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

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.     

Inflammation in Lafora Disease: Evolution with Disease Progression in Laforin and Malin Knock-out Mouse Models

Lafora progressive myoclonus epilepsy (Lafora disease, LD) is a fatal rare autosomal recessive neurodegenerative disorder characterized by the accumulation of insoluble ubiquitinated polyglucosan inclusions in the cytoplasm of neurons, which is most commonly associated with mutations in two genes: EPM2A, encoding the glucan phosphatase laforin, and EPM2B, encoding the E3-ubiquitin ligase malin. The present study analyzes possible inflammatory responses in the mouse lines Epm2a ^?/? (laforin knock-out) and Epm2b ^?/? (malin knock-out) with disease progression. Increased numbers of reactive astrocytes (expressing the GFAP marker) and microglia (expressing the Iba1 marker) together with increased expression of genes encoding cytokines and mediators of the inflammatory response occur in both mouse lines although with marked genotype differences. C3ar1 and CxCl10 messenger RNAs (mRNAs) are significantly increased in Epm2a ^?/? mice aged 12 months when compared with age-matched controls, whereas C3ar1, C4b, Ccl4, CxCl10, Il1b, Il6, Tnfα, and Il10ra mRNAs are significantly upregulated in Epm2b ^?/? at the same age. This is accompanied by increased protein levels of IL1-β, IL6, TNFα, and Cox2 particularly in Epm2b ^?/? mice. The severity of inflammatory changes correlates with more severe clinical symptoms previously described in Epm2b ^?/? mice. These findings show for the first time increased innate inflammatory responses in a neurodegenerative disease with polyglucosan intraneuronal deposits which increase with disease progression, in a way similar to what is seen in neurodegenerative diseases with abnormal protein aggregates. These findings also point to the possibility of using anti-inflammatory agents to mitigate the degenerative process in LD.     

Laforin is required for the functional activation of malin in endoplasmic reticulum stress resistance in neuronal cells

Mutations in either EPM2A, the gene encoding a dual-specificity phosphatase named laforin, or NHLRC1, the gene encoding an E3 ubiquitin ligase named malin, cause Lafora disease in humans. Lafora disease is a fatal neurological disorder characterized by progressive myoclonus epilepsy, severe neurological deterioration and accumulation of poorly branched glycogen inclusions, called Lafora bodies or polyglucosan bodies, within the cell cytoplasm. The molecular mechanism underlying the neuropathogenesis of Lafora disease remains unknown. Here, we present data demonstrating that in the cells expressing low levels of laforin protein, overexpressed malin and its Lafora disease-causing missense mutants are stably polyubiquitinated. Malin and malin mutants form ubiquitin-positive aggregates in or around the nuclei of the cells in which they are expressed. Neither wild-type malin nor its mutants elicit endoplasmic reticulum stress, although the mutants exaggerate the response to endoplasmic reticulum stress. Overexpressed laforin impairs the polyubiquitination of malin while it recruits malin to polyglucosan bodies. The recruitment and activities of laforin and malin are both required for the polyglucosan body disruption. Consistently, targeted deletion of laforin in brain cells from Epm2a knockout mice increases polyubiquitinated proteins. Knockdown of Epm2a or Nhlrc1 in neuronal Neuro2a cells shows that they cooperate to allow cells to resist ER stress and apoptosis. These results reveal that a functional laforin-malin complex plays a critical role in disrupting Lafora bodies and relieving ER stress, implying that a causative pathogenic mechanism underlies their deficiency in Lafora disease.     

Increased Endoplasmic Reticulum Stress and Decreased Proteasomal Function in Lafora Disease Models Lacking the Phosphatase Laforin

Background

Lafora progressive myoclonus epilepsy (Lafora disease; LD) is a fatal autosomal recessive neurodegenerative disorder caused by loss-of-function mutations in either the EPM2A gene, encoding the dual specificity phosphatase laforin, or the EPM2B gene, encoding the E3-ubiquitin ligase malin. Previously, we and others have shown that both proteins form a functional complex that regulates glycogen synthesis by a novel mechanism involving ubiquitination and proteasomal degradation of at least two proteins, glycogen synthase and R5/PTG. Since laforin and malin localized at the endoplasmic reticulum (ER) and their regulatory role likely extend to other proteins unrelated to glycogen metabolism, we postulated that their absence may also affect the ER-unfolded protein response pathway.

