Berge A. Minassian

Angelman syndrome (AS) is a severe neurodevelopmental disorder resulting from a deletion/mutation in maternal chromosome 15q11-13. The genes in 15q11-13 contributing to the full array of the clinical phenotype are not fully identified. This study examines whether a loss or reduction in the GABAA receptor beta3 subunit (GABRB3) gene, contained within the AS deletion region, may contribute to the overall severity of AS. Disrupting the gabrb3 gene in mice produces electroencephalographic abnormalities, seizures, and behavior that parallel those seen in AS. The seizures that are observed in these mice showed a pharmacological response profile to antiepileptic medications similar to that observed in AS. Additionally, these mice exhibited learning and memory deficits, poor motor skills on a repetitive task, hyperactivity, and a disturbed rest-activity cycle, features all common to AS. The loss of the single gene, gabrb3, in these mice is sufficient to cause phenotypic traits that have marked similarities to the clinical features of AS, indicating that impaired expression of the GABRB3 gene in humans probably contributes to the overall phenotype of Angelman syndrome. At least one other gene, the E6-associated protein ubiquitin-protein ligase (UBE3A) gene, has been implicated in AS, so the relative contribution of the GABRB3 gene alone or in combination with other genes remains to be established.

Summary Objective Epilepsy is a common neurologic disorder of childhood. To determine the genetic diagnostic yield in epileptic encephalopathy, we performed a retrospective cohort study in a single epilepsy genetics clinic. Methods We included all patients with intractable epilepsy, global developmental delay, and cognitive dysfunction seen between January 2012 and June 2014 in the Epilepsy Genetics Clinic. Electronic patient charts were reviewed for clinical features, neuroimaging, biochemical investigations, and molecular genetic investigations including targeted next‐generation sequencing of epileptic encephalopathy genes. Results Genetic causes were identified in 28% of the 110 patients: 7% had inherited metabolic disorders including pyridoxine dependent epilepsy caused by ALDH 7A1 mutation, Menkes disease, pyridox(am)ine‐5‐phosphate oxidase deficiency, cobalamin G deficiency, methylenetetrahydrofolate reductase deficiency, glucose transporter 1 deficiency, glycine encephalopathy, and pyruvate dehydrogenase complex deficiency; 21% had other genetic causes including genetic syndromes, pathogenic copy number variants on array comparative genomic hybridization, and epileptic encephalopathy related to mutations in the SCN 1A , SCN 2A , SCN 8A , KCNQ 2 , STXBP 1 , PCDH 19 , and SLC 9A6 genes. Forty‐five percent of patients obtained a genetic diagnosis by targeted next‐generation sequencing epileptic encephalopathy panels. It is notable that 4.5% of patients had a treatable inherited metabolic disease. Significance To the best of our knowledge, this is the first study to combine inherited metabolic disorders and other genetic causes of epileptic encephalopathy. Targeted next‐generation sequencing panels increased the genetic diagnostic yield from <10% to >25% in patients with epileptic encephalopathy.

We describe a disease encompassing infantile-onset movement disorder (including severe parkinsonism and nonambulation), mood disturbance, autonomic instability, and developmental delay, and we describe evidence supporting its causation by a mutation in SLC18A2 (which encodes vesicular monoamine transporter 2 [VMAT2]). VMAT2 translocates dopamine and serotonin into synaptic vesicles and is essential for motor control, stable mood, and autonomic function. Treatment with levodopa was associated with worsening, whereas treatment with direct dopamine agonists was followed by immediate ambulation, near-complete correction of the movement disorder, and resumption of development.

Rett syndrome is caused by mutations in the gene MECP2 in approximately 80% of affected individuals. We describe a previously unknown MeCP2 isoform. Mutations unique to this isoform and the absence, until now, of identified mutations specific to the previously recognized protein indicate an important role for the newly discovered molecule in the pathogenesis of Rett syndrome.

The late-infantile-onset forms are the most genetically heterogeneous group among the autosomal recessively inherited neurodegenerative disorders, the neuronal ceroid lipofuscinoses (NCLs). The Turkish variant was initially considered to be a distinct genetic entity, with clinical presentation similar to that of other forms of late-infantile-onset NCL (LINCL), including age at onset from 2 to 7 years, epileptic seizures, psychomotor deterioration, myoclonus, loss of vision, and premature death. However, Turkish variant LINCL was recently found to be genetically heterogeneous, because mutations in two genes, CLN6 and CLN8, were identified to underlie the disease phenotype in a subset of patients. After a genomewide scan with single-nucleotide-polymorphism markers and homozygosity mapping in nine Turkish families and one Indian family, not linked to any of the known NCL loci, we mapped a novel variant LINCL locus to chromosome 4q28.1-q28.2 in five families. We identified six different mutations in the MFSD8 gene (previously denoted "MGC33302"), which encodes a novel polytopic 518-amino acid membrane protein that belongs to the major facilitator superfamily of transporter proteins. MFSD8 is expressed ubiquitously, with several alternatively spliced variants. Like the majority of the previously identified NCL proteins, MFSD8 localizes mainly to the lysosomal compartment. However, the function of MFSD8 remains to be elucidated. Analysis of the genome-scan data suggests the existence of at least three more genes in the remaining five families, further corroborating the great genetic heterogeneity of LINCLs.

DNA sequence and annotation of the entire human chromosome 7, encompassing nearly 158 million nucleotides of DNA and 1917 gene structures, are presented. To generate a higher order description, additional structural features such as imprinted genes, fragile sites, and segmental duplications were integrated at the level of the DNA sequence with medical genetic data, including 440 chromosome rearrangement breakpoints associated with disease. This approach enabled the discovery of candidate genes for developmental diseases including autism.

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 K m 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.

Abstract We compared epilepsy phenotypes with genotypes of Angelman syndrome (AS), including chromosome 15q11‐13 deletions (class I), uniparental disomy (class II), methylation imprinting abnormalities (class III), and mutation in the UBE3A gene (class IV). Twenty patients were prospectively selected based on clinical cytogenetic and molecular diagnosis of AS. All patients had 6 to 72 hours of closed‐circuit television videotaping and digitized electroencephalographic (EEG) telemetry. Patients from all genotypic classes had characteristic EEGs with diffuse bifrontally dominant high‐amplitude 1‐ to 3‐Hz notched or triphasic or polyphasic slow waves, or slow and sharp waves. Class I patients had severe intractable epilepsy, most frequently with atypical absences and myoclonias and less frequently with generalized extensor tonic seizures or flexor spasms. Epileptic spasms were recorded in AS patients as old as 41 years. Aged‐matched class II, III, and IV patients had either no epilepsy or drug‐responsive mild epilepsy with relatively infrequent atypical absences, myoclonias, or atonic seizures. In conclusion, maternally inherited chromosome 15q11‐13 deletions produce severe epilepsy. Loss‐of‐function UBE3A mutations, uniparental disomy, or methylation imprint abnormalities in AS are associated with relatively mild epilepsy. Involvement of other genes in the chromosome 15q11‐13 deletion, such as GABRB3, may explain severe epilepsy in AS.

The object of this study was to determine the concordance of the anatomical location of interictal magnetoencephalographic (MEG) spike foci with the location of ictal onset zones identified by invasive ictal intracranial electroencephalographic recordings in children undergoing evaluation for epilepsy surgery. MEG was performed in 11 children with intractable, nonlesional, extratemporal, localization-related epilepsy. Subsequently, chronic invasive intracranial electroencephalographic monitoring was performed by using subdural electrodes to localize the ictal onset zone and eloquent cortex. Based on the invasive monitoring data, all children had excision of, or multiple subpial transections through, ictal onset cortex and surrounding irritative zones. In 10 of 11 patients, the anatomical location of the epileptiform discharges as determined by MEG corresponded to the ictal onset zone established by ictal intracranial recordings. In all children, the anatomical location of the somatosensory hand area, determined by functional mapping through the subdural electrode array, was the same as that delineated by MEG. Nine of 11 patients became either seizure-free or had a greater than 90% reduction in seizures after surgery, with a mean follow-up of 24 months. MEG is a powerful and accurate tool in the presurgical evaluation of children with refractory nonlesional extratemporal epilepsy.

