Zebrafish npc2 mutant. (A) Multiple species alignments demonstrated high conservation of the NPC2 protein among vertebrates. Arrows indicate positions of the mutation. (B) Expression levels of the npc2 gene in different tissues in adult fish. Expression was normalized to tissue with the lowest expression (brain). (C) Chromatograms confirmed a small deletion in the npc2 mutant. (D) Graphical representation of melt profiles in wildtypes (black), heterozygous mutants (blue), and homozygous mutants (gray) that originated from fish after incrossing npc2^±. (E) mRNA levels of the npc2 gene in various organs in npc2^–/– zebrafish relative to wildtype controls. The data are expressed as the mean ± SEM of three 9-month-old fish per group. Error bars represent the SEM. ***p < 0.001 and **p < 0.01. (F) Wildtype, heterozygous, and homozygous npc2 mutants had indistinguishable phenotypes at 5 dpf. Scale = 1 mm. (G) Morphology of adult fish. Smaller body size and weight in 8-month-old npc2^–/– fish and indistinguishable phenotype of npc2^±. Error bars represent the SD.

Nile blue staining distinguishes npc2^–/– fish from wildtype and heterozygous siblings. The strong signal could be detected in the peripheral olfactory organ in 5-day-old npc2^–/– larvae (B–B”), unlike their siblings (A–A”). (A,B) lateral view; (A’,A”,B’,B”) dorsal view; and (A”,B”) enlarged fragments of A’,B’, respectively. Arrows indicate the position of the olfactory organ. Scale bar = 0.5 mm.

Specificity of Nile blue staining in the olfactory organ in npc2^–/– larvae at 5 dpf. (A–A”) untreated npc2^–/– larvae; (B–B”) treatment with 2 mM 2-hydroxypropyl-β-cyclodextrin (2HPβCD; a drug that is used in clinical trials to treat NPC patients) decreased staining intensity to wildtype levels (Figures 2A–A”); and (C–C”) treatment with 8 mM (3β)-3-(2-[diethylamino]ethoxy)androst-5-en-17-one hydrochloride (U18666A, an inhibitor of cholesterol synthesis that is used to model NPC chemically) increased staining intensity in npc2^–/– larvae. (A–C) lateral view; (A’,A”,B’,B”,C’,C”), dorsal view; and (A”,B”,C”) enlarged fragments of A’,B’,C’, respectively. Scale bar = 0.5 mm.

Decrease in mobility and increase in thigmotaxis in npc2^–/– larvae. (A) Heatmaps of mean traces of npc2^+/+ and npc2^–/– larvae during each 5 min period. (B–E) Boxplots of total distance traveled, time spent moving, mean velocity, and thigmotaxis in npc2^+/+ and npc2^–/– larvae. (E) *p < 0.05 and **p < 0.01.

Pathological changes in soft tissues in npc2^–/– revealed by H&E staining. (A) Cells with enlarged nuclei (black arrow), hepatosteatosis with different type of fatty droplet accumulation (open arrowhead) and foam cytoplasm (asterix) were found in npc2-deficient livers. (B) Kidneys with degenerating cells (DMc) between ductal (DT) and proximal (PT) tubules. (C) Pancreas with degeneration of zymogen granules in acinar cells and cholesterol deposits. The paraffin sections are from adult npc2^–/– fish and wildtype npc2^+/+ control. Scale bar = 50 μm.

Pathological changes in the central nervous system in adult npc2^–/– zebrafish. (A) Luxol fast blue staining showed differences in myelination between wildtype and npc2^–/– fish. (B) Changes in habenula structure were present in some npc2^–/– fish, which lacked the characteristic structure of this part of the habenular tract. (C) Midbrain with vacuolization of the habenula tract (blue arrow) with fascicular retroflexus (blue arrowhead) and degenerative changes in the medial and lateral longitudinal fascicle. OB, olfactory bulb; OR, olfactory rosette; Te, telencephalon; TeO, tectum opticum; Ce, cerebellum; Me, medulla; PG, preglomerular complex; and PT, posterior tuberculum. (A) LFB staining. Scale bar = 500 μm. (B,C) H&E staining. Scale bar = 100 μm. (C) scale bar

Pathological changes in npc2^–/– sensory organs. (A) Cerebellum structure with Purkinje cells degeneration in npc2 mutant (gray arrow), (B) Olfactory rosette with olfactory nerves (white arrow). (C) Optic nerve ganglion. Cells with a foamy, pale cytoplasm, eosinophilic nuclei, and signs of degeneration (black arrows) within the optic tectum. (A) ML, molecular layer; P, Purkinje cell leyer; GL, granule cell layer; OfN, olfactory nerves; and OR, olfactory rosette; (B) OT, optic tectum; OpG, optic nerve ganglion. H&E staining. Scale bar = 50 μm.

Decrease in myelination in npc2-deficient larvae and adult fish. Scatter plots show the normalized expression of selected genes in 5 dpf larvae and in the brain in npc^–/– and wildtype zebrafish. Each circle corresponds to one zebrafish. The 18S ribosomal gene was used as a reference. At least three samples were analyzed. ***p < 0.001; **p < 0.01; and *p < 0.05.

Signs of inflammation and alterations of Ca²⁺ homeostasis in npc2^–/– zebrafish. Scatterplots show the normalized expression of selected genes in brain and liver tissue from 9-month-old npc2^–/– homozygotes and wildtype zebrafish. Each circle corresponds to one zebrafish. The 18S ribosomal gene was used as a reference. Samples from at least three fish were analyzed. ***p < 0.001, **p < 0.01, and *p < 0.05.

Treatment with 2-hydroxypropyl-β-cyclodextrin increased the expression of genes that are related to inflammation and Ca²⁺ homeostasis in 5 dpf npc2^–/– larvae to wildtype levels. Scatterplots show the normalized expression of selected genes in untreated larvae (A) and larvae that were treated with 2 mM 2HPβCD (B) at 5 dpf. Each circle corresponds to one zebrafish. The 18S ribosomal gene was used as a reference. The experiments were performed in triplicate. ***p < 0.001, **p < 0.01, *p < 0.05.