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Ubiquitin ligases regulate quantities and activities of target proteins, often pleiotropically. The malin ubiquitin E3 ligase is reported to regulate autophagy, the misfolded protein response, microRNA silencing, Wnt signaling, neuronatin-mediated endoplasmic reticulum stress, and the laforin glycogen phosphatase. Malin deficiency causes Lafora disease, pathologically characterized by neurodegeneration and accumulations of malformed glycogen (Lafora bodies). We show that reducing glycogen production in malin-deficient mice by genetically removing PTG, a glycogen synthesis activator protein, nearly completely eliminates Lafora bodies and rescues the neurodegeneration, myoclonus, seizure susceptibility, and behavioral abnormality. Glycogen synthesis downregulation is a potential therapy for the fatal adolescence onset epilepsy Lafora disease.

Lafora disease (LD) is the major teenage-onset progressive myoclonus epilepsy (PME). Insidious cognitive decline and escalating myoclonic, visual, convulsive, and other seizures follow an initial decade of normal development. Within a few years, seizures are intractable, myoclonic absences are near constant, and a disinhibited dementia has set in. A vegetative state with continuous myoclonus characterizes the final stage and most patients die in status epilepticus before age 301. The neuropathology of LD is characterized by progressive formation and growth of Lafora bodies (LB) in neuronal somata and processes, and by neurodegeneration1. LB are composed of aggregates of a variety of proteins and an abnormal form of glycogen that lacks normal glycogen’s normal branching and spherical structure essential to its solubility. The abnormal glycogen, called polyglucosan, makes up over 70% of a LB2. Whether LB are pathogenic, or a mere epiphenotype, remains uncertain. LD is caused by loss of function of either of two interacting enzymes, malin, a ubiquitin E3 ligase, and laforin, a phosphatase3. Malin regulates the amount of laforin, and laforin regulates glycogen phosphorylation. The latter is essential to normal glycogen structure, through mechanisms that remain poorly defined3–5. Based mostly on cell culture experiments, several additional functions, unrelated to glycogen metabolism, have been tentatively attributed to laforin and malin, including tau kinase dephosphorylation, Wnt signaling regulation, and others6–9. It is possible that loss of one or more of these functions, rather than effects on glycogen metabolism and LB formation, underlie the neurodegeneration and PME of LD. PTG is an adaptor protein that mediates dephosphorylation of the glycogen synthesizing (glycogen synthase; GS) and degrading (glycogen phosphorylase; GP) enzymes by the pleiotropic phosphatase PP1, which activates GS, inactivates GP, and thus increases glycogen production10. We recently hypothesized that, malstructured though they are, polyglucosans are glycogen, and reducing glycogen synthesis might reduce LB formation, which, if LB cause the disease, might prevent LD. As a test of this hypothesis, we removed PTG from the laforin-deficient mouse model of LD (laforin knockout; LKO) by crossing PTG knockout mice with the LD mice. This resulted in drastic reduction of LB and rescued the neurodegeneration of the LD mice11. While these results supported the view that LB are pathogenic, there remained the possibility that PTG has adaptor or other functions outside of glycogen metabolism and that its removal prevented neurodegeneration through pathways unrelated to glycogen metabolism and LB. Furthermore, the study was of short duration and did not assess whether the correction of the neurodegeneration is maintained. To address these two issues, we removed GS itself from LKO mice and found that the absence of glycogen synthesis alone was sufficient to prevent LB formation, neurodegeneration, and seizure susceptibility, and do so long-term.

Objective: Glycogen, the largest cytosolic macromolecule, acquires solubility, essential to its function, through extreme branching. Lafora bodies are aggregates of polyglucosan, a long, linear, poorly branched, and insoluble form of glycogen. Lafora bodies occupy vast numbers of neuronal dendrites and perikarya in Lafora disease in time-dependent fashion, leading to intractable and fatal progressive myoclonus epilepsy. Lafora disease is caused by deficiency of either the laforin glycogen phosphatase or the malin E3 ubiquitin ligase. The 2 leading hypotheses of Lafora body formation are: (1) increased glycogen synthase activity extends glycogen strands too rapidly to allow adequate branching, resulting in polyglucosans; and (2) increased glycogen phosphate leads to glycogen conformational change, unfolding, precipitation, and conversion to polyglucosan. Recently, it was shown that in the laforin phosphatase-deficient form of Lafora disease, there is no increase in glycogen synthase, but there is a dramatic increase in glycogen phosphate, with subsequent conversion of glycogen to polyglucosan. Here, we determine whether Lafora bodies in the malin ubiquitin ligase-deficient form of the disease are due to increased glycogen synthase or increased glycogen phosphate. Methods: We generated malin-deficient mice and tested the 2 hypotheses. Results: Malin-deficient mice precisely replicate the pathology of Lafora disease with Lafora body formation in skeletal muscle, liver, and brain, and in the latter in the pathognomonic perikaryal and dendritic locations. Glycogen synthase quantity and activity are unchanged. There is a highly significant increase in glycogen phosphate. Interpretation: We identify a single common modification, glycogen hyperphosphorylation, as the root cause of Lafora body pathogenesis. ANN NEUROL, 2010