Investigation of pathophysiology and therapeutic strategies in PMM2-CDG model zebrafish
Cline, Abigail Elizabeth
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Congenital Disorder of Glycosylation PMM2-CDG results from mutations in PMM2, which encodes the phosphomannomutase that converts mannose-6-P to mannose-1-P. Patients have wide-spectrum clinical abnormalities associated with impaired protein N-glycosylation. Though widely proposed that PMM2 deficiency depletes mannose-1-P, a precursor of GDP-mannose, and consequently suppresses lipid-linked oligosaccharide (LLO) levels needed for N-glycosylation, these deficiencies have not been demonstrated in patients or any animal model. In order to explore the developmental and biochemical consequences of PMM2 deficiency, we generated and characterized a morpholino-based PMM2-CDG model in zebrafish. Morphant embryos had developmental abnormalities consistent with PMM2-CDG patients, including craniofacial defects and impaired motility associated with altered motor neurogenesis within the spinal cord. Significantly, global N-linked glycosylation and LLO levels were reduced in pmm2 morphants. Although insufficient to reduce mannose-1-P and GDP-mannose levels, Pmm2 depletion unexpectedly caused accumulation of mannose-6-P, shown earlier to promote LLO cleavage in vitro. In pmm2 morphants, the free glycan by-products of LLO cleavage increased nearly two-fold. Suppression of the mannose-6-P synthesizing enzyme, mannose phosphate isomerase, within the pmm2 background normalized mannose-6-P levels and certain aspects of the craniofacial phenotype, and abrogated pmm2-dependent LLO cleavage. Overexpression of pmm1 mRNA, a homologous enzyme to PMM2, within the pmm2 background not only increased phosphomannomutase activity, but also corrected abnormal phenotypes and deficient glycosylation profiles noted in pmm2 morphants. In summary, we employed the first zebrafish model of PMM2-CDG to uncover novel cellular insights not possible with other systems, including a mannose-6-P accumulation mechanism for under-glycosylation.