Methodology/Principal Findings

Here, we demonstrate that siRNA silencing of laforin in Hek293 and SH-SY5Y cells increases their sensitivity to agents triggering ER-stress, which correlates with impairment of the ubiquitin-proteasomal pathway and increased apoptosis. Consistent with these findings, analysis of tissue samples from a LD patient lacking laforin, and from a laforin knockout (Epm2a-/-) mouse model of LD, demonstrates constitutive high expression levels of ER-stress markers BIP/Grp78, CHOP and PDI, among others.

Conclusions/Significance

We demonstrate that, in addition to regulating glycogen synthesis, laforin and malin play a role protecting cells from ER-stress, likely contributing to the elimination of unfolded proteins. These data suggest that proteasomal dysfunction and ER-stress play an important role in the pathogenesis of LD, which may offer novel therapeutic approaches for this fatal neurodegenerative disorder.     

Lafora disease: from genotype to phenotype

The progressive myoclonic epilepsy of Lafora or Lafora disease (LD) is a neurodegenerative disorder characterized by recurrent seizures and cognitive deficits. With typical onset in the late childhood or early adolescence, the patients show progressive worsening of the disease symptoms, leading to death in about 10 years. It is an autosomal recessive disorder caused by the loss-of-function mutations in the EPM2A gene, coding for a protein phosphatase (laforin) or the NHLRC1 gene coding for an E3 ubiquitin ligase (malin). LD is characterized by the presence of abnormally branched water insoluble glycogen inclusions known as Lafora bodies in the neurons and other tissues, suggesting a role for laforin and malin in glycogen metabolic pathways. Mouse models of LD, developed by targeted disruption of the Epm2a or Nhlrc1 gene, recapitulated most of the symptoms and pathological features as seen in humans, and have offered insight into the pathomechanisms. Besides the formation of Lafora bodies in the neurons in the presymptomatic stage, the animal models have also demonstrated perturbations in the proteolytic pathways, such as ubiquitin-proteasome system and autophagy, and inflammatory response. This review attempts to provide a comprehensive coverage on the genetic defects leading to the LD in humans, on the functional properties of the laforin and malin proteins, and on how defects in any one of these two proteins result in a clinically similar phenotype. We also discuss the disease pathologies as revealed by the studies on the animal models and, finally, on the progress with therapeutic attempts albeit in the animal models.     

Laforin–Malin Complex Degrades Polyglucosan Bodies in Concert with Glycogen Debranching Enzyme and Brain Isoform Glycogen Phosphorylase

In Lafora disease (LD), the deficiency of either EPM2A or NHLRC1, the genes encoding the phosphatase laforin and E3 ligase, respectively, causes massive accumulation of less-branched glycogen inclusions, known as Lafora bodies, also called polyglucosan bodies (PBs), in several types of cells including neurons. The biochemical mechanism underlying the PB accumulation, however, remains undefined. We recently demonstrated that laforin is a phosphatase of muscle glycogen synthase (GS1) in PBs, and that laforin recruits malin, together reducing PBs. We show here that accomplishment of PB degradation requires a protein assembly consisting of at least four key enzymes: laforin and malin in a complex, and the glycogenolytic enzymes, glycogen debranching enzyme 1 (AGL1) and brain isoform glycogen phosphorylase (GPBB). Once GS1-synthesized polyglucosan accumulates into PBs, laforin recruits malin to the PBs where laforin dephosphorylates, and malin degrades the GS1 in concert with GPBB and AGL1, resulting in a breakdown of polyglucosan. Without fountional laforin–malin complex assembled on PBs, GPBB and AGL1 together are unable to efficiently breakdown polyglucosan. All these events take place on PBs and in cytoplasm. Deficiency of each of the four enzymes causes PB accumulation in the cytoplasm of affected cells. Demonstration of the molecular mechanisms underlying PB degradation lays a substantial biochemical foundation that may lead to understanding how PB metabolizes and why mutations of either EPM2A or NHLRC1 in humans cause LD. Mutations in AGL1 or GPBB may cause diseases related to PB accumulation.     

