Overelaborated synaptic architecture and reduced synaptomatrix glycosylation in a Drosophila classic galactosemia disease model

Abstract

Classic galactosemia (CG) is an autosomal recessive disorder resulting from loss of galactose-1-phosphate uridyltransferase (GALT), which catalyzes conversion of galactose-1-phosphate and uridine diphosphate (UDP)-glucose to glucose-1-phosphate and UDP-galactose, immediately upstream of UDP-N-acetylgalactosamine and UDP-N-acetylglucosamine synthesis. These four UDP-sugars are essential donors for driving the synthesis of glycoproteins and glycolipids, which heavily decorate cell surfaces and extracellular spaces. In addition to acute, potentially lethal neonatal symptoms, maturing individuals with CG develop striking neurodevelopmental, motor and cognitive impairments. Previous studies suggest that neurological symptoms are associated with glycosylation defects, with CG recently being described as a congenital disorder of glycosylation (CDG), showing defects in both N- and O-linked glycans. Here, we characterize behavioral traits, synaptic development and glycosylated synaptomatrix formation in a GALT-deficient Drosophila disease model. Loss of Drosophila GALT (dGALT) greatly impairs coordinated movement and results in structural overelaboration and architectural abnormalities at the neuromuscular junction (NMJ). Dietary galactose and mutation of galactokinase (dGALK) or UDP-glucose dehydrogenase (sugarless) genes are identified, respectively, as critical environmental and genetic modifiers of behavioral and cellular defects. Assaying the NMJ extracellular synaptomatrix with a broad panel of lectin probes reveals profound alterations in dGALT mutants, including depletion of galactosyl, N-acetylgalactosamine and fucosylated horseradish peroxidase (HRP) moieties, which are differentially corrected by dGALK co-removal and sugarless overexpression. Synaptogenesis relies on trans-synaptic signals modulated by this synaptomatrix carbohydrate environment, and dGALT-null NMJs display striking changes in heparan sulfate proteoglycan (HSPG) co-receptor and Wnt ligand levels, which are also corrected by dGALK co-removal and sugarless overexpression. These results reveal synaptomatrix glycosylation losses, altered trans-synaptic signaling pathway components, defective synaptogenesis and impaired coordinated movement in a CG neurological disease model.

Keywords: Congenital disorder of glycosylation (CDG); Galactokinase; HSPG; Neuromuscular junction; Synaptogenesis; Trans-synaptic signaling; WNT; sugarless.

Fig. 1.

Loss of dGALT impairs coordinated movement and disrupts NMJ architecture. (A) Schematic diagram of the Leloir pathway. Galactose is phosphorylated by Drosophila galactokinase (dGALK) and then Drosophila galactose-1-P uridylyltransferase (dGALT) catalyzes the synthesis of glucose-1-P (glc-1-P) and UDP-galactose (UDP-gal) from UDP-glucose (UDP-glc) and galactose-1P (gal-1-P). UDP-glucose is a substrate for UDP-glucose dehydrogenase [encoded by sugarless (sgl)], catalyzing conversion to UDP-glucuronate, essential for proteoglycan biosynthesis. dGALE, uridine diphosphate galactose-4-epimerase. (B) Fold differences in the time required (controls set at 1) for wandering L3 to rollover from inverted to upright position for control (dGALT^C2), dGALT null (dGALT^ΔAP2), transgenic rescue (dGALT^ΔAP2; UH1-Gal4/UAS-hGALT) and rescue control (dGALT^ΔAP2; UH1-Gal4/+). Data are normalized to respective control. (C) Representative NMJs imaged with anti-horseradish-peroxidase (HRP; green) and anti-Discs-large (DLG; red) in wandering L3 in dGALT mutants (bottom row) with complete loss (dGALT^ΔAP2), ubiquitous (UH1-Gal4 driven) and tissue-specific (i.e. elav- or 24-Gal4-driven) knockdown. Respective controls for each condition are shown in the top row. (D,E) Quantification of differences in NMJ bouton (D) and branch (E) number for all genotypes, normalized to appropriate controls. Sample size: ≥ten animals per genotype. Error bars show s.e.m. with significance indicated: *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001.

Fig. 2.

High-galactose diet phenocopies dGALT-null movement and NMJ defects. (A) Differences in the time required for wandering L3 larvae to rollover from inverted to upright position: control (dGALT^C2) and dGALT-null (dGALT^ΔAP2) flies were fed food that was either galactose-free or supplemented with 200 mM galactose. (B) Representative NMJs imaged with anti-horseradish peroxidase (HRP; green) and anti-Discs-large (DLG; red) in wandering L3 for the above four conditions. (C–E) Quantification of synaptic bouton number (C), branch number (D) and inter-bouton spacing distance (E) for all four conditions, normalized to appropriate controls. Sample size: ≥ten animals per genotype. Error bars show s.e.m. with significance indicated: *P<0.05, **P<0.01, ***P<0.001.

Fig. 3.

