Genetic Depletion of the Malin E3 Ubiquitin Ligase in Mice Leads to Lafora Bodies and the Accumulation of Insoluble Laforin

Abstract

Approximately 90% of cases of Lafora disease, a fatal teenage-onset progressive myoclonus epilepsy, are caused by mutations in either the EPM2A or the EPM2B genes that encode, respectively, a glycogen phosphatase called laforin and an E3 ubiquitin ligase called malin. Lafora disease is characterized by the formation of Lafora bodies, insoluble deposits containing poorly branched glycogen or polyglucosan, in many tissues including skeletal muscle, liver, and brain. Disruption of the Epm2b gene in mice resulted in viable animals that, by 3 months of age, accumulated Lafora bodies in the brain and to a lesser extent in heart and skeletal muscle. Analysis of muscle and brain of the Epm2b ؊/؊ mice by Western blotting indicated no effect on the levels of glycogen synthase, PTG (type 1 phosphatase-targeting subunit), or debranching enzyme, making it unlikely that these proteins are targeted for destruction by malin, as has been proposed. Total laforin protein was increased in the brain of Epm2b ؊/؊ mice and, most notably, was redistributed from the soluble, low speed supernatant to the insoluble low speed pellet, which now contained 90% of the total laforin. This result correlated with elevated insolubility of glycogen and glycogen synthase. Because up-regulation of laforin cannot explain Lafora body formation, we conclude that malin functions to maintain laforin associated with soluble glycogen and that its absence causes sequestration of laforin to an insoluble polysaccharide fraction where it is functionally inert.

The Laforin-Malin Complex Negatively Regulates Glycogen Synthesis by Modulating Cellular Glucose Uptake via Glucose Transporters

“…Previous studies that investigated the possible role of laforin/ malin in glycogen metabolism focused on the enzymes involved in glycogen synthesis or its degradation (11,14,42,(44)(45)(46). Studies from laforin-or malin-deficient animals have demonstrated that none of the critical enzymes show any difference in their level or activity (11,44).…”

“…Thus, a role for laforin in carbohydrate metabolism and in the disposition of Lafora bodies was proposed (5,18,49). Besides Lafora bodies, glycogen content has also been found at higher levels in animals that were deficient for laforin or malin (11,43). Intriguingly, the glycogen reserve in LD animal models shows a higher phosphate content (11,43), and laforin has been shown to dephosphorylate glycogen (43,44).…”

“…Besides Lafora bodies, glycogen content has also been found at higher levels in animals that were deficient for laforin or malin (11,43). Intriguingly, the glycogen reserve in LD animal models shows a higher phosphate content (11,43), and laforin has been shown to dephosphorylate glycogen (43,44). A recent report suggested that glycogen phosphorylation possibly represents an error in a catalytic step in glycogen synthesis and that its removal by laforin could be a damage control mechanism (45).…”

“…However, whether the laforin-malin complex regulates glycogenesis or glycogenolysis, or both of these processes, is yet to be resolved. For example, a role for laforin and malin in regulating the cellular level of glycogen synthase (GS) (48) and R5/PTG (subunit of protein phosphate 1) has been proposed (14,46), but the cellular levels (and activities) of GS and R5/PTG were found to be unaltered in laforin-and malin-deficient mouse models (11,44). Thus, the specific pathway through which the laforin-malin complex is able to regulate glycogen metabolic process is yet to be unequivocally established.…”

Phenotype variations in Lafora progressive myoclonus epilepsy: possible involvement of genetic modifiers?

“…10 The laforin-malin complex was shown to negatively regulate the activity of PTG by promoting its degradation via the proteasome. 10, 24 As the loss of laforin or malin results in the genesis of Lafora bodies 25,26 and suppression of PTG in an LD mouse model rescued, most of the disease symptoms and pathologies, 27 it is reasonable to expect that the variant of PTG that associate with the milder phenotype of LD might show a diminished activity towards glycogen synthesis. Biochemical studies have shown that the LD-linked PTG variant indeed displayed decreased ability to promote the glycogen synthesis.…”