Lafora’s disease is one of five inherited progressive myoclonus epilepsy syndromes. It is an autosomal-recessive disorder with onset in late childhood or adolescence. Characteristic seizures include myoclonic and occipital lobe seizures with visual hallucinations, scotomata, and photoconvulsions. The course of the disease consists of worsening seizures and an inexorable decline in mental and other neurologic functions that result in dementia and death within 10 years of onset. Pathology reveals pathognomonic polyglucosan inclusions that are not seen in any other progressive myoclonus epilepsy. Lafora’s disease is one of several neurologic conditions associated with brain polyglucosan bodies. Why Lafora’s polyglucosan bodies alone are associated with epilepsy is unknown and is discussed in this article. Up to 80% of patients with Lafora’s disease have mutations in the EPM2A gene. Although common mutations are rare, simple genetic tests to identify most mutations have been established. At least one other still-unknown gene causes Lafora’s disease. The EPM2A gene codes for the protein laforin, which localizes at the plasma membrane and the rough endoplasmic reticulum and functions as a dual-specificity phosphatase. Work toward establishing the connection between laforin and Lafora’s disease polyglucosans is underway, as are attempts to replace it into the central nervous system of patients with Lafora’s 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. 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 (1Roach P.J. Curr. Mol. Med. 2002; 2: 101-120Crossref PubMed Scopus (357) Google Scholar). 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 α-1,4-glycosidic linkages between glucose residues, with branch points introduced by α-1,6-glycosidic linkages. The frequency of branching determines the topology of glycogen and distinguishes it from the carbohydrate moiety of starch (2Ball S. Guan H.P. James M. Myers A. Keeling P. Mouille G. Buleon A. Colonna P. Preiss J. Cell. 1996; 86: 349-352Abstract Full Text Full Text PDF PubMed Scopus (399) Google Scholar). 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 (3Gunja-Smith Z. Marshall J.J. Mercier C. Smith E.E. Whelan W.J. FEBS Lett. 1970; 12: 101-104Crossref PubMed Scopus (164) Google Scholar). Glycogen is composed of successive layers, or tiers, of glucose residues; a full size molecule consists of 12 tiers, with Mr = ∼107 and a diameter of ∼40 nm (4Shearer J. Graham T.E. Exerc. Sport Sci. Rev. 2004; 32: 120-126Crossref PubMed Scopus (46) Google Scholar). 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 (5Lomako J. Lomako W.M. Whelan W.J. Marchase R.B. FEBS Lett. 1993; 329: 263-267Crossref PubMed Scopus (30) Google Scholar, 6Lomako J. Lomako W.M. Kirkman B.R. Whelan W.J. Biofactors. 1994; 4: 167-171PubMed Google Scholar, 7Fontana J.D. FEBS Lett. 1980; 109: 85-92Crossref PubMed Scopus (35) Google Scholar). The mechanism by which the phosphate is introduced remains unclear. One suggestion (5Lomako J. Lomako W.M. Whelan W.J. Marchase R.B. FEBS Lett. 1993; 329: 263-267Crossref PubMed Scopus (30) Google Scholar) 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 (8Ritte G. Heydenreich M. Mahlow S. Haebel S. Kotting O. Steup M. FEBS Lett. 2006; 580: 4872-4876Crossref PubMed Scopus (148) Google Scholar), but extensive bioinformatic analysis of mammalian genomes has failed to reveal any analogous enzyme. Reduced branching of glycogen is associated with the accumulation of insoluble polysaccharide or polyglucosan in several disease states (9Cavanagh J.B. Brain Res. 1999; 29: 265-295Crossref Scopus (217) Google Scholar, 10Nakajima H. Raben N. Hamaguchi T. Yamasaki T. Curr. Mol. Med. 2002; 2: 197-212Crossref PubMed Scopus (73) Google Scholar, 11DiMauro S. Lamperti C. Muscle Nerve. 2001; 24: 984-999Crossref PubMed Scopus (113) Google Scholar). In Andersen disease and Tarui disease, it is thought that an imbalance between elongating and branching enzymatic activities explains the formation of polyglucosan. Lafora disease (LD) 2The abbreviations used are: LD, Lafora disease; LB, Lafora bodies; GS, glycogen synthase; AGL, amylo-1,6-glucosidase, 4-α-glucanotransferase; PTG, protein targeting to glycogen; LSS, low speed supernatant; LSP, low speed pellet; G6P, glucose-6-phosphate; E3, ubiquitin-protein isopeptide ligase. (OMIM254780) is an autosomal recessive form of juvenile onset progressive myoclonus epilepsy. Patients appear relatively normal until early adolescence when seizures and rapid mental deterioration ensue resulting in death within 10 years (12Ganesh S. Puri R. Singh S. Mittal S. Dubey D. J. Hum. Genet. 2006; 51: 1-8Crossref PubMed Scopus (117) Google Scholar, 13Chan E.M. Andrade D.M. Franceschetti S. Minassian B. Adv. Neurol. 2005; 95: 47-57PubMed Google Scholar, 14Delgado-Escueta A.V. Curr. Neurol. Neurosci. Rep. 2007; 7: 428-433Crossref PubMed Scopus (78) Google Scholar). The hallmark of the disease is the presence of intracellular inclusions known as Lafora bodies (LB), the main constituent of which is polyglucosan. LB are found in many tissues, including skeletal muscle and brain. To date, mutations in two genes have been shown to account for ∼90% of LD cases. Approximately 60% of LD cases can be attributed to mutations in the EPM2A (epilepsy progressive myoclonus type 2A) gene, which encodes, by sequence, a dual specificity protein phosphatase named laforin (12Ganesh S. Puri R. Singh S. Mittal S. Dubey D. J. Hum. Genet. 2006; 51: 1-8Crossref PubMed Scopus (117) Google Scholar, 15Minassian B.A. Lee J.R. Herbrick J.A. Huizenga J. Soder S. Mungall A.J. Dunham I. Gardner R. Fong C.Y. Carpenter S. Jardim L. Satishchandra P. Andermann E. Snead O.C. 3rd, Lopes-Cendes I. Tsui L.C. Delgado-Escueta A.V. Rouleau G.A. Scherer S.W. Nat. Genet. 1998; 20: 171-174Crossref PubMed Scopus (418) Google Scholar, 16Ianzano L. Zhang J. Chan E.M. Zhao X.C. Lohi H. Scherer S.W. Minassian B.A. Hum. Mut. 2005; 26: 397Crossref PubMed Scopus (54) Google Scholar). Laforin also contains a highly conserved carbohydrate-binding module subtype 20 (17Boraston A.B. Bolam D.N. Gilbert H.J. Davies G.J. Biochem. J. 2004; 382: 769-781Crossref PubMed Scopus (1580) Google Scholar) that interacts with glycogen and the polyglucosan found in patients with LD (18Chan E.M. Ackerley C.A. Lohi H. Ianzano L. Cortez M.A. Shannon P. Scherer S.W. Minassian B.A. Hum. Mol. Genet. 2004; 13: 1117-1129Crossref PubMed Scopus (88) Google Scholar, 19Wang J. Stuckey J.A. Wishart M.J. Dixon J.E. J. Biol. Chem. 2002; 277: 2377-2380Abstract Full Text Full Text PDF PubMed Scopus (145) Google Scholar). The second gene, mutated in ∼30% of LD patients is EPM2B (NHLRC1), which encodes malin, an E3 ubiquitin ligase (20Chan E.M. Young E.J. Ianzano L. Munteanu I. Zhao X. Christopoulos C.C. Avanzini G. Elia M. Ackerley C.A. Jovic N.J. Bohlega S. Andermann E. Rouleau G.A. Delgado-Escueta A.V. Minassian B.A. Scherer S.W. Nat. Genet. 2003; 35: 125-127Crossref PubMed Scopus (267) Google Scholar). Two mouse models of LD have been developed (18Chan E.M. Ackerley C.A. Lohi H. Ianzano L. Cortez M.A. Shannon P. Scherer S.W. Minassian B.A. Hum. Mol. Genet. 2004; 13: 1117-1129Crossref PubMed Scopus (88) Google Scholar, 21Ganesh S. Delgado-Escueta A.V. Sakamoto T. Avila M.R. Machado-Salas J. Hoshii Y. Akagi T. Gomi H. Suzuki T. Amano K. Agarwala K.L. Hasegawa Y. Bai D.S. Ishihara T. Hashikawa T. Itohara S. Cornford E.M. Niki H. Yamakawa K. Hum. Mol. Genet. 2002; 11: 1251-1262Crossref PubMed Scopus (191) Google Scholar). Disruption of the mouse Epm2a gene resulted in viable homozygous null mice that had many, but not all, of the features of the human disease (21Ganesh S. Delgado-Escueta A.V. Sakamoto T. Avila M.R. Machado-Salas J. Hoshii Y. Akagi T. Gomi H. Suzuki T. Amano K. Agarwala K.L. Hasegawa Y. Bai D.S. Ishihara T. Hashikawa T. Itohara S. Cornford E.M. Niki H. Yamakawa K. Hum. Mol. Genet. 2002; 11: 1251-1262Crossref PubMed Scopus (191) Google Scholar). LB begin to appear at 4 months of age, followed by a much more robust accumulation of polyglucosan after 9 months when neurological defects were detectable. The second mouse model utilized transgenic overexpression of a dominant negative form of laforin generated by mutating the catalytic Cys266 to Ser (18Chan E.M. Ackerley C.A. Lohi H. Ianzano L. Cortez M.A. Shannon P. Scherer S.W. Minassian B.A. Hum. Mol. Genet. 2004; 13: 1117-1129Crossref PubMed Scopus (88) Google Scholar). The mice developed LB in muscle, liver, and neurons, and by immunogold electron microscopy, laforin was shown to be in the proximity of the polyglucosan deposits. Since the identification of laforin and malin, significant effort has been directed at identifying their physiological targets in an attempt to define the molecular basis for the disease. Several potential targets for malin have been proposed, including laforin (22Gentry M.S. Worby C.A. Dixon J.E. Proc. Natl. Acad. Sci. U. S. A. 2005; 102: 8501-8506Crossref PubMed Scopus (195) Google Scholar), glycogen synthase (GS) (23Vilchez D. Ros S. Cifuentes D. Pujadas L. Valles J. Garcia-Fojeda B. Criado-Garcia O. Fernandez-Sanchez E. Medrano-Fernandez I. Dominguez J. Garcia-Rocha M. Soriano E. Rodriguez de Cordoba S. Guinovart J.J. Nat. Neurosci. 2007; 10: 1407-1413Crossref PubMed Scopus (277) Google Scholar), the glycogen debranching enzyme, amylo-1,6-glucosidase,4-α-glucanotransferase (AGL) (24Cheng A. Zhang M. Gentry M.S. Worby C.A. Dixon J.E. Saltiel A.R. Gene Dev. 2007; 21: 2399-2409Crossref PubMed Scopus (81) Google Scholar), and the type 1 phosphatase regulatory subunit protein targeting to glycogen (PTG) (23Vilchez D. Ros S. Cifuentes D. Pujadas L. Valles J. Garcia-Fojeda B. Criado-Garcia O. Fernandez-Sanchez E. Medrano-Fernandez I. Dominguez J. Garcia-Rocha M. Soriano E. Rodriguez de Cordoba S. Guinovart J.J. Nat. Neurosci. 2007; 10: 1407-1413Crossref PubMed Scopus (277) Google Scholar, 25Worby C.A. Gentry M.S. Dixon J.E. J. Biol. Chem. 2008; 283: 4069-4076Abstract Full Text Full Text PDF PubMed Scopus (126) Google Scholar) (see “Discussion”). Candidate substrates for laforin have been more elusive. We recently demonstrated that in vitro laforin could release the covalent phosphate present in glycogen (26Tagliabracci V.S. Turnbull J. Wang W. Girard J.M. Zhao X. Skurat A.V. Delgado-Escueta A.V. Minassian B.A. Depaoli-Roach A.A. Roach P.J. Proc. Natl. Acad. Sci. U. S. A. 2007; 104: 19262-19266Crossref PubMed Scopus (163) Google Scholar). Furthermore, we showed that glycogen isolated from Epm2a–/– mice contained elevated levels of covalent phosphate, supporting the argument that laforin acts as a glycogen phosphatase in vivo. In the present study, we have extended this work to demonstrate that the absence of laforin leads to hyperphosphorylated glycogen that has abnormal physical properties and that over time develops into a poorly branched, insoluble polysaccharide that tends to aggregate, paralleling the formation of LB in LD. Chemicals and Reagents—Potato amylopectin, malachite green oxalate, and α-amylase (Bacillus species) were from Sigma-Aldrich. Amyloglucosidase (Aspergillus niger) was from Fluka. Anti-GS antibody was from Cell Signaling Technology; anti-laforin and anti-branching enzyme antibodies were from Abnova; anti-glyceraldehyde-3-phosphate dehydrogenase antibody was from Biodesign; anti-AGL antibody was from Abgent; anti-glycogenin-1 antibody was as previously described (27Skurat A.V. Cao Y. Roach P.J. J. Biol. Chem. 1993; 268: 14701-14707Abstract Full Text PDF PubMed Google Scholar); and anti-PTG antibody was a generous gift from Dr. Alan Salteil (University of Michigan, Ann Arbor, MI). Animals—The Epm2a–/– mice have been described (21Ganesh S. Delgado-Escueta A.V. Sakamoto T. Avila M.R. Machado-Salas J. Hoshii Y. Akagi T. Gomi H. Suzuki T. Amano K. Agarwala K.L. Hasegawa Y. Bai D.S. Ishihara T. Hashikawa T. Itohara S. Cornford E.M. Niki H. Yamakawa K. Hum. Mol. Genet. 2002; 11: 1251-1262Crossref PubMed Scopus (191) Google Scholar). Animals 3 months or 9–12 months of age were killed by cervical dislocation, the heads were decapitated directly into liquid nitrogen, and the other tissues were rapidly excised, immersed in liquid nitrogen, and stored at –80 °C until use. Glycogen Purification—Glycogen for use in branching assays, electron microscopy, ethanol solubility assays, and covalent phosphate determination was purified as described previously (26Tagliabracci V.S. Turnbull J. Wang W. Girard J.M. Zhao X. Skurat A.V. Delgado-Escueta A.V. Minassian B.A. Depaoli-Roach A.A. Roach P.J. Proc. Natl. Acad. Sci. U. S. A. 2007; 104: 19262-19266Crossref PubMed Scopus (163) Google Scholar). Because of the lack of water solubility of the 9–12-month-old Epm2a–/– glycogen samples, both knock-out and wild type samples were boiled for 3–5 min in a water bath to fully redissolve the glycogen pellets. 6.7 mm LiCl was used to aid the precipitation of glycogen in ethanol after dialysis. Glycogen Determination—Glycogen was quantitated in total tissue or in the low speed supernatant (LSS) and the low speed pellet (LSP) after low speed centrifugation (8000 × g) by measuring glucose equivalents after digestion with amyloglucosidase (28Bergmeyer H.U. Methods of Enzymatic Analysis. 2nd English Ed. Academic Press, New York1974: 1196-1201Google Scholar, 29Suzuki Y. Lanner C. Kim J.H. Vilardo P.G. Zhang H. Yang J. Cooper L.D. Steele M. Kennedy A. Bock C.B. Scrimgeour A. Lawrence Jr., J.C. DePaoli-Roach A.A. Mol. Cell. Biol. 2001; 21: 2683-2694Crossref PubMed Scopus (132) Google Scholar). Branching Determination—The degree of branching of polysaccharides was analyzed by the protocol of Krisman (30Krisman C.R. Anal. Biochem. 