Loss of dGALT activity compromises the NMJ glycosylated synaptomatrix. Representative wandering L3 NMJs imaged with anti-Fasciclin-II (FASII; red) in all cases and co-labeled with lectins (A) Erythrina cristagalli (ECL; green), (B) Wisteria floribunda (WFA; green) and (C) anti-horseradish-peroxidase (HRP; green) in genetic controls (precise-excision dGALT^C2) and dGALT nulls (imprecise-excision dGALT^ΔAP2). Quantification shows normalized ECL (≥15 NMJs) (A′), WFA (≥19 NMJs) (B′) and anti-HRP (≥16 NMJs) (C′) intensities. Error bars show s.e.m. with significance indicated as **P<0.01.

Fig. 4.

Null dGALT NMJs exhibit an altered Wnt trans-synaptic signaling pathway. (A) Representative NMJs imaged with anti-horseradish-peroxidase (HRP; green) and anti-Dally-like-protein (Dlp; red) in genetic control (dGALT^C2), dGALT-null (dGALT^ΔAP2) and rescue (dGALT^ΔAP2; UH1-Gal4/UAS-hGALT) larvae. Right: quantification of Dlp intensity normalized to control (dGALT^C2). (B) Representative NMJs probed with anti-HRP (green) and anti-Wingless (Wg; red) in the same genotypes. Right: quantification of Wg intensity normalized to control. Sample size: ≥ten NMJs. Error bars show s.e.m. with significance indicated: *P<0.05, **P<0.01, ***P<0.001.

Fig. 5.

Co-removal of dGALK prevents dGALT movement and NMJ structural defects. (A) Protein-protein interaction network for dGALT generated with the Search Tool for Retrieval of Interacting Genes (STRING). Line thickness represents the strength of predicted interactions. (B) Representative NMJs imaged with anti-horseradish-peroxidase (HRP; green) and anti-Discs-large (DLG; red) in control (dGALT^C2), dGALK-null (dGALK^ΔEXC9), dGALT-null (dGALT^ΔAP2) and double-null mutant (dGALT^ΔAP2; dGALK^ΔEXC9) larvae. (C) Normalized time required for wandering L3 to rollover for all four genotypes. (D) Quantification of NMJ bouton number and (E) inter-bouton distance, normalized to control. Sample size: ≥seven animals for each genotype. Error bars show s.e.m. with significance indicated: *P<0.05, **P<0.01, ***P<0.001, not significant (P>0.05, N.S.).

Fig. 6.

Co-removal of dGALK prevents the loss of galactosylation in the dGALT-null synaptomatrix. Representative NMJs imaged with anti-Fasciclin-II (FASII; red) and (A) Erythrina cristagalli lectin (ECL; green) or (B) Wisteria floribunda agglutinin (WFA; green) in control (dGALT^C2), dGALK-null (dGALK^ΔEXC9), dGALT-null (dGALT^ΔAP2) and double-null (dGALT^ΔAP2; dGALK^ΔEXC9) larvae. Normalized quantification of ECL (A′) and WFA (B′) intensities. Sample size: ≥six NMJs. Error bars show s.e.m. with significance indicated: ***P<0.001, not significant (P>0.05, N.S.).

Fig. 7.

Restoration of Wnt trans-synaptic signaling components in dGALT-null NMJs. Representative NMJs imaged with anti-horseradish peroxidase (HRP; green) and anti-Dally-like-protein (Dlp; red) (A) or anti-Wingless (Wg; red) (B) in control (dGALT^C2), dGALT null (dGALT^ΔAP2), dGALT null with driver alone (dGALT^ΔAP2; UH1/+), dGALT; dGALK double null (dGALT^ΔAP2; dGALK^ΔEXC9) and dGALT null with sgl overexpression (dGALT^ΔAP2; UH1-Gal4/UAS-hGALT). Quantification of Dlp (C) and Wg (D) intensity normalized to genetic control (dGALT^C2). Sample size: ≥eight NMJs (Dlp) and ≥five NMJs (Wg) per genotype. Error bars show s.e.m. with significance indicated: *P<0.05, **P<0.01, ***P<0.001.

Fig. 8.

sgl overexpression prevents dGALT motor, NMJ and HRP glycosylation defects. (A) Representative NMJs imaged with anti-horseradish-peroxidase (HRP; green) and anti-Discs-large (DLG; red) for control (dGALT^C2), dGALT null with driver alone (dGALT^ΔAP2; UH1/+), sgl overexpression (UAS-sgl/UH1-Gal4) and dGALT null with sgl overexpression (dGALT; UAS-sgl/UH1-Gal4). (B) Normalized time required for the wandering L3 to rollover for all four genotypes. (C) Quantification of synaptic bouton number and (D) inter-bouton spacing distance. (E) Sample NMJs imaged with anti-Fasciclin-II (FASII; red) and anti-HRP (green). (F) Normalized quantification of HRP intensity. Sample size: ≥seven NMJs. Error bars show s.e.m. with significance indicated: *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001, not significant (P>0.05, N.S.).