Establishment and characterization of Neu1-knockout zebrafish and its abnormal clinical phenotypes

Abstract

Mammalian sialidase Neu1 is involved in various physiological functions, including cell adhesion, differentiation, cancer metastasis, and diabetes through lysosomal catabolism and desialylation of glycoproteins at the plasma membrane. Various animal models have been established to further explore the functions of vertebrate Neu1. The present study focused on zebrafish (Danio rerio) belonging to Cypriniformes as an experimental animal model with neu1 gene deficiency. The results revealed that the zebrafish Neu1 desialyzed both a2-3 and a2-6 sialic acid linkages from oligosaccharides and glycoproteins at pH 4.5, and it is highly conserved with other fish species and mammalian Neu1. Further, Neu1-knockout zebrafish (Neu1-KO) was established through CRISPR/Cas9 genome editing. Neu1-KO fish exhibited slight abnormal embryogenesis with the accumulation of pleural effusion; however, no embryonic lethality was observed. Although Neu1-KO fish were able to be maintained as homozygous, they showed smaller body length and weight than the wild type (WT) fish, and muscle atrophy and curvature of the vertebra were observed in adult Neu1-KO fish (8 months). The expression patterns of myod and myog transcription factors regulating muscle differentiation varied between Neu1-KO and WT fish embryo. Expression of lysosomal-related genes, including ctsa,lamp1a, and tfeb were upregulated in adult Neu1-KO muscle as compared to WT. Furthermore, the expression pattern of genes involved in bone remodeling (runx2a, runx2b, and mmp9) was decreased in Neu1-KO fish. These phenotypes were quite similar to those of Neu1-KO mice and human sialidosis patients, indicating the effectiveness of the established Neu1-KO zebrafish for the study of vertebrate Neu1 sialidase.

Neu1 deficiency induces abnormal emotional behavior in zebrafish

“…We assessed the alterations of sialoglycoconjugates in Neu1-KO zebrafish brain by lectin blotting using MAM (Sia α2-3 linkage specific) and SSA lectin (Sia α2-6 linkage specific). Although the recombinant zebrafish Neu1 has been reported to recognize both Sia α2-3 and α2-6 linkages in oligosaccharides and glycoproteins 26 , we could not detect a clear difference in the glycosylation pattern between Neu1-KO and WT zebrafish brains in our MAM and SSA lectin blot analysis (Fig. 7 a,b).…”

“…8 d) accompanied by an upregulation of lamp1a ( p < 0.05), but not lamp1b (Fig. 8 e,f), showing the same pattern as Neu1-KO zebrafish muscle 26 . In mammals, TFEB is known to activate the translocalization of lysosomal enzymes to the plasma membrane 38 , with NEU1 being a negative regulator of lysosomal exocytosis through the desialylation of LAMP1 17 .…”

“…A previous study reported that older Neu1-KO zebrafish (> 11 months old) exhibited spinal bending, inducing a suppression in their swimming ability 26 . Thus, this study employed younger Neu1-KO zebrafish that had not developed any such symptoms and were considered suitable for behavioral analysis.…”

“…1 ). Interestingly, the glycosphingolipid (GSL) pattern in the Neu1-KO zebrafish brain was shown to drastically differ from that in the WT brain, despite gangliosides not being substrates for zebrafish Neu1 26 . TLC analysis of the neutral lipid fraction showed an accumulation of lactosylceramide (LacCer) and globoside Gb4 in Neu1-KO zebrafish, whereas these two GSLs were almost undetectable in WT ( p < 0.05, Fig.…”

“…Fish Neu1 is known to be localized at the lysosome and its enzymatic properties, such as optimal pH and substrate specificity, have been reported to be shared among fish species. Recently, we established a Neu1 knockout zebrafish (Neu1-KO) strain using the CRISPR/Cas9 genome editing method 26 . The Neu1-KO zebrafish was shown to possess 5 nucleotide deletions in the first exon, which caused a frameshift, resulting in the interruption of the translation of the Neu1 polypeptide.…”