1962; 4: 17-23Crossref PubMed Scopus (421) Google Scholar). Electron Microscopy—Purified glycogen (15–25 μg), whether from whole tissue or after separation by low speed centrifugation and subsequent purification, was spotted on a Formvar-coated grid and allowed to settle for 30–60 s, at which time a drop of NanoVan (Nanoprobes) was added and wicked off 30 s later. The glycogen was viewed with a Technia G12 Biotwin transmission electron microscope (FEI, Hillsboro, OR) equipped with an AMT CCD camera (Advanced Microscopy Techniques, Danvers, MA) at 80 kEV and 150,000× magnification at the Indiana University Electron Microscopy Center. Particle diameters were measured, and a histogram was constructed depicting the size distribution of the particles for different age groups and genotypes. Ethanol Solubility Assay—Purified skeletal muscle glycogen was subjected to treatment with wild type laforin and C266S laforin as described previously (26Tagliabracci V.S. Turnbull J. Wang W. Girard J.M. Zhao X. Skurat A.V. Delgado-Escueta A.V. Minassian B.A. Depaoli-Roach A.A. Roach P.J. Proc. Natl. Acad. Sci. U. S. A. 2007; 104: 19262-19266Crossref PubMed Scopus (163) Google Scholar). The reaction was terminated by boiling for 10 min in a water bath, cooled, and then centrifuged to remove denatured protein. The supernatant was dialyzed overnight against water, and the glycogen was recovered by ethanol precipitation, at which time we noticed that the glycogen treated with active laforin precipitated like wild type glycogen, without the need for added LiCl. This glycogen was also used for electron microscopy studies as described above. Western Blotting and Glycogen Synthase Activity Assays—Skeletal muscle or brain was homogenized in 10 volumes of buffer (31Pederson B.A. Csitkovits A.G. Simon R. Schroeder J.M. Wang W. Skurat A.V. Roach P.J. Biochem. Biophys. Res. Commun. 2003; 305: 826-830Crossref PubMed Scopus (25) Google Scholar) and subjected to low speed centrifugation at 8000 × g for 10 min. The LSP was resuspended in the initial volume of buffer. GS activity in LSS and LSP was measured as previously described (32Thomas J.A. Schlender K.K. Larner J. Anal. Biochem. 1968; 25: 486-499Crossref PubMed Scopus (963) Google Scholar). Comparable amounts of the LSS and the LSP samples were used for Western blot analysis. The remainder of the LSS was subjected to high speed centrifugation at 100,000 × g for 90 min. The high speed pellet was resuspended by sonication in one-fifth of the original homogenization volume and subjected to Western blot analysis for PTG. Glycogenin was detected after incubation of both the LSP and the high speed pellet with or without ∼100 μg/ml α-amylase for 30 min and subjected to Western blot analysis. Statistics—The data are displayed as the means ± S.E. Statistical significance was evaluated using an unpaired Student t test and considered significant at p < 0.05. Glycogen and Glycogen Phosphate Levels Increase with Age in the Absence of Laforin—Previous analyses of 3–4-month-old Epm2a–/– mice (26Tagliabracci V.S. Turnbull J. Wang W. Girard J.M. Zhao X. Skurat A.V. Delgado-Escueta A.V. Minassian B.A. Depaoli-Roach A.A. Roach P.J. Proc. Natl. Acad. Sci. U. S. A. 2007; 104: 19262-19266Crossref PubMed Scopus (163) Google Scholar) revealed no major changes in glycogen level or glycogen metabolizing enzyme activities but a clear increase in the glycogen phosphate content. However, LD is a progressive disorder, and in the Epm2a–/– mice neurological symptoms were only apparent in older, 9-month-old animals (21Ganesh S. Delgado-Escueta A.V. Sakamoto T. Avila M.R. Machado-Salas J. Hoshii Y. Akagi T. Gomi H. Suzuki T. Amano K. Agarwala K.L. Hasegawa Y. Bai D.S. Ishihara T. Hashikawa T. Itohara S. Cornford E.M. Niki H. Yamakawa K. Hum. Mol. Genet. 2002; 11: 1251-1262Crossref PubMed Scopus (191) Google Scholar). We therefore compared young animals with mice 9–12 months of age. Analysis of muscle glycogen isolated from 3-month-old animals reproduced our earlier findings (26Tagliabracci V.S. Turnbull J. Wang W. Girard J.M. Zhao X. Skurat A.V. Delgado-Escueta A.V. Minassian B.A. Depaoli-Roach A.A. Roach P.J. Proc. Natl. Acad. Sci. U. S. A. 2007; 104: 19262-19266Crossref PubMed Scopus (163) Google Scholar), with no difference in glycogen levels between wild type and Epm2a–/– animals and an ∼4-fold increase in the phosphate content (Fig. 1, A and B). In the older animals, there was a 3-fold elevation in total glycogen content and a further increase in phosphate to approximately six times wild type. Phosphate content of glycogen was unchanged as wild type animals aged from 3 to 9–12 months. Brain glycogen levels were already increased at 3 months and progressed to a 5-fold elevation in the old animals (Fig. 1C). Because of the much lower amounts of glycogen recoverable from brain, it was not feasible to measure phosphate content. The greater sensitivity of brain glycogen accumulation to the absence of laforin could reflect differences in brain metabolism and explain the more serious consequences in this tissue. Age-dependent Changes in Chemical and Physical Properties of Glycogen in Epm2a–/– Mice—LD is characterized by poorly branched glycogen-like polymers in the LB (12Ganesh S. Puri R. Singh S. Mittal S. Dubey D. J. Hum. Genet. 2006; 51: 1-8Crossref PubMed Scopus (117) Google Scholar, 13Chan E.M. Andrade D.M. Franceschetti S. Minassian B. Adv. Neurol. 2005; 95: 47-57PubMed Google Scholar, 14Delgado-Escueta A.V. Curr. Neurol. Neurosci. Rep. 2007; 7: 428-433Crossref PubMed Scopus (78) Google Scholar). We therefore monitored the degree of branching of glycogen purified from laforin-deficient mice by recording the visible absorption spectrum in the presence of iodine. Iodine intercalates into polysaccharide helices to give a characteristic spectrum whose absorption maximum is influenced by the degree of branching and hence the length of uninterrupted helical segments (30Krisman C.R. Anal. Biochem. 1962; 4: 17-23Crossref PubMed Scopus (421) Google Scholar). This technique readily distinguishes wild type mouse glycogen from the amylopectin of plant starch (average branching one in ∼30 glucoses) whose absorption maximum is at a much higher wavelength (Fig. 2A). Mammalian glycogen typically has branches every ∼12 glucose residues. The iodine spectra of muscle glycogen from 3- or 9–12-month-old wild type mice are superimposable (triangles and diamonds), indicating no change in branching with age in normal animals. Glycogen from 3-month-old Epm2a–/– mice had an iodine spectrum slightly but significantly shifted to longer wavelength (Fig. 2, A and B). However, the corresponding spectrum for 9–12-month-old knock-out animals was greatly altered, approaching that of amylopectin and indicative of a substantial reduction in the number of branches. Removal of the phosphate by laforin treatment did not alter the iodine spectrum (data not shown). Poorly branched glucose polymers, like amylopectin, are less soluble in water than more branched polysaccharides. Indeed, during isolation (see “Experimental Procedures”), it was more difficult to dissolve precipitated glycogen from the 9–12-month-old Epm2a–/– mice than glycogen from any of the other mouse groups analyzed. Likewise, after storage of 9–12-month-old mouse glycogen solutions at –20 °C, thawing resulted in a precipitate that required heating to redissolve. Similar treatment of glycogen from wild type or 3-month-old Epm2a–/– mice did not result in precipitated material. Polysaccharides like glycogen are insoluble in ethanol, a property that is useful for their separation from ethanol soluble constituents. Thus, our normal isolation protocol for glycogen from tissues (see “Experimental Procedures”) utilizes precipitation in 66% (v/v) ethanol. We observed that glycogen from Epm2a–/– mice, regardless of age, was much more soluble in 66% ethanol than wild type glycogen and did not come readily out of solution (data not shown). The addition of traces of a salt, like LiCl, has traditionally been used to ensure complete precipitation of polysaccharides from ethanol, and this was effective for the glycogen from both the 3-month-old and the 9–12-month-old Epm2a–/– mice. Glycogen from 9–12-month-old Epm2a–/– mice was exposed to either the catalytically inactive C266S mutant laforin or wild type laforin followed by the addition of ethanol to 66%. Treatment with inactive laforin had no effect, with the glycogen remaining in solution (Fig. 2C, tube i). Treatment with active laforin, which removes most of the phosphate, resulted in a polysaccharide that precipitated normally (Fig. 2C, tube ii). The addition of LiCl to both samples hastened precipitation of glycogen, although the phosphorylated glycogen formed more flocculent material that, under gravity, was pelleted at a slower rate (Fig. 2C, tubes iii and iv). The visible material was not salt because it was absent in the control sample lacking any glycogen (Fig. 2C, tube v). Age-dependent Changes in Glycogen Structure in Epm2a–/– Mice—The high molecular weight of glycogen makes it large enough to be analyzed by electron microscopy, and the literature contains numerous examples of such visualization (33Drochmans P. J. Ultra Res. 1962; 6: 141-163Crossref PubMed Scopus (221) Google Scholar, 34Rybicka K.K. Tissue Cell. 1996; 28: 253-265Crossref PubMed Scopus (103) Google Scholar). For glycogen in skeletal muscle, the particles have been described as rosettes that have a characteristic granular appearance. Analysis of the glycogen purified from the muscle of wild type mice by transmission electron microscopy indicated particles reminiscent of these structures, whether from old or young animals (Fig. 3, A and B). Glycogen from 3-month-old Epm2a–/– mice had a generally similar appearance, (Fig. 3C). In contrast, glycogen from the 9–12-month-old mice had a strikingly distinct appearance, with a more defined boundary, less granularity, and a more even density (Fig. 3D). Furthermore, many fields had large regions where particles appear to have coalesced or aggregated, either before or during preparation of the grids. The size distribution of the particles was estimated by measuring apparent particle diameter. Glycogen particles from 3-month-old wild type mice had an average diameter of 27.1 nm, in the range reported for native muscle glycogen particles (Fig. 3E) (4Shearer J. Graham T.E. Exerc. Sport Sci. Rev. 2004; 32: 120-126Crossref PubMed Scopus (46) Google Scholar). With age, there appeared to be a shift in the population to a slightly larger size (average 35.1 nm) but retaining the approximately Gaussian distribution seen in the younger animals. Glycogen particles from the 3-month-old knock-out animals were a little smaller (average diameter, 24.2 nm) but still normally distributed. The comparable analysis of the particles from old Epm2a–/– mice was hampered by the presence of the large conglomerates, within which only hints of individual particles were visible. Particles in regions outside of the conglomerates had much greater variability in size, with many particles of diameter well beyond that anticipated for a normal glycogen particle (Fig. 3D). This could be due to a different density of the polysaccharide or more likely an aggregation of individual molecules that cannot be resolved by this analysis. The glycogen purified from the old knock-out mice was characterized by having the highest phosphate content and much reduced branching. This glycogen was subjected to treatment with laforin to remove phosphate or the catalytically inactive laforin mutant C266S. After removing protein and buffer, the glycogen was analyzed by electron microscopy. Glycogen from the control sample had an appearance comparable with that shown in Fig. 3D (Fig. 4A). Treatment with active laforin caused a significant change in appearance, leading to a population of particles with much reduced size and individual appearance tending more to that of wild type. However, the granularity of the normal particles was not completely restored. Because the removal of phosphate by laforin did not affect branching (previous section), we conclude that the phosphate is primarily responsible for the abnormal morphology of the glycogen from the 9–12-month-old animals, in particular the presence of larger particles and aggregates. With the possibility that abnormal glycogen phosphorylation might affect the aggregation of glycogen, we wondered whether our standard sample preparation for enzyme analysis, in which low speed centrifugation removes gross cellular debris from tissue extracts, may have removed some of the glycogen. This separation is not an issue for samples directly processed for measurement of glycogen. We found that in extracts from the muscle of 9–12-month-old Epm2a–/– mice, ∼60% of the glycogen was recovered in the LSP, compared with ∼25% for wild type muscle (Fig. 5A). In brain extracts, over 75% of the glycogen was in the pellet from knock-out mouse extracts (Fig. 5B). The increased glycogen in the old knock-out mice was associated with the LSP because the glycogen level in the LSS was the same as wild type. Glycogen purified from the LSP and LSS fractions of muscle extracts of the 9–12-month-old Epm2a–/– animals was analyzed by electron microscopy (Fig. 5, C and D). During this procedure, it was noted that the LSP glycogen was significantly harder to dissolve in water than the LSS fraction. The pellet fraction was highly enriched for the morphologically abnormal structures described in Fig. 3, whereas in the supernatant the particles were predominantly of more wild type appearance. The glycogen in the pellet also had significantly higher glycogen phosphate content (Fig. 5E). Th

