Lafora disease is a progressive myoclonus epilepsy with onset typically in the second decade of life and death within 10 years. Lafora bodies, deposits of abnormally branched, insoluble glycogen-like polymers, form in neurons, muscle, liver, and other tissues. Approximately half of the cases of Lafora disease result from mutations in the EPM2A gene, which encodes laforin, a member of the dual-specificity protein phosphatase family that additionally contains a glycogen binding domain. The molecular basis for the formation of Lafora bodies is completely unknown. Glycogen, a branched polymer of glucose, contains a small amount of covalently linked phosphate whose origin and function are obscure. We report here that recombinant laforin is able to release this phosphate in vitro, in a time-dependent reaction with an apparent Km for glycogen of 4.5 mg/ml. Mutations of laforin that disable the glycogen binding domain also eliminate its ability to dephosphorylate glycogen. We have also analyzed glycogen from a mouse model of Lafora disease, Epm2a ؊/؊ mice, which develop Lafora bodies in several tissues. Glycogen isolated from these mice had a 40% increase in the covalent phosphate content in liver and a 4-fold elevation in muscle. We propose that excessive phosphorylation of glycogen leads to aberrant branching and Lafora body formation. This study provides a molecular link between an observed biochemical property of laforin and the phenotype of a mouse model of Lafora disease. The results also have important implications for glycogen metabolism generally.polyglucosan ͉ Lafora bodies ͉ Epm2a ͉ phosphate

Lafora disease is a progressive myoclonus epilepsy with onset in the teenage years followed by neurodegeneration and death within 10 years. A characteristic is the widespread formation of poorly branched, insoluble glycogen-like polymers (polyglucosan) known as Lafora bodies, which accumulate in neurons, muscle, liver, and other tissues. Approximately half of the cases of Lafora disease result from mutations in the EPM2A gene, which encodes laforin, a member of the dual specificity protein phosphatase family that is able to release the small amount of covalent phosphate normally present in glycogen. In studies of Epm2a ؊/؊ mice that lack laforin, we observed a progressive change in the properties and structure of glycogen that paralleled the formation of Lafora bodies. At three months, glycogen metabolism remained essentially normal, even though the phosphorylation of glycogen has increased 4-fold and causes altered physical properties of the polysaccharide. By 9 months, the glycogen has overaccumulated by 3-fold, has become somewhat more phosphorylated, but, more notably, is now poorly branched, is insoluble in water, and has acquired an abnormal morphology visible by electron microscopy. These glycogen molecules have a tendency to aggregate and can be recovered in the pellet after low speed centrifugation of tissue extracts. The aggregation requires the phosphorylation of glycogen. The aggregrated glycogen sequesters glycogen synthase but not other glycogen metabolizing enzymes. We propose that laforin functions to suppress excessive glycogen phosphorylation and is an essential component of the metabolism of normally structured glycogen.Glycogen is a branched polymer of glucose that acts as a repository of energy used in times of need (1). Liver and skeletal muscle contain the two largest deposits in mammals, but heart, brain, adipose, as well as many other tissues synthesize the polymer. Polymerization occurs via ␣-1,4-glycosidic linkages between glucose residues, with branch points introduced by ␣-1,6-glycosidic linkages. The frequency of branching determines the topology of glycogen and distinguishes it from the carbohydrate moiety of starch (2). A unique three-dimensional structure of glycogen cannot be determined experimentally because of the polydispersity of the molecule, but a widely accepted model of its structure has been proposed (3). Glycogen is composed of successive layers, or tiers, of glucose residues; a full size molecule consists of 12 tiers, with M r ϭ ϳ10 7 and a diameter of ϳ40 nm (4). Glycogen has been reported to contain small amounts of covalently linked phosphate, on the order of 0.064% by weight or 0.121% mol/mol (5-7). The mechanism by which the phosphate is introduced remains unclear. One suggestion (5) has been the existence of a glucose-1-phosphate transferase that would form C1-C6 bridging phosphodiesters, but this enzyme has not been further defined at the molecular level. In plant amylopectin, C1 and C3 phosphomonoesters have been shown to be formed by dikinase enzymes (8), but...

Lafora progressive myoclonus epilepsy is characterized by pathognomonic endoplasmic reticulum (ER)-associated polyglucosan accumulations. We previously discovered that mutations in EPM2A cause Lafora disease. Here, we identify a second gene associated with this disease, NHLRC1 (also called EPM2B), which encodes malin, a putative E3 ubiquitin ligase with a RING finger domain and six NHL motifs. Laforin and malin colocalize to the ER, suggesting they operate in a related pathway protecting against polyglucosan accumulation and epilepsy.

Lafora disease is the most common teenage-onset neurodegenerative disease, the main teenage-onset form of progressive myoclonus epilepsy (PME), and one of the severest epilepsies. Pathologically, a starch-like compound, polyglucosan, accumulates in neuronal cell bodies and overtakes neuronal small processes, mainly dendrites. Polyglucosan formation is catalyzed by glycogen synthase, which is activated through dephosphorylation by glycogen-associated protein phosphatase-1 (PP1). Here we remove PTG, one of the proteins that target PP1 to glycogen, from mice with Lafora disease. This results in near-complete disappearance of polyglucosans and in resolution of neurodegeneration and myoclonic epilepsy. This work discloses an entryway to treating this fatal epilepsy and potentially other glycogen storage diseases.

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