Investigation of Neural Pathogenic Mechanisms Associated with Congenital Disorders of Glycosylation

PROJECT SUMMARY The Congenital Disorders of Glycosylation (CDG) are a growing group of inherited diseases caused by pathogenic variants in genes involved in protein and lipid glycosylation. Our limited understanding of the mechanisms driving disease pathogenesis highlights how little is known about glycan-mediated regulation of neural development. To begin defining these mechanisms, we developed a novel zebrafish mutant for the most common CDG, PMM2-CDG. PMM2-CDG results from variants in phosphomannomutase 2 (PMM2), an enzyme essential to synthesize N-glycosylation precursors. Defects in PMM2 limit production of GDP- mannose, impairing both protein glycosylation and sugar phosphate metabolism. Clinically this results in skeletal dysplasia, cerebellar atrophy and ataxia. The connection between misglycosylated proteins, disrupted sugar metabolism and PMM2-CDG phenotypes is poorly understood. However, through developmental phenotyping and biochemical analyses of pmm2 mutants we identified proprotein convertases and matrix metalloproteinases (Mmps) as candidate drivers of disease. Analyses of cartilage defects revealed a block in chondrogenesis caused by impaired processing of the cell adhesion molecule N-cadherin. N-cadherin is itself normally glycosylated, but the activities of two enzymes that cleave N-cadherin (furin and Mmp2) are disrupted in mutants. Using transgenic fish that fluorescently label Bergmann glia and Purkinje cells we recently uncovered alterations in the cerebellum that correspond with abnormal processing of additional adhesion molecules, like E-cadherin, suggesting protease dependent mechanisms also impair neural development. Parallel efforts to examine the role of metabolic dysfunction identified multiple metabolites altered in pmm2 mutants, including increased abundance of the polyol sorbitol. Sorbitol levels are also elevated in PMM2-CDG patients, and its level correlates with disease severity. A role for sorbitol in neural dysfunction is supported by the fact that reducing its level with the aldose reductase inhibitor epalrestat improves swim behavior in pmm2 mutants. Additional work in vitro has shown sorbitol induced osmotic stress activates the mTOR pathway via Jun kinase (JNK), impacting cellular survival and proliferation. New studies show both JNK and mTOR are activated in pmm2 mutant zebrafish. Taken together, our data suggest multiple pathogenic mechanisms contribute to PMM2-CDG neuronal pathogenesis, one related to altered protease function and processing of cell adhesion molecules, and the other to metabolite-mediated cellular stress. This grant will investigate each mechanism with long-term goals of defining how defects in CDG genes cause neural pathology. To broadly examine glycan-mediated regulation of cerebellar development, we will use new transgenic tools to investigate mechanisms underlying neuronal pathology of another common CDG, PIGA-CDG, which involves abnormal formation of GPI-linked proteins.