Epilepsy afflicts 1% of humans and 5% of dogs. We report a canine epilepsy mutation and evidence for the existence of repeat-expansion disease outside humans. A canid-specific unstable dodecamer repeat in the Epm2b (Nhlrc1) gene recurrently expands, causing a fatal epilepsy and contributing to the high incidence of canine epilepsy. Tracing the repeat origins revealed two successive events, starting 50 million years ago, unique to canid evolution. A genetic test, presented here, will allow carrier and presymptomatic diagnosis and disease eradication. Clinicopathologic characterization establishes affected animals as a model for Lafora disease, the most severe teenage-onset human epilepsy.

Lafora progressive myoclonus epilepsy, caused by defective laforin or malin, insidiously present in normal teenagers with cognitive decline, followed by rapidly intractable epilepsy, dementia and death. Pathology reveals neurodegeneration with neurofibrillary tangle formation and Lafora bodies (LBs). LBs are deposits of starch-like polyglucosans, insufficiently branched and hence insoluble glycogen molecules resulting from glycogen synthase (GS) overactivity relative to glycogen branching enzyme activity. We previously made the unexpected observation that laforin, in the absence of which polyglucosans accumulate, specifically binds polyglucosans. This suggested that laforin's role is to detect polyglucosan appearances during glycogen synthesis and to initiate mechanisms to downregulate GS. Glycogen synthase kinase 3 (GSK3) is the principal inhibitor of GS. Dephosphorylation of GSK3 at Ser 9 activates GSK3 to inhibit GS through phosphorylation at multiple sites. Glucose-6-phosphate is a potent allosteric activator of GS. Glucose-6-phosphate levels are high when the amount of glucose increases and its activation of GS overrides any phospho-inhibition. Here, we show that laforin is a GSK3 Ser 9 phosphatase, and therefore capable of inactivating GS through GSK3. We also show that laforin interacts with malin and that malin is an E3 ubiquitin ligase that binds GS. We propose that laforin, in response to appearance of polyglucosans, directs two negative feedback pathways: polyglucosan-laforin-GSK3-GS to inhibit GS activity and polyglucosan-laforin-malin-GS to remove GS through proteasomal degradation.

In a mouse mutagenesis screen, we isolated a mutant, Myshkin ( Myk ), with autosomal dominant complex partial and secondarily generalized seizures, a greatly reduced threshold for hippocampal seizures in vitro, posttetanic hyperexcitability of the CA3-CA1 hippocampal pathway, and neuronal degeneration in the hippocampus. Positional cloning and functional analysis revealed that Myk /+ mice carry a mutation (I810N) which renders the normally expressed Na + ,K + -ATPase α3 isoform inactive. Total Na + ,K + -ATPase activity was reduced by 42% in Myk /+ brain. The epilepsy in Myk /+ mice and in vitro hyperexcitability could be prevented by delivery of additional copies of wild-type Na + ,K + -ATPase α3 by transgenesis, which also rescued Na + ,K + -ATPase activity. Our findings reveal the functional significance of the Na + ,K + -ATPase α3 isoform in the control of epileptiform activity and seizure behavior.

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.

