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Noebels JL, Avoli M, Rogawski MA, et al., editors. Jasper's Basic Mechanisms of the Epilepsies [Internet]. 4th edition. Bethesda (MD): National Center for Biotechnology Information (US); 2012.

Cover of Jasper's Basic Mechanisms of the Epilepsies

Jasper's Basic Mechanisms of the Epilepsies [Internet]. 4th edition.

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Progressive myoclonus epilepsy of Lafora

, , and .

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,1 ,2 and 3.*

1 Neurology Service, Fundación Jiménez Díaz and CIBERER Madrid, Spain
2 Division of Neurology, Department of Paediatrics, The Hospital for Sick Children, Toronto, Canada
3 Indian Institute of Technology, Kanpur, India
*Correspondence E-mail: ten.acinofelet@asotarres

Lafora disease is an autosomal recessive form of progressive myoclonus epilepsy characterized by a severe course that leads to death in 5–10 years in most patients. Patients present myoclonic, absence and generalized tonic-clonic seizures at onset, tipically at around age 14–15 years. As the disease progresses, difficulties in speech generation and gait as well as cognitive decline appear. Seizures soon become intractable and due to a rapidly progressing dementia patients become severely incapacitated and die. Lafora bodies are the characteristic hallmark and consist of an abnormal, poorly branched, intracellular glucose polymer accumulating in many tissues including heart, skeletal muscle, liver, and brain. They can be observed on optic microscopy as periodic acid-Schiff-positive (PAS+) cytoplasmic inclusions. Lafora bodies thus resemble glycogen with reduced branching, suggesting an alteration in glycogen metabolism as the cause of their accumulation. Since the discovery of the localization of the first gene for Lafora disease in 1995, major advances have led to a better understanding of the basic mechanisms involved in this adolescent-onset and deadly disease.

MOLECULAR GENETICS AND GENOTYPE-PHENOTYPE CORRELATIONS

Linkage analysis and homozygosity mapping initially localized a major gene for Lafora disease to chromosome 6q24.1 Subsequently, the first gene, known as EPM2A, encoding laforin was identified.2,3 Mutation analysis has revealed a marked allelic heterogeneity in EPM2A. In spite of this remarkable allelic heterogeneity, one mutation, R241stop, was found in approximately 40% of Lafora disease patients in one study of Lafora disease patients in Spain.4 This study also suggested that both founder effect and recurrence contributed to the relatively high prevalence of R241stop mutation in Spain.

Chan et al5 identified a second gene associated with Lafora disease, NHLRC1 or EPM2B, which encodes malin, a putative E3 ubiquitin ligase with a RING finger domain and six NHL motifs. Malin was found to colocalize with laforin and it was suggested that the malin-laforin interplay protected against polyglucosan body formation and epilepsy.

Defects in EPM2A and EPM2B account for more than 95% of cases and more than 100 mutations have been reported. A study of 77 families with Lafora disease found that 54 (70.1%) had mutations in EPM2A, 21 (27.3%) in EPM2B, and 2 (2.6%) had no mutations in either gene. The course of the disease was longer in patients with EPM2B mutations compared to patients with EPM2A mutations, suggesting that patients with EPM2B-associated Lafora disease tend to have a slightly milder clinical course and a slower progression.6 This finding was also reported in an Italian family with a EPM2B mutation and a remarkably slow and mild course and in a series of Italian patients.7,8 Singh et al reported similar findings.9

In a meta-analysis performed in 2009,10 nearly 100 distinct mutations were identified in the two genes in over 200 independent LD families. Nearly half of them were missense mutations and deletions accounted for one-quarter. The proportion of patients with EPM2A and EPM2B mutations varies among countries and, whereas in Spain EPM2A mutations are more common, in Italy and France EPM2B mutations predominate. In India and in the Arabic countries the mutations are distributed evenly.

FUNCTIONAL STUDIES: MECHANISMS OF LAFORA BODY FORMATION AND NEURODEGENERATION

Lafora bodies consist of an abnormal glycogen known as polyglucosan which has a low number of branches and very long chains of glucose. Lafora bodies resemble starch and have a low solubility presenting a tendency to accumulate. Glycogen is synthesized through the action of glycogen synthase, responsible for chain elongation, and glycogen branching enzyme, responsible for chain branching. Glycogen is eliminated through digestion by glycogen phosphorylase and glycogen debranching enzyme. Reversible phosphorylation modulates nearly every step of glycogenesis and glycogenolysis. PTG (protein targeting to glycogen) is an indirect activator of glycogen synthase and an indirect inhibitor of both glycogen phosphorylase and glycogen phosphorylase kinase, the enzyme that activates glycogen phosphorylase. PTG performs this reciprocal activation of synthesis and inhibition of breakdown by binding the protein phosphatase PP1 through its C-terminus, binding glycogen, and through a common region in its N-terminus binding glycogen synthase, glycogen phosphorylase, or glycogen phosphorylase kinase, thus targeting PP1 to each of these enzymes. PP1 dephosphorylates each of the three enzymes, activating glycogen synthase and inhibiting glycogen phosphorylase and glycogen phosphorylase kinase.11