Regulation of the autophagic PI3KC3 complex by laforin/malin E3-ubiquitin ligase,two proteins involved in Lafora disease

Lafora progressive myoclonus epilepsy is a fatal rare neurodegenerative disorder characterized by the accumulation of insoluble abnormal glycogen deposits in the brain and peripheral tissues. Mutations in at least two genes are responsible for the disease: EPM2A, encoding the glucan phosphatase laforin, and EPM2B, encoding the RING-type E3-ubiquitin ligase malin. Both laforin and malin form a functional complex in which laforin recruits the substrates to be ubiquitinated by malin. We and others have described that, in cellular and animal models of this disease, there is an autophagy impairment which leads to the accumulation of dysfunctional mitochondria. In addition, we established that the autophagic defect occurred at the initial steps of autophagosome formation. In this work, we present evidence that in cellular models of the disease there is a decrease in the amount of phosphatidylinositol-3P. This is probably due to defective regulation of the autophagic PI3KC3 complex, in the absence of a functional laforin/malin complex. In fact, we demonstrate that the laforin/malin complex interacts physically and co-localizes intracellularly with core components of the PI3KC3 complex (Beclin1, Vps34 and Vps15), and that this interaction is specific and results in the polyubiquitination of these proteins. In addition, the laforin/malin complex is also able to polyubiquitinate ATG14L and UVRAG. Finally, we show that overexpression of the laforin/malin complex increases PI3KC3 activity. All these results suggest a new role of the laforin/malin complex in the activation of autophagy via regulation of the PI3KC3 complex and explain the defect in autophagy described in Lafora disease.     

Increased laforin and laforin binding to glycogen underlie Lafora body formation in malin-deficient Lafora disease

The solubility of glycogen, essential to its metabolism, is a property of its shape, a sphere generated through extensive branching during synthesis. Lafora disease (LD) is a severe teenage-onset neurodegenerative epilepsy and results from multiorgan accumulations, termed Lafora bodies (LB), of abnormally structured aggregation-prone and digestion-resistant glycogen. LD is caused by loss-of-function mutations in the EPM2A or EPM2B gene, encoding the interacting laforin phosphatase and malin E3 ubiquitin ligase enzymes, respectively. The substrate and function of malin are unknown; an early counterintuitive observation in cell culture experiments that it targets laforin to proteasomal degradation was not pursued until now. The substrate and function of laforin have recently been elucidated. Laforin dephosphorylates glycogen during synthesis, without which phosphate ions interfere with and distort glycogen construction, leading to LB. We hypothesized that laforin in excess or not removed following its action on glycogen also interferes with glycogen formation. We show in malin-deficient mice that the absence of malin results in massively increased laforin preceding the appearance of LB and that laforin gradually accumulates in glycogen, which corresponds to progressive LB generation. We show that increasing the amounts of laforin in cell culture causes LB formation and that this occurs only with glycogen binding-competent laforin. In summary, malin deficiency causes increased laforin, increased laforin binding to glycogen, and LB formation. Furthermore, increased levels of laforin, when it can bind glycogen, causes LB. We conclude that malin functions to regulate laforin and that malin deficiency at least in part causes LB and LD through increased laforin binding to glycogen.     

Impaired autophagy in Lafora disease

Lafora disease (LD) is a progressive, lethal, autosomal recessive, neurodegenerative disorder that manifests with myoclonus epilepsy. LD is characterized by the presence of intracellular inclusion bodies called Lafora bodies (LB), in brain, spinal cord and other tissues. More than 50 percent of LD is caused by mutations in EPM2A that encodes laforin. Here we review our recent findings that revealed that laforin regulates autophagy. We consider how autophagy compromise may predispose to LB formation and neurodegeneration in LD, and discuss future investigations suggested by our data.Key words: autophagy, glycogen metabolism, Lafora disease, laforin, malin, neurodegeneration     

