Lafora disease (LD) is an autosomal recessive and incurable disease with characters of progressive myoclonus epilepsy (PME), severe neurological deterioration, and the accumulation of starch-like glycogen inclusion bodies (LB) mainly in the brain, muscle, and liver. Defects in two genes: EPM2A, which encodes a dual specificity phosphatase named Laforin and NHLRC, which encodes an E3 ligase named Malin, are identified to be directly linked to the disease. However, the molecular pathological cause of LD is unknown. Two common pathological causes for developing neurodegenerative diseases are diverse abnormal aggregates formed inside or outside of affected neuronal cells and endoplasmic reticulum (ER) stress-induced neuronal apoptosis. We found that the homodimer of Laforin is critical for its optimal phosphatase activity and the majority of missense mutants carried by LD patients impair the mutants'ability to form homodimer, consequently loosing phosphatase activity as well. These mutants elicit unfolded protein response (UPR) when expressed in non-neuronal or neuronal cells;however, they are inefficiently degraded by proteasome and are retained intracellularly as detergent-resistant ubiquitin-positive aggregates around nuclei. Once expressed in neuronal cells, mutants make the cells vulnerable to death induced by ER stressor thapsigargin. In contrast to the mutants, wild type Laforin, predominantly located in the ER, functions as a guardian sensing ER stress and resists ER stress-induced cell death. Therefore, the aggregates and ER stress that are initiated by the mutation in EPM2A gene could be two major pathological causes for LD development. By molecular genetic approaches of overexpression, tetrocycline-regulation, knockdown, and knockout of Epm2a in vitro and in vivo in Epm2a knockout mouse model, we will delineate the molecular mechanism of LD. Based on our preliminary data in vitro and in vivo, we hypothesize that Laforin functions in mobilizing endogenous glucose for emergent supply, preventing glycogen accumulation, easing ER stress, and protecting cells from stress-induced apoptosis. The molecular mechanism of LD is likely that EPM2A mutant-triggered ER stress in combination with loss of Laforin protective role in ER stress, preventive role in glycogen accumulation cause progressive epilepsy and severe neuronal degeneration. These findings provide very important insight not only for developing therapeutic agents for the treatment of the so far incurable LD patients but also for extending Laforin study in other stress- induced diseases such as stresses of hypoglycemia, ischemia, and hypoxia. PUBLIC HEALTH RELEVANCE: This research proposal will test the hypothesis that Epm2a mutant-formed aggregate, mutant triggered- endoplasmic reticulum (ER) stress, loss of wild type Epm2a (laforin) protective role in ER stress, and defect in laforin preventive role in glycogen accumulation and neuronal cell apoptosis are major pathologic causes for development of Lafora disease (LD). Our preliminary data demonstrated that laforin senses ER stress, mobilizes endogenous glucose for emergent glucose supply, prevents glycogen accumulation, and protects cells from stress-induced apoptosis. Our proposal will provide very important evidences for delineating the molecular mechanism of LD by molecular genetic approach in vitro and in vivo in Epm2a knockout mouse model. The protective role of laforin in ER stress and possibly other stresses such as hypoglycemia, ischemia, and hypoxia, will extend laforin function to other stress-induced diseases. Therefore, the proposed study could establish new protective rationales to push forward laforin study in other stress-related disorders. Knowledge gained from this project could greatly improve the care of Lafora patients and provide very important insight for developing therapeutic agents for the treatment of the so far incurable LD patients.