Lafora disease (LD) is an autosomal recessive progressive myoclonus epilepsy due to mutations in the EPM2A (laforin) and EPM2B (malin) genes, with no substantial genotype-phenotype differences between the two. Founder effects and recurrent mutations are common, and mostly isolated to specific ethnic groups and/or geographical locations. Pathologically, LD is characterized by distinctive polyglucosans, which are formations of abnormal glycogen. Polyglucosans, or Lafora bodies (LB) are typically found in the brain, periportal hepatocytes of the liver, skeletal and cardiac myocytes, and in the eccrine duct and apocrine myoepithelial cells of sweat glands. Mouse models of the disease and other naturally occurring animal models have similar pathology and phenotype. Hypotheses of LB formation remain controversial, with compelling evidence and caveats for each hypothesis. However, it is clear that the laforin and malin functions regulating glycogen structure are key. With the exception of a few missense mutations LD is clinically homogeneous, with onset in adolescence. Symptoms begin with seizures, and neurological decline follows soon after. The disease course is progressive and fatal, with death occurring within 10 years of onset. Antiepileptic drugs are mostly non-effective, with none having a major influence on the progression of cognitive and behavioral symptoms. Diagnosis and genetic counseling are important aspects of LD, and social support is essential in disease management. Future therapeutics for LD will revolve around the pathogenesics of the disease. Currently, efforts at identifying compounds or approaches to reduce brain glycogen synthesis appear to be highly promising.

The neuronal ceroid lipofuscinoses (NCLs), the most common neurodegenerative disorders of childhood, are characterized by the accumulation of autofluorescent storage material mainly in neurons. Although clinically rather uniform, variant late-infantile onset NCL (vLINCL) is genetically heterogeneous with four major underlying genes identified so far. We evaluated the genetic background underlying vLINCL in 119 patients, and specifically analysed the recently reported CLN7/MFSD8 gene for mutations in 80 patients. Clinical data were collected from the CLN7/MFSD8 mutation positive patients. Eight novel CLN7/MFSD8 mutations and seven novel mutations in the CLN1/PPT1, CLN2/TPP1, CLN5, CLN6 and CLN8 genes were identified in patients of various ethnic origins. A significant group of Roma patients originating from the former Czechoslovakia was shown to bear the c.881C>A (p.Thr294Lys) mutation in CLN7/MFSD8, possibly due to a founder effect. With one exception, the CLN7/MFSD8 mutation positive patients present a phenotype indistinguishable from the other vLINCL forms. In one patient with an in-frame amino acid substitution mutation in CLN7/MFSD8, the disease onset was later and the disease course less aggressive than in variant late-infantile NCL. Our findings raise the total number of CLN7/MFSD8 mutations to 14 with the majority of families having private mutations. Our study confirms that CLN7/MFSD8 defects are not restricted to the Turkish population, as initially anticipated, but are a relatively common cause of NCL in different populations. CLN7/MFSD8 should be considered a diagnostic alternative not only in variant late-infantile but also later onset NCL forms with a more protracted disease course. A significant number of NCL patients in Turkey exist, in which the underlying genetic defect remains to be determined.

Lafora disease is a severe, autosomal recessive, progressive myoclonus epilepsy. The disease usually manifests in previously healthy adolescents, and death commonly occurs within 10 years of symptom onset. Lafora disease is caused by loss-of-function mutations in EPM2A or NHLRC1, which encode laforin and malin, respectively. The absence of either protein results in poorly branched, hyperphosphorylated glycogen, which precipitates, aggregates and accumulates into Lafora bodies. Evidence from Lafora disease genetic mouse models indicates that these intracellular inclusions are a principal driver of neurodegeneration and neurological disease. The integration of current knowledge on the function of laforin–malin as an interacting complex suggests that laforin recruits malin to parts of glycogen molecules where overly long glucose chains are forming, so as to counteract further chain extension. In the absence of either laforin or malin function, long glucose chains in specific glycogen molecules extrude water, form double helices and drive precipitation of those molecules, which over time accumulate into Lafora bodies. In this article, we review the genetic, clinical, pathological and molecular aspects of Lafora disease. We also discuss traditional antiseizure treatments for this condition, as well as exciting therapeutic advances based on the downregulation of brain glycogen synthesis and disease gene replacement. Lafora disease is an autosomal recessive, progressive myoclonus epilepsy caused by loss of function of laforin or malin, leading to impaired glycogen metabolism. The authors review the clinical and molecular features of Lafora disease and discuss current and emerging treatment options.

Abstract The neuronal ceroid lipofuscinoses (NCL) are neurodegenerative conditions that associate cognitive decline, progressive cerebellar atrophy, retinopathy, and myoclonic epilepsy. NCL result from the excessive accumulation of neuronal and extraneuronal lipopigments, despite having diverse underlying biochemical aetiologies. Here we review the clinical presentation, pathophysiology and genetics of these conditions as well as the approach to diagnosis and management.

The transition from a pediatric to adult health care system is challenging for many youths with epilepsy and their families. Recently, the Ministry of Health and Long-Term Care of the Province of Ontario, Canada, created a transition working group (TWG) to develop recommendations for the transition process for patients with epilepsy in the Province of Ontario. Herein we present an executive summary of this work. The TWG was composed of a multidisciplinary group of pediatric and adult epileptologists, psychiatrists, and family doctors from academia and from the community; neurologists from the community; nurses and social workers from pediatric and adult epilepsy programs; adolescent medicine physician specialists; a team of physicians, nurses, and social workers dedicated to patients with complex care needs; a lawyer; an occupational therapist; representatives from community epilepsy agencies; patients with epilepsy; parents of patients with epilepsy and severe intellectual disability; and project managers. Three main areas were addressed: (1) Diagnosis and Management of Seizures; 2) Mental Health and Psychosocial Needs; and 3) Financial, Community, and Legal Supports. Although there are no systematic studies on the outcomes of transition programs, the impressions of the TWG are as follows. Teenagers at risk of poor transition should be identified early. The care coordination between pediatric and adult neurologists and other specialists should begin before the actual transfer. The transition period is the ideal time to rethink the diagnosis and repeat diagnostic testing where indicated (particularly genetic testing, which now can uncover more etiologies than when patients were initially evaluated many years ago). Some screening tests should be repeated after the move to the adult system. The seven steps proposed herein may facilitate transition, thereby promoting uninterrupted and adequate care for youth with epilepsy leaving the pediatric system.

Lafora disease (LD) is a fatal intractable adolescence-onset progressive myoclonus epilepsy. Recently, two single-case studies reported drastic reductions in seizures and myoclonus with the AMPA antagonist perampanel and improved activities of daily living. We proceeded to study the effect of perampanel on 10 patients with genetically confirmed LD with disease duration ranging from 2 to 27 years. Open-label perampanel was added to ongoing medications to a mean dose of 6.7 mg/day. Seizures, myoclonus, functional disability, and cognition scores were measured for the third and ninth months following initiation and compared with those of the month prior to the start of therapy. Three patients withdrew because of inefficacy or side effects. Four had significant reduction in seizures of greater than 74% from baseline. Seven had major improvement in myoclonus with group-adjusted sum score of myoclonus intensity reduced from 7.01 at baseline to 5.67 and 5.18 at 3 and 9 months, respectively. There was no significant improvement in disability and cognition. While not universal, perampanel adjunctive therapy appears to confer particular benefit not commonly seen with other antiepileptic drugs on seizures and myoclonus in LD. Improvement in the extremely disabling myoclonus of LD is a major benefit in this devastating disease.

Lafora disease (LD) is a fatal, autosomal recessive, glycogen-storage disorder that manifests as severe epilepsy. LD results from mutations in the gene encoding either the glycogen phosphatase laforin or the E3 ubiquitin ligase malin. Individuals with LD develop cytoplasmic, aberrant glycogen inclusions in nearly all tissues that more closely resemble plant starch than human glycogen. This Minireview discusses the unique window into glycogen metabolism that LD research offers. It also highlights recent discoveries, including that glycogen contains covalently bound phosphate and that neurons synthesize glycogen and express both glycogen synthase and glycogen phosphorylase.

Epilepsy includes a number of medical conditions with recurrent seizures as common denominator. The large number of different syndromes and seizure types as well as the highly variable inter-individual response to the therapies makes management of this condition often challenging. In the last two decades, a genetic etiology has been revealed in more than half of all epilepsies and single gene defects in ion channels or neurotransmitter receptors have been associated with most inherited forms of epilepsy, including some focal and lesional forms as well as specific epileptic developmental encephalopathies. Several genetic tests are now available, including targeted assays up to revolutionary tools that have made sequencing of all coding (whole exome) and non-coding (whole genome) regions of the human genome possible. These recent technological advances have also driven genetic discovery in epilepsy and increased our understanding of the molecular mechanisms of many epileptic disorders, eventually providing targets for precision medicine in some syndromes, such as Dravet syndrome, pyroxidine-dependent epilepsy, and glucose transporter 1 deficiency. However, these examples represent a relatively small subset of all types of epilepsy, and to date, precision medicine in epilepsy has primarily focused on seizure control, and other clinical aspects, such as neurodevelopmental and neuropsychiatric comorbidities, have yet been possible to address. We herein summarize the most recent advances in genetic testing and provide up-to-date approaches for the choice of the correct test for some epileptic disorders and tailored treatments that are already applicable in some monogenic epilepsies. In the next years, the most probably scenario is that epilepsy treatment will be very different from the currently almost empirical approach, eventually with a "precision medicine" approach applicable on a large scale.

Lafora's disease is a progressive myoclonus epilepsy with pathognomonic inclusions (polyglucosan bodies) caused by mutations in the EPM2A gene. EPM2A codes for laforin, a protein with unknown function. Mutations have been reported in the last three of the gene's exons. To date, the first exon has not been determined conclusively. It has been predicted based on genomic DNA sequence analysis including comparison with the mouse homologue.1) To detect new mutations in exon 1 and establish the role of this exon in Lafora's disease. 2) To generate hypotheses about the biological function of laforin based on bioinformatic analyses.1) PCR conditions and components were refined to allow amplification and sequencing of the first exon of EPM2A. 2) Extensive bioinformatic analyses of the primary structure of laforin were completed.1) Seven new mutations were identified in the putative exon 1. 2) Laforin is predicted not to localize to the cell membrane or any of the organelles. It contains all components of the catalytic active site of the family of dual-specificity phosphatases. It contains a sequence predicted to encode a carbohydrate binding domain (coded by exon 1) and two putative glucohydrolase catalytic sites.The identification of mutations in exon 1 of EPM2A establishes its role in the pathogenesis of Lafora's disease. The presence of potential carbohydrate binding and cleaving domains suggest a role for laforin in the prevention of accumulation of polyglucosans in healthy neurons.

