Abstracts

Rationale: Of all the severe and intractable epilepsies, Lafora disease (LD) is among the most severe, and is inevitably fatal. Mutations in two genes have been identified that cause LD, EPM2A (laforin) and EPM2B (malin). Identification of the genetic basis for LD has opened up a new era in our understanding of the cause of LD, leading to rapid progress in the field. Mutations in either of these genes cause glycogen to transform into malformed (starch-like), aggregated inclusions called Lafora bodies (LBs). LBs overtake the cytoplasm of dendrites, and drive the progressive refractory seizure disorder. The human EPM2A gene encodes the phosphatase laforin and recessive mutations in EPM2A result in Lafora disease (LD). In the absence of laforin activity, glycogen transforms into hyper-phosphorylated, water-insoluble, starch-like Lafora bodies that drive neuronal apoptosis, neurodegeneration, and eventual death of LD patients. We previously defined that the physiological function of laforin is to dephosphorylate glycogen, yet the mechanism of glycogen dephosphorylation by laforin was unknown. Additionally, LD patient missense mutations are dispersed throughout laforin, bringing to question the structural mechanism(s) of disease.Methods: We recently determined the crystal structure of human laforin at 2.4 Å bound to oligosaccharides with a phospho-glucan product at the active site. Using a number of biochemical assays, we define patient specific mechanisms that drive Lafora disease progression.Results: The structure reveals an integrated tertiary structure of the carbohydrate binding module and dual specificity phosphatase domains as well as an antiparallel dimer mediated by the phosphatase domain that results in a tetramodular architecture, positioning the two active sites ~31 Å from each other. We utilized the crystal structure and three solution-based, biophysical techniques along with biochemical analyses of LD patient mutations and structured guided mutations to probe this unique tertiary and quaternary structure. We define a cooperative mechanism of action for laforin as well as establish the effect of LD disease mutations, thereby providing atomic level insights that connect basic glycogen metabolism to human neurodegenerative disease.Conclusions: Cumulatively, this work allows us to provide a patient specific, molecular diagnosis of each LD laforin mutation. This personalized, molecular diagnosis will prove invaluable as our groups and others move towards a LD therapeutic and/or cure.