pathogenesis.…”

“…Due to its biological features and easy genetic manipulation, Zebrafish is commonly used as a model organism in neuronal (Bandmann and Burton, 2010; Quelle-Regaldie et al, 2021a; Quelle-Regaldie et al, 2021b) and metabolic diseases (Hölttä-Vuori et al, 2010; Ka and Jin, 2021). Previous zebrafish models for NPC include morphant knockdown (Schwend et al, 2011; Louwette et al, 2013), and mutant models for npc1 and npc2 carrying mutations in the first exons in the N-terminal domain (Lin et al, 2018; Tseng et al, 2018; Tseng et al, 2021; Wiweger et al, 2021). However, zebrafish models with mutations in the C-terminal region of the protein have not yet been developed.…”