Altered synaptic architecture and glycosylated synaptomatrix composition in aDrosophilaclassic 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.

Acute and long-term outcomes in a Drosophila melanogaster model of classic galactosemia occur independently of galactose-1-phosphate accumulation

“…On the surface, our findings presented here appear to contradict a recent report by Jumbo-Lucioni and colleagues who used the dGALT ΔAP2 and the dGALK exc9 alleles provided by our laboratory to test the role of GALK loss on synapse morphology and a larval movement phenotype in GALT-deficient Drosophila (Jumbo-Lucioni et al, 2014a). Their report stated that homozygosity for the dGALK exc9 allele relieved many of the phenotypes they measured.…”

“…However, the larval outcomes described by Jumbo-Lucioni et al were different from the phenotypes measured here. Of note, some of the larval phenotypes assessed by Jumbo-Lucioni and colleagues (Jumbo-Lucioni et al, 2014a) were also apparently phenocopied in control ( GALT+ ) animals exposed to galactose, which is contrary to the larval and adult phenotypes we have observed previously and describe here (Kushner et al, 2010; Ryan et al, 2012). To be clear, even exposure of wild-type Drosophila to high (200 mM) levels of galactose does not mimic the larval death and adult climbing and female fecundity outcomes described here (Fig.…”

Classical galactosaemia: novel insights in IgG N-glycosylation and N-glycan biosynthesis

“…7,35 It is possible that the bisecting GlcNAcs could also influence glycan processing. To summarise our current and previous observation, we present a schemata of the pathway illustrating the biosynthesis of lipid-linked oligosaccharides (LLO) in the endoplasmic reticulum (ER) (Figure 6a) and diversification of N-glycan biosynthesis in medial-Golgi (Figure 6b).…”

“…Abnormalities were identified at the neuromuscular junction, as well as depletion of galactosyl/N-acetylgalactosamine and fucosylated moieties, which are suggested to result from limited UDP-sugar bioavailability. 7 Defective glycosylation is known to impair neurodevelopment and neurological function. 8 The use of glycan profiling is now increasingly used as a prognostic and diagnostic biomarker in a number of diseases, including cancer, diabetes and rheumatoid arthritis.…”

Classical Galactosaemia and CDG, the N-Glycosylation Interface. A Review

“…UDP-galactose substrate deficiency is one of the proposed contributing pathophysiological mechanisms (Gibson et al 1995;Lai et al 2003;Parkinson et al 2013;Jumbo-Lucioni et al 2014).…”