Malin and laforin co-localize in endoplasmic reticulum (ER) and form centrosomal aggregates when treated with proteasomal inhibitors in both neuronal and non-neuronal cells. Laforin/malin aggregates co-localize with gamma-tubulin and cause redistribution of alpha-tubulin. The centrosomal accumulation of malin, possibly with the help of laforin, may enhance the ubiquitination of its substrates and facilitate their efficient degradation by proteasome. Defects in malin or laforin may thus lead to increased levels of misfolded and/or target proteins, which may eventually affect the physiological processes of the neuron. Thus, defects in protein degradation and clearance are likely to be the primary trigger in the physiopathology of LD.12

The first piece of evidence of the possible role of a dysfunction in glycogen synthesis in the accumulation of polyglucosans came from a study of transgenic mice overexpressing glycogen synthase. The authors proposed an imbalance between glycogen synthase and branching enzyme as the mechanism involved in the production of polyglucosan bodies.13

Laforin is a protein of 331 amino acids with two domains, a dual-specificity phosphatase domain and a carbohydrate binding domain. Laforin forms part of a multiprotein complex associated with intracellular glycogen particles, and is involved in the regulation of glycogen metabolism. Laforin interacts with itself and with PTG, the glycogen targeting regulatory subunit R5 of protein phosphatase 1 (PP1). R5 is the human homolog of the murine PTG, a protein that acts as a molecular scaffold assembling PP1 with its substrate, glycogen synthase, at the intracellular glycogen particles. The majority of EPM2A missense mutations found in LD patients result in lack of phosphatase activity, absence of binding to glycogen and lack of interaction with PTG.14

Polyglucosan formation is catalyzed by glycogen synthase, which is activated through dephosphorylation by glycogen-associated protein phosphatase-1 (PP1). PTG, one of the proteins that target PP1 to glycogen, was removed from laforin knock-out mice. This resulted in near-complete disappearance of polyglucosans and in resolution of neurodegeneration and myoclonic epilepsy. Blocking of PTG could in this way be a form of treating Lafora disease.15

Malin is a single subunit E3 ubiquitin (Ub) ligase. Its RING domain is necessary and sufficient to mediate ubiquitination. Additionally, malin interacts with and polyubiquitinates laforin, leading to its degradation. Missense mutations in malin that are present in Lafora disease patients abolish its ability to polyubiquitinate and signal the degradation of laforin. Laforin is thus a physiologic substrate of malin.16

The laforin-malin complex suppresses glycogen synthesis in neurons and its malfunction would explain the accumulation of a poorly branched glycogen.17 Either the abnormal glucose polymer, a malfunction of other pathways where laforin and malin are involved or both would result in progressive neurodegeneration and epileptic seizures. The laforin-malin complex also downregulates PTG-induced glycogen synthesis through a mechanism involving ubiquitination and degradation of PTG. The interaction between laforin and malin is a regulated process that is modulated by the AMP-activated protein kinase (AMPK).18 Another theory proposes that excessive phosphorylation of glycogen leads to aberrant branching and Lafora body formation.19

MOUSE MODELS BEARING ENGINEERED MUTATIONS IN EPM2A AND EPM2B

To study the pathology of Lafora disease and the functions of laforin, Ganesh et al disrupted the Epm2a gene in mice.20 At two months of age, homozygous null mutants developed widespread degeneration of neurons, most of which occurred in the absence of Lafora bodies. Dying neurons characteristically exhibited swelling in the endoplasmic reticulum, Golgi networks and mitochondria in the absence of apoptotic bodies or fragmentation of DNA. The Lafora bodies, present both in neuronal and non-neural tissues, were found positive for ubiquitin and advanced glycation end-products only in neurons, suggesting different pathological consequences for Lafora inclusions in neuronal tissues. The authors concluded that Lafora disease is a primary neurodegenerative disorder that may utilize a non-apoptotic mechanism of cell death.

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, the authors concluded 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.21

These mouse models permit a more clear understanding of the pathogenic mechanisms involved in Lafora disease in humans and provide tools to unravel the complex mechanisms involved in the formation of Lafora bodies.

In conclusion, major advances have been achieved in the knowledge of the basic mechanisms involved in Lafora disease since the initial molecular genetic studies of 15 years ago. Studies have mainly focused on the mechanisms of Lafora body formation and reveal some clues on how neurodegeneration is produced. However, we do not know how the molecular defects found in Lafora disease result in the production of epileptic seizures: the role of Lafora bodies is still in question and it may be that seizures are the consequence of other dysfunctions. The next steps in research on Lafora disease should lead to the development of experimental test systems to define better therapies.

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Footnotes

DISCLOSURE

The authors declare no conflicts of interest.

Copyright © 2012, Michael A Rogawski, Antonio V Delgado-Escueta, Jeffrey L Noebels, Massimo Avoli and Richard W Olsen.

All Jasper's Basic Mechanisms of the Epilepsies content, except where otherwise noted, is licensed under a Creative Commons Attribution-NonCommercial-NoDerivs 3.0 Unported license, which permits copying, distribution and transmission of the work, provided the original work is properly cited, not used for commercial purposes, nor is altered or transformed.

Bookshelf ID: NBK98134PMID: 22787674

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