Hyperphosphorylation and Aggregation of Tau in Laforin-deficient Mice, an Animal Model for Lafora Disease

Lafora progressive myoclonous epilepsy (Lafora disease; LD) is caused by mutations in the EPM2A gene encoding a dual specificity protein phosphatase named laforin. Our analyses on the Epm2a gene knock-out mice, which developed most of the symptoms of LD, reveal the presence of hyperphosphorylated Tau protein (Ser³⁹⁶ and Ser²⁰²) as neurofibrillary tangles (NFTs) in the brain. Intriguingly, NFTs were also observed in the skeletal muscle tissues of the knock-out mice. The hyperphosphorylation of Tau was associated with increased levels of the active form of GSK3β. The observations on Tau protein were replicated in cell lines using laforin overexpression and knockdown approaches. We also show here that laforin and Tau proteins physically interact and that the interaction was limited to the phosphatase domain of laforin. Finally, our in vitro and in vivo assays demonstrate that laforin dephosphorylates Tau, and therefore laforin is a novel Tau phosphatase. Taken together, our study suggests that laforin is one of the critical regulators of Tau protein, that the NFTs could underlie some of the symptoms seen in LD, and that laforin can contribute to the NFT formation in Alzheimer disease and other tauopathies.Lafora disease (LD)² is an autosomal recessive and a fatal form of progressive myoclonus epilepsy characterized by the presence of Lafora polyglucosan bodies in the affected tissues (1). The symptoms of LD include stimulus-sensitive epilepsy, dementia, ataxia, and rapid neurologic deterioration (1, 2). LD is caused by mutations in the EPM2A gene encoding laforin, a dual specificity protein phosphatase, or in the NHLRC1 gene encoding malin, an E3 ubiquitin ligase (3–7). Both laforin and malin are ubiquitously expressed (3, 5), associated with the endoplasmic reticulum (4, 7), form aggresome upon proteasomal blockade (7), and clear misfolded protein through ubiquitin-proteasome (8). Laforin has two functional domains: a phosphatase domain (dual specificity phosphatase domain; DSPD) and a carbohydrate binding domain (CBD) (9). The CBD helps laforin to target to the glycogen particle and to the Lafora bodies (9, 10), and the DSPD of laforin dephosphorylates carbohydrate moieties (11). Recent studies have further shown that laforin and malin together regulate the cellular levels of PTG, the adaptor protein targeting to glycogen, and that the loss of either malin or laforin results in increased levels of PTG that eventually lead to excessive glycogen deposition (12–14). Although this model explains the genesis of Lafora bodies, the molecular etiology of LD is yet to be understood. For example, unlike this cell line study (12), the presence of Lafora bodies does not lead to neuronal cell death in the two murine models of LD (10, 15), and no difference in the level of PTG was seen in laforin-deficient mice (16).³ However, widespread degeneration of neurons was seen in laforin-deficient mouse with the absence of Lafora bodies, suggesting that the polyglucosan bodies may not play a primary role in the epileptogenesis (15). The laforin dominant-negative transgenic mice line also developed Lafora bodies but had no signs of neurodegeneration or epileptic seizures (10). Thus, the neurodegenerative changes are likely to underlie the etiology of some of the LD symptoms (1). The mouse model developed by the knockdown of the Epm2a gene exhibited a majority of the symptoms known in LD, including the ataxia, spontaneous myoclonic seizures, EEG epileptiform activity, and impaired behavioral responses (15). The knock-out animals showed a number of degenerative changes that include swelling and/or loss of morphological features of mitochondria, endoplasmic reticulum, Golgi apparatus, and the neuronal processes (15). Preliminary histochemical investigations have also suggested the possible presence of neurofibrillary tangles (NFTs) in the knock-out mice (17). In this study, we have characterized the biochemical properties of Tau protein in the animal model of LD and identified laforin as an interacting partner of Tau. Our study identifies laforin to be one of the critical regulators of Tau protein and suggests that the Tau pathology might underlie some of the symptoms seen in LD.     