Abstract 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

Autosomal recessively inherited progressive myoclonus epilepsies (PMEs) include Lafora disease, Unverricht-Lundborg disease, the neuronal ceroid lipofuscinoses, type I sialidosis (cherry-red spot myoclonus), action myoclonus-renal failure syndrome, and type III Gaucher disease. Almost all the autosomal recessively inherited PMEs are lysosomal diseases, with the exception of Lafora disease in which neither the accumulating material nor the gene products are in lysosomes. Progress in identifying the causative defects of PME is near-complete. Much work lies ahead to resolve the pathobiology and neurophysiology of this group of devastating disorders.

One quadrillion synapses are laid in the first two years of postnatal construction of the human brain, which are then pruned until age 10 to 500 trillion synapses composing the final network. Genetic epilepsies are the most common neurological diseases with onset during pruning, affecting 0.5% of 2-10-year-old children, and these epilepsies are often characterized by spontaneous remission. We previously described a remitting epilepsy in the Lagotto romagnolo canine breed. Here, we identify the gene defect and affected neurochemical pathway. We reconstructed a large Lagotto pedigree of around 34 affected animals. Using genome-wide association in 11 discordant sib-pairs from this pedigree, we mapped the disease locus to a 1.7 Mb region of homozygosity in chromosome 3 where we identified a protein-truncating mutation in the Lgi2 gene, a homologue of the human epilepsy gene LGI1. We show that LGI2, like LGI1, is neuronally secreted and acts on metalloproteinase-lacking members of the ADAM family of neuronal receptors, which function in synapse remodeling, and that LGI2 truncation, like LGI1 truncations, prevents secretion and ADAM interaction. The resulting epilepsy onsets at around seven weeks (equivalent to human two years), and remits by four months (human eight years), versus onset after age eight in the majority of human patients with LGI1 mutations. Finally, we show that Lgi2 is expressed highly in the immediate post-natal period until halfway through pruning, unlike Lgi1, which is expressed in the latter part of pruning and beyond. LGI2 acts at least in part through the same ADAM receptors as LGI1, but earlier, ensuring electrical stability (absence of epilepsy) during pruning years, preceding this same function performed by LGI1 in later years. LGI2 should be considered a candidate gene for common remitting childhood epilepsies, and LGI2-to-LGI1 transition for mechanisms of childhood epilepsy remission.

Endoplasmic reticulum (ER) stress has been implicated in glucose-induced beta cell dysfunction. However, its causal role has not been established in vivo. Our objective was to determine the causal role of ER stress and its link to oxidative stress in glucose-induced beta cell dysfunction in vivo. Healthy Wistar rats were infused i.v. with glucose for 48 h to achieve 20 mmol/l hyperglycaemia with or without the co-infusion of the superoxide dismutase mimetic tempol (TPO), or the chemical chaperones 4-phenylbutyrate (PBA) or tauroursodeoxycholic acid (TUDCA). This was followed by assessment of beta cell function and measurement of ER stress markers and superoxide in islets. Glucose infusion for 48 h increased mitochondrial superoxide and ER stress markers and impaired beta cell function. Co-infusion of TPO, which we previously found to reduce mitochondrial superoxide and prevent glucose-induced beta cell dysfunction, reduced ER stress markers. Similar to findings with TPO, co-infusion of PBA, which decreases mitochondrial superoxide, prevented glucose-induced beta cell dysfunction in isolated islets. TUDCA was also effective. Also similar to findings with TPO, PBA prevented beta cell dysfunction during hyperglycaemic clamps in vivo and after hyperglycaemia (15 mmol/l) for 96 h. Here, we causally implicate ER stress in hyperglycaemia-induced beta cell dysfunction in vivo. We show that: (1) there is a positive feedback cycle between oxidative stress and ER stress in glucose-induced beta cell dysfunction, which involves mitochondrial superoxide; and (2) this cycle can be interrupted by superoxide dismutase mimetics as well as chemical chaperones, which are of potential interest to preserve beta cell function in type 2 diabetes.

Laforin or malin deficiency causes Lafora disease, characterized by altered glycogen metabolism and teenage-onset neurodegeneration with intractable and invariably fatal epilepsy. Plant starches possess small amounts of metabolically essential monophosphate esters. Glycogen contains similar phosphate amounts, which are thought to originate from a glycogen synthase error side reaction and therefore lack any specific function. Glycogen is also believed to lack monophosphates at glucosyl carbon C6, an essential phosphorylation site in plant starch metabolism. We now show that glycogen phosphorylation is not due to a glycogen synthase side reaction, that C6 is a major glycogen phosphorylation site, and that C6 monophosphates predominate near centers of glycogen molecules and positively correlate with glycogen chain lengths. Laforin or malin deficiency causes C6 hyperphosphorylation, which results in malformed long-chained glycogen that accumulates in many tissues, causing neurodegeneration in brain. Our work advances the understanding of Lafora disease pathogenesis and suggests that glycogen phosphorylation has important metabolic function.

Lafora disease (LD) is a fatal progressive myoclonus epilepsy characterized neuropathologically by aggregates of abnormally structured glycogen and proteins (Lafora bodies [LBs]), and neurodegeneration. Whether LBs could be prevented by inhibiting glycogen synthesis and whether they are pathogenic remain uncertain. We genetically eliminated brain glycogen synthesis in LD mice. This resulted in long-term prevention of LB formation, neurodegeneration, and seizure susceptibility. This study establishes that glycogen synthesis is requisite for LB formation and that LBs are pathogenic. It opens a therapeutic window for potential treatments in LD with known and future small molecule inhibitors of glycogen synthesis.

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. Ann Neurol 2014;75:442–446

The most common progressive myoclonus epilepsies are the late infantile and late infantile-variant neuronal ceroid lipofuscinoses (onset before the age of 6 years), Unverricht-Lundborg disease (onset after the age of 6 years) and Lafora disease. Lafora disease is a distinct disorder with uniform course: onset in teenage years, followed by progressively worsening myoclonus, seizures, visual hallucinations and cognitive decline, leading to a vegetative state in status myoclonicus and death within 10 years. Biopsy reveals Lafora bodies, which are pathognomonic and not seen with any other progressive myoclonus epilepsies. Lafora bodies are aggregates of polyglucosans, poorly constructed glycogen molecules with inordinately long strands that render them insoluble. Lafora disease is caused by mutations in the EPM2A or EPM2B genes, encoding the laforin phosphatase and the malin ubiquitin ligase, respectively, two cytoplasmically active enzymes that regulate glycogen construction, ensuring symmetric expansion into a spherical shape, essential to its solubility. In this work, we report a new progressive myoclonus epilepsy associated with Lafora bodies, early-onset Lafora body disease, map its locus to chromosome 4q21.21, identify its gene and mutation and characterize the relationship of its gene product with laforin and malin. Early-onset Lafora body disease presents early, at 5 years, with dysarthria, myoclonus and ataxia. The combination of early-onset and early dysarthria strongly suggests late infantile-variant neuronal ceroid lipofuscinosis, not Lafora disease. Pathology reveals no ceroid lipofuscinosis, but Lafora bodies. The subsequent course is a typical progressive myoclonus epilepsy, though much more protracted than any infantile neuronal ceroid lipofuscinosis, or Lafora disease, patients living into the fourth decade. The mutation, c.781T>C (Phe261Leu), is in a gene of unknown function, PRDM8. We show that the PRDM8 protein interacts with laforin and malin and causes translocation of the two proteins to the nucleus. We find that Phe261Leu-PRDM8 results in excessive sequestration of laforin and malin in the nucleus and that it therefore likely represents a gain-of-function mutation that leads to an effective deficiency of cytoplasmic laforin and malin. We have identified a new progressive myoclonus epilepsy with Lafora bodies, early-onset Lafora body disease, 101 years after Lafora disease was first described. The results to date suggest that PRDM8, the early-onset Lafora body disease protein, regulates the cytoplasmic quantities of the Lafora disease enzymes.

Abstract Lafora disease ( LD ) is a fatal progressive epilepsy essentially caused by loss‐of‐function mutations in the glycogen phosphatase laforin or the ubiquitin E3 ligase malin. Glycogen in LD is hyperphosphorylated and poorly hydrosoluble. It precipitates and accumulates into neurotoxic Lafora bodies ( LB s). The leading LD hypothesis that hyperphosphorylation causes the insolubility was recently challenged by the observation that phosphatase‐inactive laforin rescues the laforin‐deficient LD mouse model, apparently through correction of a general autophagy impairment. We were for the first time able to quantify brain glycogen phosphate. We also measured glycogen content and chain lengths, LB s, and autophagy markers in several laforin‐ or malin‐deficient mouse lines expressing phosphatase‐inactive laforin. We find that: (i) in laforin‐deficient mice, phosphatase‐inactive laforin corrects glycogen chain lengths, and not hyperphosphorylation, which leads to correction of glycogen amounts and prevention of LB s; (ii) in malin‐deficient mice, phosphatase‐inactive laforin confers no correction; (iii) general impairment of autophagy is not necessary in LD . We conclude that laforin's principle function is to control glycogen chain lengths, in a malin‐dependent fashion, and that loss of this control underlies LD .

