Fig 2: Knock Down of GLT8D1 and Overexpression of Mutant GLT8D1 Produce Toxicity and Motor Impairment in Zebrafish Embryos(A, B, and D) Mean distance moved (mm) during three light-dark cycles at 5 days post-fertilization is reduced following injection with 1.5 ng GLT8D1 splice blocking morpholino (A) compared to 1.5-ng control morpholino (n = 160 from 3 clutches); and following injection with 1-nl mature mRNA encoding human GLT8D1-R92C (B) (n = 96 from 2 clutches) or GLT8D1-G78W (D) (n = 48 from one clutch) compared to 1-nl mature mRNA encoding wild-type human GLT8D1.(C) Injection of 1-nl mature mRNA encoding human GLT8D1-R92C significantly reduces survival of zebrafish embryos at 24 hours and 48 hours compared to mature mRNA encoding wild-type human GLT8D1. WT, wild-type; SB, splice blocking morpholino. ∗p < 0.05; ∗∗p < 0.01; ∗∗∗∗p < 0.0001.Error bars represent SEM (A, B, and D) or SD (C).

Fig 3: When Overexpressed in HEK293 and N2A Cells, GLT8D1 Is Localized to the Golgi Network and Produces Significant Toxicity(A) The Golgi network was imaged in N2A (top) and HEK293 (bottom) cells using anti-TGN46 (red); GLT8D1-GFP fusion protein was imaged using anti-JL8 (green). Nuclear counterstain (Hoechst) is shown in blue. Scale bar, 50 μm.(B–E) Overexpression of mutated GLT8D1 increases cytotoxicity (B and C) and reduces metabolic activity (D and E) in HEK293 (B and D) and N2A (C and E) cells compared to overexpression of wild-type protein; all experiments included 6 biological replicates and either 2 (MTT assays) or 4 (LDH assays) technical replicates per biological replicate. ∗p < 0.05.Error bars represent mean and SD.

Fig 4: ALS-Linked GLT8D1-R92C Mutation Impairs Glycosyltransferase Activity(A) FLAG-tagged WT, GLT8D1-G78W, and GLT8D1-R92C proteins were immunopurified in 1M NaCl-containing buffer to maximize dissociation of interacting partners and eluted in native conditions prior to analysis by SDS-PAGE and Coomassie staining. Input represents ∼0.1% of whole-cell protein extracts loaded onto anti-FLAG coated beads. Arrow indicates FLAG-GLT8D1 proteins.(B) Standard curve for UDP-Glo assay illustrates linear correlation for <500 μmol/min UDP.(C) Initial reaction velocity at fixed enzyme concentration with variable substrate concentration; Michaelis-Menten curves were fitted with nonlinear regression. WT, wild-type; FT, flow-through.