Laforin, a dual-specificity phosphatase involved in Lafora disease, is phosphorylated at Ser25 by AMP-activated protein kinase

Lafora progressive myoclonus epilepsy [LD (Lafora disease)] is a fatal autosomal recessive neurodegenerative disorder caused by loss-of-function mutations in either the EPM2A gene, encoding the dual-specificity phosphatase laforin, or the EPM2B gene, encoding the E3-ubiquitin ligase malin. Previously, we and others showed that laforin and malin form a functional complex that regulates multiple aspects of glycogen metabolism, and that the interaction between laforin and malin is enhanced by conditions activating AMPK (AMP-activated protein kinase). In the present study, we demonstrate that laforin is a phosphoprotein, as indicated by two-dimensional electrophoresis, and we identify Ser(25) as the residue involved in this modification. We also show that Ser(25) is phosphorylated both in vitro and in vivo by AMPK. Lastly, we demonstrate that this residue plays a critical role for both the phosphatase activity and the ability of laforin to interact with itself and with previously established binding partners. The results of the present study suggest that phosphorylation of laforin-Ser(25) by AMPK provides a mechanism to modulate the interaction between laforin and malin. Regulation of this complex is necessary to maintain normal glycogen metabolism. Importantly, Ser(25) is mutated in some LD patients (S25P), and our results begin to elucidate the mechanism of disease in these patients.     

Neuronatin-mediated Aberrant Calcium Signaling and Endoplasmic Reticulum Stress Underlie Neuropathology in Lafora Disease

Lafora disease (LD) is a teenage-onset inherited progressive myoclonus epilepsy characterized by the accumulations of intracellular inclusions called Lafora bodies and caused by mutations in protein phosphatase laforin or ubiquitin ligase malin. But how the loss of function of either laforin or malin causes disease pathogenesis is poorly understood. Recently, neuronatin was identified as a novel substrate of malin that regulates glycogen synthesis. Here we demonstrate that the level of neuronatin is significantly up-regulated in the skin biopsy sample of LD patients having mutations in both malin and laforin. Neuronatin is highly expressed in human fetal brain with gradual decrease in expression in developing and adult brain. However, in adult brain, neuronatin is predominantly expressed in parvalbumin-positive GABAergic interneurons and localized in their processes. The level of neuronatin is increased and accumulated as insoluble aggregates in the cortical area of LD brain biopsy samples, and there is also a dramatic loss of parvalbumin-positive GABAergic interneurons. Ectopic expression of neuronatin in cultured neuronal cells results in increased intracellular Ca²⁺, endoplasmic reticulum stress, proteasomal dysfunction, and cell death that can be partially rescued by malin. These findings suggest that the neuronatin-induced aberrant Ca²⁺ signaling and endoplasmic reticulum stress might underlie LD pathogenesis.     

Relationship between glycogen accumulation and the laforin dual specificity phosphatase

Laforin, encoded by the EPM2A gene, is a dual specificity protein phosphatase that has a functional glycogen-binding domain. Mutations in the EPM2A gene account for around half of the cases of Lafora disease, an autosomal recessive neurodegenerative disorder, characterized by progressive myoclonus epilepsy. The hallmark of the disease is the presence of Lafora bodies, which contain polyglucosan, a poorly branched form of glycogen, in neurons and other tissues. We examined the level of laforin protein in several mouse models in which muscle glycogen accumulation has been altered genetically. Mice with elevated muscle glycogen have increased laforin as judged by Western analysis. Mice completely lacking muscle glycogen or with 10% normal muscle glycogen had reduced laforin. Mice defective in the GAA gene encoding lysosomal alpha-glucosidase (acid maltase) overaccumulate glycogen in the lysosome but did not have elevated laforin. We propose, therefore, that laforin senses cytosolic glycogen accumulation which in turn determines the level of laforin protein.     