Developmental epileptic encephalopathies (DEEs) are genetically heterogeneous severe childhood-onset epilepsies with developmental delay or cognitive deficits. In this study, we explored the pathogenic mechanisms of DEE-associated de novo mutations in the CACNA1A gene.We studied the functional impact of four de novo DEE-associated CACNA1A mutations, including the previously described p.A713T variant and three novel variants (p.V1396M, p.G230V, and p.I1357S). Mutant cDNAs were expressed in HEK293 cells, and whole-cell voltage-clamp recordings were conducted to test the impacts on CaV 2.1 channel function. Channel localization and structure were assessed with immunofluorescence microscopy and three-dimensional (3D) modeling.We find that the G230V and I1357S mutations result in loss-of-function effects with reduced whole-cell current densities and decreased channel expression at the cell membrane. By contrast, the A713T and V1396M variants resulted in gain-of-function effects with increased whole-cell currents and facilitated current activation (hyperpolarized shift). The A713T variant also resulted in slower current decay. 3D modeling predicts conformational changes favoring channel opening for A713T and V1396M.Our findings suggest that both gain-of-function and loss-of-function CACNA1A mutations are associated with similarly severe DEEs and that functional validation is required to clarify the underlying molecular mechanisms and to guide therapies.

Progressive Myoclonus Epilepsies (PMEs) are a group of uncommon clinically and genetically heterogeneous disorders characterised by myoclonus, generalized epilepsy, and neurological deterioration, including dementia and ataxia. PMEs may have infancy, childhood, juvenile or adult onset, but usually present in late childhood or adolescence, at variance from epileptic encephalopathies, which start with polymorphic seizures in early infancy. Neurophysiologic recordings are suited to describe faithfully the time course of the shock-like muscle contractions which characterize myoclonus. A combination of positive and negative myoclonus is typical of PMEs. The gene defects for most PMEs (Unverricht-Lundborg disease, Lafora disease, several forms of neuronal ceroid lipofuscinoses, myoclonus epilepsy with ragged-red fibers [MERRF], and type 1 and 2 sialidoses) have been identified. PMEs are uncommon disorders, difficult to diagnose in the absence of extensive experience. Thus, aetiology is undetermined in many patients, despite the advance in molecular medicine. Treatment of PMEs remains essentially symptomaticof seizures and myoclonus, together with palliative, supportive, and rehabilitative measures. The response to therapy may initially be relatively favourable, afterwards however, seizures may become more frequent, and progressive neurologic decline occurs. The prognosis of a PME depends on the specific disease. The history of PMEs revealed that the international collaboration and sharing experience is the right way to proceed. This emerging picture and biological insights will allow us to find ways to provide the patients with meaningful treatment.

Lafora disease (LD, OMIM #254780) is a rare, recessively inherited neurodegenerative disease with adolescent onset, resulting in progressive myoclonus epilepsy which is fatal usually within ten years of symptom onset. The disease is caused by loss-of-function mutations in either of the two genes EPM2A (laforin) or EPM2B (malin). It characteristically involves the accumulation of insoluble glycogen-derived particles, named Lafora bodies (LBs), which are considered neurotoxic and causative of the disease. The pathogenesis of LD is therefore centred on the question of how insoluble LBs emerge from soluble glycogen. Recent data clearly show that an abnormal glycogen chain length distribution, but neither hyperphosphorylation nor impairment of general autophagy, strictly correlates with glycogen accumulation and the presence of LBs. This review summarizes results obtained with patients, mouse models, and cell lines and consolidates apparent paradoxes in the LD literature. Based on the growing body of evidence, it proposes that LD is predominantly caused by an impairment in chain-length regulation affecting only a small proportion of the cellular glycogen. A better grasp of LD pathogenesis will further develop our understanding of glycogen metabolism and structure. It will also facilitate the development of clinical interventions that appropriately target the underlying cause of LD.

Glycogen branching enzyme 1 (GBE1) plays an essential role in glycogen biosynthesis by generating α-1,6-glucosidic branches from α-1,4-linked glucose chains, to increase solubility of the glycogen polymer. Mutations in the GBE1 gene lead to the heterogeneous early-onset glycogen storage disorder type IV (GSDIV) or the late-onset adult polyglucosan body disease (APBD). To better understand this essential enzyme, we crystallized human GBE1 in the apo form, and in complex with a tetra- or hepta-saccharide. The GBE1 structure reveals a conserved amylase core that houses the active centre for the branching reaction and harbours almost all GSDIV and APBD mutations. A non-catalytic binding cleft, proximal to the site of the common APBD mutation p.Y329S, was found to bind the tetra- and hepta-saccharides and may represent a higher-affinity site employed to anchor the complex glycogen substrate for the branching reaction. Expression of recombinant GBE1-p.Y329S resulted in drastically reduced protein yield and solubility compared with wild type, suggesting this disease allele causes protein misfolding and may be amenable to small molecule stabilization. To explore this, we generated a structural model of GBE1-p.Y329S and designed peptides ab initio to stabilize the mutation. As proof-of-principle, we evaluated treatment of one tetra-peptide, Leu-Thr-Lys-Glu, in APBD patient cells. We demonstrate intracellular transport of this peptide, its binding and stabilization of GBE1-p.Y329S, and 2-fold increased mutant enzymatic activity compared with untreated patient cells. Together, our data provide the rationale and starting point for the screening of small molecule chaperones, which could become novel therapies for this disease.

Dravet syndrome (DS) is a neurodevelopmental genetic disorder caused by mutations in the SCN1A gene encoding the α subunit of the NaV1.1 voltage-gated sodium channel that controls neuronal action potential firing. The high density of this mutated channel in GABAergic interneurons results in impaired inhibitory neurotransmission and subsequent excessive activation of excitatory neurons. The syndrome is associated with severe childhood epilepsy, autistic behaviors, and sudden unexpected death in epilepsy. Here, we compared the rescue effects of an adeno-associated viral (AAV) vector coding for the multifunctional β1 sodium channel auxiliary subunit (AAV-NaVβ1) with a control vector lacking a transgene. We hypothesized that overexpression of NaVβ1 would facilitate the function of residual voltage-gated channels and improve the DS phenotype in the Scn1a+/− mouse model of DS. AAV-NaVβ1 was injected into the cerebral spinal fluid of neonatal Scn1a+/− mice. In untreated control Scn1a+/− mice, females showed a higher degree of mortality than males. Compared with Scn1a+/− control mice, AAV-NaVβ1-treated Scn1a+/− mice displayed increased survival, an outcome that was more pronounced in females than males. In contrast, behavioral analysis revealed that male, but not female, Scn1a+/− mice displayed motor hyperactivity, and abnormal performance on tests of fear and anxiety and learning and memory. Male Scn1a+/− mice treated with AAV-NaVβ1 showed reduced spontaneous seizures and normalization of motor activity and performance on the elevated plus maze test. These findings demonstrate sex differences in mortality in untreated Scn1a+/− mice, an effect that may be related to a lower level of intrinsic inhibitory tone in female mice, and a normalization of aberrant behaviors in males after central nervous system administration of AAV-NaVβ1. The therapeutic efficacy of AAV-NaVβ1 in a mouse model of DS suggests a potential new long-lasting biological therapeutic avenue for the treatment of this catastrophic epilepsy.

Abstract Mitochondrial acyl-coenzyme A species are emerging as important sources of protein modification and damage. Succinyl-CoA ligase (SCL) deficiency causes a mitochondrial encephalomyopathy of unknown pathomechanism. Here, we show that succinyl-CoA accumulates in cells derived from patients with recessive mutations in the tricarboxylic acid cycle (TCA) gene succinyl-CoA ligase subunit-β ( SUCLA2 ), causing global protein hyper-succinylation. Using mass spectrometry, we quantify nearly 1,000 protein succinylation sites on 366 proteins from patient-derived fibroblasts and myotubes. Interestingly, hyper-succinylated proteins are distributed across cellular compartments, and many are known targets of the (NAD + )-dependent desuccinylase SIRT5. To test the contribution of hyper-succinylation to disease progression, we develop a zebrafish model of the SCL deficiency and find that SIRT5 gain-of-function reduces global protein succinylation and improves survival. Thus, increased succinyl-CoA levels contribute to the pathology of SCL deficiency through post-translational modifications.

Lafora disease is a fatal progressive myoclonus epilepsy. At root, it is due to constant acquisition of branches that are too long in a subgroup of glycogen molecules, leading them to precipitate and accumulate into Lafora bodies, which drive a neuroinflammatory response and neurodegeneration. As a potential therapy, we aimed to downregulate glycogen synthase, the enzyme responsible for glycogen branch elongation, in mouse models of the disease. We synthesized an antisense oligonucleotide (Gys1-ASO) that targets the mRNA of the brain-expressed glycogen synthase 1 gene (Gys1). We administered Gys1-ASO by intracerebroventricular injection and analysed the pathological hallmarks of Lafora disease, namely glycogen accumulation, Lafora body formation, and neuroinflammation. Gys1-ASO prevented Lafora body formation in young mice that had not yet formed them. In older mice that already exhibited Lafora bodies, Gys1-ASO inhibited further accumulation, markedly preventing large Lafora bodies characteristic of advanced disease. Inhibition of Lafora body formation was associated with prevention of astrogliosis and strong trends towards correction of dysregulated expression of disease immune and neuroinflammatory markers. Lafora disease manifests gradually in previously healthy teenagers. Our work provides proof of principle that an antisense oligonucleotide targeting the GYS1 mRNA could prevent, and halt progression of, this catastrophic epilepsy.

The ubiquitin system impacts most cellular processes and is altered in numerous neurodegenerative diseases. However, little is known about its role in neurodegenerative diseases due to disturbances of glycogen metabolism such as Lafora disease (LD). In LD, insufficiently branched and long-chained glycogen forms and precipitates into insoluble polyglucosan bodies (Lafora bodies), which drive neuroinflammation, neurodegeneration and epilepsy. LD is caused by mutations in the gene encoding the glycogen phosphatase laforin or the gene coding for the laforin interacting partner ubiquitin E3 ligase malin. The role of the malin-laforin complex in regulating glycogen structure remains with full of gaps. In this review we bring together the disparate body of data on these two proteins and propose a mechanistic hypothesis of the disease in which malin-laforin's role to monitor and prevent over-elongation of glycogen branch chains, which drive glycogen molecules to precipitate and accumulate into Lafora bodies. We also review proposed connections between Lafora bodies and the ensuing neuroinflammation, neurodegeneration and intractable epilepsy. Finally, we review the exciting activities in developing therapies for Lafora disease based on replacing the missing genes, slowing the enzyme – glycogen synthase – that over-elongates glycogen branches, and introducing enzymes that can digest Lafora bodies. Much more work is needed to fill the gaps in glycogen metabolism in which laforin and malin operate. However, knowledge appears already adequate to advance disease course altering therapies for this catastrophic fatal disease.

