Role of β-glucosidase 2 in aberrant glycosphingolipid metabolism: model of glucocerebrosidase deficiency in zebrafish

Lelieveld, L.T.; Mirzaian, M.; Kuo, C.L.; Artola Perez de Azanza, M.E.; Guimaraes Da Lomba Ferraz, M.J.; Peter, R.E.A.; Akiyama, H.; Greimel, P.; Berg, R.J.B.H.N. van den; Overkleeft, H.S.; Boot, R.G.; Meijer, A.; Aerts, J.M.F.G.

doi:doi:10.1194/jlr.RA119000154

β-glucosidases (GBA1 [glucocerebrosidase], GBA2, and GBA3) are ubiquitous, essential enzymes. Lysosomal GBA1 and cytosol-facing GBA2 degrade glucosylceramide (GlcCer); GBA1 deficiency causes Gaucher disease (GD), a lysosomal storage disorder characterized by lysosomal accumulation of GlcCer, which is partly converted to glucosylsphingosine (GlcSph). GBA1 and GBA2 also may transfer glucose from GlcCer to cholesterol, yielding glucosylated cholesterol (GlcChol). Here, we aimed to clarify the role of zebrafish Gba2 in glycosphingolipid metabolism during Gba1 deficiency in zebrafish (Danio rerio), which are able to survive total Gba1 deficiency. We developed Gba1 and Gba2 zebrafish knockouts (gba1^-/- and gba2^-/-, respectively) using CRISPR/Cas9, modulated glucosidases genetically and pharmacologically, studied GlcCer metabolism in individual larvae, and explored the feasibility of pharmacologic or genetic interventions. Activity-based probes...Show moreβ-glucosidases (GBA1 [glucocerebrosidase], GBA2, and GBA3) are ubiquitous, essential enzymes. Lysosomal GBA1 and cytosol-facing GBA2 degrade glucosylceramide (GlcCer); GBA1 deficiency causes Gaucher disease (GD), a lysosomal storage disorder characterized by lysosomal accumulation of GlcCer, which is partly converted to glucosylsphingosine (GlcSph). GBA1 and GBA2 also may transfer glucose from GlcCer to cholesterol, yielding glucosylated cholesterol (GlcChol). Here, we aimed to clarify the role of zebrafish Gba2 in glycosphingolipid metabolism during Gba1 deficiency in zebrafish (Danio rerio), which are able to survive total Gba1 deficiency. We developed Gba1 and Gba2 zebrafish knockouts (gba1^-/- and gba2^-/-, respectively) using CRISPR/Cas9, modulated glucosidases genetically and pharmacologically, studied GlcCer metabolism in individual larvae, and explored the feasibility of pharmacologic or genetic interventions. Activity-based probes and quantification of relevant glycolipid metabolites confirmed enzyme deficiency. GlcSph increased in gba1^-/- larvae (0.09 pmol/fish) but did not increase more in gba1^-/-:gba2^-/- larvae. GlcCer was comparable in gba1^-/- and wild-type (WT) larvae but increased in gba2^-/- and gba1^-/-:gba2^-/- larvae. Independent of Gba1 status, GlcChol was low in all gba2^-/- larvae (0.05 vs. 0.18. pmol/fish in WT). Pharmacologic inactivation of zebrafish Gba1 comparably increased GlcSph. Inhibition of glucosylceramide synthase in Gba1-deficient larvae reduced GlcCer and GlcSph, and concomitant inhibition of glucosylceramide synthase and Gba2 with iminosugars also reduced excessive GlcChol. Finally, overexpression of human GBA1 and injection of recombinant GBA1 both decreased GlcSph. We determined that zebrafish larvae offer an attractive model to study glucosidase actions in glycosphingolipid metabolism in vivo, and we identified distinguishing characteristics of zebrafish Gba2 deficiency. Show less

Leiden University Scholarly Publications

View statistics

Documents

In Collections

Role of β-glucosidase 2 in aberrant glycosphingolipid metabolism: model of glucocerebrosidase deficiency in zebrafish