Malin decreases glycogen accumulation by promoting the degradation of protein targeting to glycogen (PTG)

Lafora disease (LD) is an autosomal recessive neurodegenerative disease that results in progressive myoclonus epilepsy and death. LD is caused by mutations in either the E3 ubiquitin ligase malin or the dual specificity phosphatase laforin. A hallmark of LD is the accumulation of insoluble glycogen in the cytoplasm of cells from most tissues. Glycogen metabolism is regulated by phosphorylation of key metabolic enzymes. One regulator of this phosphorylation is protein targeting to glycogen (PTG/R5), a scaffold protein that binds both glycogen and many of the enzymes involved in glycogen synthesis, including protein phosphatase 1 (PP1), glycogen synthase, phosphorylase, and laforin. Overexpression of PTG markedly increases glycogen accumulation, and decreased PTG expression decreases glycogen stores. To investigate if malin and laforin play a role in glycogen metabolism, we overexpressed PTG, malin, and laforin in tissue culture cells. We found that expression of malin or laforin decreased PTG-stimulated glycogen accumulation by 25%, and co-expression of malin and laforin abolished PTG-stimulated glycogen accumulation. Consistent with this result, we found that malin ubiquitinates PTG in a laforin-dependent manner, both in vivo and in vitro, and targets PTG for proteasome-dependent degradation. These results suggest an additional mechanism, involving laforin and malin, in regulating glycogen metabolism.     

Muscle Glycogen Remodeling and Glycogen Phosphate Metabolism following Exhaustive Exercise of Wild Type and Laforin Knockout Mice

Glycogen, the repository of glucose in many cell types, contains small amounts of covalent phosphate, of uncertain function and poorly understood metabolism. Loss-of-function mutations in the laforin gene cause the fatal neurodegenerative disorder, Lafora disease, characterized by increased glycogen phosphorylation and the formation of abnormal deposits of glycogen-like material called Lafora bodies. It is generally accepted that the phosphate is removed by the laforin phosphatase. To study the dynamics of skeletal muscle glycogen phosphorylation in vivo under physiological conditions, mice were subjected to glycogen-depleting exercise and then monitored while they resynthesized glycogen. Depletion of glycogen by exercise was associated with a substantial reduction in total glycogen phosphate and the newly resynthesized glycogen was less branched and less phosphorylated. Branching returned to normal on a time frame of days, whereas phosphorylation remained suppressed over a longer period of time. We observed no change in markers of autophagy. Exercise of 3-month-old laforin knock-out mice caused a similar depletion of glycogen but no loss of glycogen phosphate. Furthermore, remodeling of glycogen to restore the basal branching pattern was delayed in the knock-out animals. From these results, we infer that 1) laforin is responsible for glycogen dephosphorylation during exercise and acts during the cytosolic degradation of glycogen, 2) excess glycogen phosphorylation in the absence of laforin delays the normal remodeling of the branching structure, and 3) the accumulation of glycogen phosphate is a relatively slow process involving multiple cycles of glycogen synthesis-degradation, consistent with the slow onset of the symptoms of Lafora disease.     

Laforin Prevents Stress-Induced Polyglucosan Body Formation and Lafora Disease Progression in Neurons

Glycogen, the largest cytosolic macromolecule, is soluble because of intricate construction generating perfect hydrophilic-surfaced spheres. Little is known about neuronal glycogen function and metabolism, though progress is accruing through the neurodegenerative epilepsy Lafora disease (LD) proteins laforin and malin. Neurons in LD exhibit Lafora bodies (LBs), large accumulations of malconstructed insoluble glycogen (polyglucosans). We demonstrated that the laforin–malin complex reduces LBs and protects neuronal cells against endoplasmic reticulum stress-induced apoptosis. We now show that stress induces polyglucosan formation in normal neurons in culture and in the brain. This is mediated by increased glucose-6-phosphate allosterically hyperactivating muscle glycogen synthase (GS1) and is followed by activation of the glycogen digesting enzyme glycogen phosphorylase. In the absence of laforin, stress-induced polyglucosans are undigested and accumulate into massive LBs, and in laforin-deficient mice, stress drastically accelerates LB accumulation and LD. The mechanism through which laforin–malin mediates polyglucosan degradation remains unclear but involves GS1 dephosphorylation by laforin. Our work uncovers the presence of rapid polyglucosan metabolism as part of the normal physiology of neuroprotection. We propose that deficiency in the degradative phase of this metabolism, leading to LB accumulation and resultant seizure predisposition and neurodegeneration, underlies LD.     

