Abstract
ABSTRACTCongenital disorders of glycosylation (CDGs) constitute a rapidly growing family of human diseases resulting from heritable mutations in genes driving the production and modification of glycoproteins. The resulting symptomatic hypoglycosylation causes multisystemic defects that include severe neurological impairments, revealing a particularly critical requirement for tightly regulated glycosylation in the nervous system. The most common CDG, CDG-Ia (PMM2-CDG), arises from phosphomannomutase type 2 (PMM2) mutations. Here, we report the generation and characterization of the first Drosophila CDG-Ia model. CRISPR-generated pmm2-null Drosophila mutants display severely disrupted glycosylation and early lethality, whereas RNAi-targeted knockdown of neuronal PMM2 results in a strong shift in the abundance of pauci-mannose glycan, progressive incoordination and later lethality, closely paralleling human CDG-Ia symptoms of shortened lifespan, movement impairments and defective neural development. Analyses of the well-characterized Drosophila neuromuscular junction (NMJ) reveal synaptic glycosylation loss accompanied by defects in both structural architecture and functional neurotransmission. NMJ synaptogenesis is driven by intercellular signals that traverse an extracellular synaptomatrix and are co-regulated by glycosylation and matrix metalloproteinases (MMPs). Specifically, trans-synaptic signaling by the Wnt protein Wingless (Wg) depends on the heparan sulfate proteoglycan (HSPG) co-receptor Dally-like protein (Dlp), which is regulated by synaptic MMP activity. Loss of synaptic MMP2, Wg ligand, Dlp co-receptor and downstream trans-synaptic signaling occurs with PMM2 knockdown. Taken together, this Drosophila CDG disease model provides a new avenue for the dissection of cellular and molecular mechanisms underlying neurological impairments and is a means by which to discover and test novel therapeutic treatment strategies.
Highlights
Congenital disorders of glycosylation (CDGs), which are caused by mutation of genes encoding glycosylation pathway proteins, are classified into two categories (Freeze et al, 2015): CDG-I disease states include defects in carbohydrate production, lipid-linked oligosaccharide (LLO) formation and attachment of glycan chains to amino acids; CDG-II disease states include defects in the modification and/or maturation of glycan chains after protein attachment
We show that Drosophila pmm2 is highly conserved, and we manipulate gene function by making mutants with clustered regularly-interspaced short palindromic repeat (CRISPR)/Cas9 genome editing and tissue-targeted transgenic RNA interference (RNAi)
A total of 15 mutations were produced, all verified with direct sequencing: eight frameshifts, four insertions, two deletions and one missense mutation
Summary
Congenital disorders of glycosylation (CDGs), which are caused by mutation of genes encoding glycosylation pathway proteins, are classified into two categories (Freeze et al, 2015): CDG-I disease states include defects in carbohydrate production, lipid-linked oligosaccharide (LLO) formation and attachment of glycan chains to amino acids; CDG-II disease states include defects in the modification and/or maturation of glycan chains after protein attachment. The most common CDG is a CDG-I called CDG-Ia or PMM2-CDG and results from mutations in phosphomannomutase 2 (PMM2), which converts mannose-6-phosphate to mannose-1phosphate, the obligatory precursor for GDP-mannose production and N-linked glycosylation (Andreotti et al, 2014; Freeze et al, 2014, 2015). Individuals with CDG-Ia present with a spectrum of neurological symptoms (Jaeken, 2013), ranging from severe neurological impairments with early death, to mild defects with slight psychomotor delay (Grünewald, 2009; Marquardt and Denecke, 2003). No effective treatments are available, with the only treatment option being symptom management (Grünewald, 2009; Monin et al, 2014; Stefanits et al, 2014)
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