Abnormal metabolism of glycogen phosphate as a cause for Lafora disease

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.     

The laforin-malin complex negatively regulates glycogen synthesis by modulating cellular glucose uptake via glucose transporters

Lafora disease (LD), an inherited and fatal neurodegenerative disorder, is characterized by increased cellular glycogen content and the formation of abnormally branched glycogen inclusions, called Lafora bodies, in the affected tissues, including neurons. Therefore, laforin phosphatase and malin ubiquitin E3 ligase, the two proteins that are defective in LD, are thought to regulate glycogen synthesis through an unknown mechanism, the defects in which are likely to underlie some of the symptoms of LD. We show here that laforin's subcellular localization is dependent on the cellular glycogen content and that the stability of laforin is determined by the cellular ATP level, the activity of 5'-AMP-activated protein kinase, and the affinity of malin toward laforin. By using cell and animal models, we further show that the laforin-malin complex regulates cellular glucose uptake by modulating the subcellular localization of glucose transporters; loss of malin or laforin resulted in an increased abundance of glucose transporters in the plasma membrane and therefore excessive glucose uptake. Loss of laforin or malin, however, did not affect glycogen catabolism. Thus, the excessive cellular glucose level appears to be the primary trigger for the abnormally higher levels of cellular glycogen seen in LD.     

A PTG variant contributes to a milder phenotype in Lafora disease

Lafora disease is an autosomal recessive form of progressive myoclonus epilepsy with no effective therapy. Although the outcome is always unfavorable, onset of symptoms and progression of the disease may vary. We aimed to identify modifier genes that may contribute to the clinical course of Lafora disease patients with EPM2A or EPM2B mutations. We established a list of 43 genes coding for proteins related to laforin/malin function and/or glycogen metabolism and tested common polymorphisms for possible associations with phenotypic differences using a collection of Lafora disease families. Genotype and haplotype analysis showed that PPP1R3C may be associated with a slow progression of the disease. The PPP1R3C gene encodes protein targeting to glycogen (PTG). Glycogen targeting subunits play a major role in recruiting type 1 protein phosphatase (PP1) to glycogen-enriched cell compartments and in increasing the specific activity of PP1 toward specific glycogenic substrates (glycogen synthase and glycogen phosphorylase). Here, we report a new mutation (c.746A>G, N249S) in the PPP1R3C gene that results in a decreased capacity to induce glycogen synthesis and a reduced interaction with glycogen phosphorylase and laforin, supporting a key role of this mutation in the glycogenic activity of PTG. This variant was found in one of two affected siblings of a Lafora disease family characterized by a remarkable mild course. Our findings suggest that variations in PTG may condition the course of Lafora disease and establish PTG as a potential target for pharmacogenetic and therapeutic approaches.     

Ubiquitin conjugating enzyme E2-N and sequestosome-1 (p62) are components of the ubiquitination process mediated by the malin–laforin E3-ubiquitin ligase complex

Lafora disease (LD, OMIM254780, ORPHA501) is a rare neurodegenerative form of epilepsy related to mutations in two proteins: laforin, a dual specificity phosphatase, and malin, an E3-ubiquitin ligase. Both proteins form a functional complex, where laforin recruits specific substrates to be ubiquitinated by malin. However, little is known about the mechanism driving malin–laforin mediated ubiquitination of its substrates. In this work we present evidence indicating that the malin–laforin complex interacts physically and functionally with the ubiquitin conjugating enzyme E2-N (UBE2N). This binding determines the topology of the chains that the complex is able to promote in the corresponding substrates (mainly K63-linked polyubiquitin chains). In addition, we demonstrate that the malin–laforin complex interacts with the selective autophagy adaptor sequestosome-1 (p62). Binding of p62 to the malin–laforin complex allows its recognition by LC3, a component of the autophagosomal membrane. In addition, p62 enhances the ubiquitinating activity of the malin–laforin E3-ubiquitin ligase complex. These data enrich our knowledge on the mechanism of action of the malin–laforin complex as an E3-ubiquitin ligase and reinforces the role of this complex in targeting substrates toward the autophagy pathway.