ABSTRACT Glycogen is the largest cytosolic macromolecule and is kept in solution through a regular system of short branches allowing hydration. This structure was thought to solely require balanced glycogen synthase and branching enzyme activities. Deposition of overlong branched glycogen in the fatal epilepsy Lafora disease (LD) indicated involvement of the LD gene products laforin and the E3 ubiquitin ligase malin in regulating glycogen structure. Laforin binds glycogen, and LD-causing mutations disrupt this binding, laforin–malin interactions and malin's ligase activity, all indicating a critical role for malin. Neither malin's endogenous function nor location had previously been studied due to lack of suitable antibodies. Here, we generated a mouse in which the native malin gene is tagged with the FLAG sequence. We show that the tagged gene expresses physiologically, malin localizes to glycogen, laforin and malin indeed interact, at glycogen, and malin's presence at glycogen depends on laforin. These results, and mice, open the way to understanding unknown mechanisms of glycogen synthesis critical to LD and potentially other much more common diseases due to incompletely understood defects in glycogen metabolism.

Lafora disease (LD) is a fatal and the most common form of adolescent-onset progressive epilepsy. Fulminant endoplasmic reticulum (ER)-associated depositions of starch-like long-stranded, poorly branched glycogen molecules [known as polyglucosans, which accumulate to form Lafora bodies (LBs)] are seen in neuronal perikarya and dendrites, liver, skeletal muscle and heart. The disease is caused by loss of function of the laforin dual-specificity phosphatase or the malin E3 ubiquitin ligase. Towards understanding the pathogenesis of polyglucosans in LD, we generated a transgenic mouse overexpressing inactivated laforin to trap normal laforin's unknown substrate. The trap was successful and LBs formed in liver, muscle, neuronal perikarya and dendrites. Using immunogold electron microscopy, we show that laforin is found in close proximity to the ER surrounding the polyglucosan accumulations. In neurons, it compartmentalizes to perikaryon and dendrites and not to axons. Importantly, it binds polyglucosans, establishing for the first time a direct association between the disease-defining storage product and disease protein. It preferentially binds polyglucosans over glycogen in vivo and starch over glycogen in vitro, suggesting that laforin's role begins after the appearance of polyglucosans and that the laforin pathway is involved in monitoring for and then preventing the formation of polyglucosans. In addition, we show that the laforin interacting protein, EPM2AIP1, also localizes on the polyglucosan masses, and we confirm laforin's intense binding to LBs in human LD biopsy material.

Lafora disease (LD) is the most common teenage-onset progressive myoclonus epilepsy. It is caused by recessive mutations in the <i>EPM2A</i> or <i>EPM2B</i> genes. The authors describe a family with three affected members with no mutations in either gene. Linkage and haplotype analyses exclude both loci from causative involvement in this family. Therefore, a third LD locus is predicted. Its identification will be a crucial element in the understanding of the biochemical pathway underlying the generation of Lafora bodies and LD.

Neurons throughout the brain suddenly discharging synchronously and recurrently cause primarily generalized seizures. Discharges localized awhile in one part of the brain cause focal-onset seizures. A genetically determined generalized hyperexcitability had been predicted in primarily generalized seizures, but surprisingly the first epilepsy gene discovered, CHRNA4, was in a focal (frontal lobe)-onset syndrome. Another surprise with CHRNA4 was its encoding of an ion channel present throughout the brain. The reason why CHRNA4 causes focal-onset seizures is unknown. Recently, the second focal (temporal lobe)-onset epilepsy gene, LGI1 (unknown function), was discovered. CHRNA4 led the way to mutation identifications in 15 ion channel genes, most causing primarily generalized epilepsies. Potassium channel mutations cause benign familial neonatal convulsions. Sodium channel mutations cause generalized epilepsy with febrile seizures plus or, if more severe, severe myoclonic epilepsy of infancy. Chloride and calcium channel mutations are found in rare families with the common syndromes childhood absence epilepsy and juvenile myoclonic epilepsy (JME). Mutations in the EFHC1 gene (unknown function) occur in other rare JME families, and yet in other families, associations are present between JME (or other generalized epilepsies) and single nucleotide polymorphisms in the BRD2 gene (unknown function) and the malic enzyme 2 (ME2) gene. Hippocrates predicted the genetic nature of the 'sacred' disease. Genes underlying the 'malevolent' forces seizing 1% of humans have now been revealed. These, however, still account for a mere fraction of the genetic contribution to epilepsy. Exciting years are ahead, in which the genetics of this extremely common, and debilitating, neurological disorder will be solved.

Summary Purpose: Dravet syndrome (DS) is an aggressive epileptic encephalopathy. Pharmacoresistant seizures of several types plague most patients with DS throughout their lives. Gait difficulties are a common, but inconsistent finding. The majority of cases are caused by mutations in the SCN1A gene, but little information is available about how particular mutations influence the adult phenotype. The purpose of this study is to correlate different types of SCN1A mutations and (1) seizure control, (2) occurrence of convulsive status epilepticus (cSE), and (3) the presence of crouch gait in adult patients. Methods: In a cohort of 10 adult patients with DS caused by SCN1A mutations, we investigated seizure frequency, history of cSE, and gait. All patients were identified in the epilepsy clinic between 2009 and 2011. SCN1A mutations were divided into four different groups based on location or effect of the mutation. Retrospective chart review and recent physical examination were completed in all cases. Key Findings: All patients had a pathogenic mutation in the SCN1A gene. Four SCN1A mutations have not been described previously. Greater than 90% seizure reduction was observed (compared to childhood frequency) in six of seven patients with missense mutations in the pore‐forming region (PFR) of the Na v 1.1 protein (group A) and nonsense mutations (group B). One patient with a splice‐site mutation (group C) and another with a mutation outside the PFR (group D) became free of all types of seizures. cSE after the age of 19 years was observed in only one patient. Crouch gait, without spasticity, is identified as an element of the adult DS phenotype. However, only one half of our adult DS cohort demonstrated crouch gait. This feature was observed in five of seven patients from groups A and B. Significance: This study shows that seizure control improves and cSE become less frequent in DS as patients age, independent of their SCN1A mutation type. Complete seizure freedom was seen in two patients (groups C and D). Finally, this study shows that in DS, crouch gait can be observed in up to 50% of adults with SCN1A mutation. Although no definite statistical correlations could be made due to the small number of patients, it is interesting to note that crouch gait was observed only in those patients with nonsense mutations or mutations in the PFR. Future studies with larger cohorts will be required to formally assess an association of gait abnormalities with particular SCN1A mutations.

We describe the molecular basis of a distinctive syndrome characterized by infantile stress-induced episodic weakness, ataxia, and sensorineural hearing loss, with permanent areflexia and optic nerve pallor. Whole exome sequencing identified a deleterious heterozygous c.2452 G&gt;A, p.(E818K) variant in the ATP1A3 gene and structural analysis predicted its protein-destabilizing effect. This variant has not been reported in context with rapid-onset dystonia parkinsonism and alternating hemiplegia of childhood, the 2 main diseases associated with ATP1A3. The clinical presentation in the family described here differs categorically from these diseases in age of onset, clinical course, cerebellar over extrapyramidal movement disorder predominance, and peripheral nervous system involvement. While this paper was in review, a highly resembling phenotype was reported in additional patients carrying the same c.2452 G&gt;A variant. Our findings substantiate this variant as the cause of a unique inherited autosomal dominant neurologic syndrome that constitutes a third allelic disease of the ATP1A3 gene.

•Starch evolved from glycogen in the host cytosol after plastid endosymbiosis. •A chlamydial debranching enzyme was recruited for crystalline polysaccharide synthesis. •A GWD evolved to enable mobilization of amylopectin crystals. •The novel dikinase step built on preexisting glycogen phosphorylation in eukaryotes. •The dikinases coupled starch catabolism to cyanobacterial carbon supply. •Transition from glycogen to starch maximized cytosolic carbon-sink strength. In this opinion article we propose a scenario detailing how two crucial components have evolved simultaneously to ensure the transition of glycogen to starch in the cytosol of the Archaeplastida last common ancestor: (i) the recruitment of an enzyme from intracellular Chlamydiae pathogens to facilitate crystallization of α-glucan chains; and (ii) the evolution of novel types of polysaccharide (de)phosphorylating enzymes from preexisting glycogen (de)phosphorylation host pathways to allow the turnover of such crystals. We speculate that the transition to starch benefitted Archaeplastida in three ways: more carbon could be packed into osmotically inert material; the host could resume control of carbon assimilation from the chlamydial pathogen that triggered plastid endosymbiosis; and cyanobacterial photosynthate export could be integrated in the emerging Archaeplastida. In this opinion article we propose a scenario detailing how two crucial components have evolved simultaneously to ensure the transition of glycogen to starch in the cytosol of the Archaeplastida last common ancestor: (i) the recruitment of an enzyme from intracellular Chlamydiae pathogens to facilitate crystallization of α-glucan chains; and (ii) the evolution of novel types of polysaccharide (de)phosphorylating enzymes from preexisting glycogen (de)phosphorylation host pathways to allow the turnover of such crystals. We speculate that the transition to starch benefitted Archaeplastida in three ways: more carbon could be packed into osmotically inert material; the host could resume control of carbon assimilation from the chlamydial pathogen that triggered plastid endosymbiosis; and cyanobacterial photosynthate export could be integrated in the emerging Archaeplastida.

Epilepsy will affect nearly 3% of people at some point during their lifetime. Previous copy number variants (CNVs) studies of epilepsy have used array-based technology and were restricted to the detection of large or exonic events. In contrast, whole-genome sequencing (WGS) has the potential to more comprehensively profile CNVs but existing analytic methods suffer from limited accuracy. We show that this is in part due to the non-uniformity of read coverage, even after intra-sample normalization. To improve on this, we developed PopSV, an algorithm that uses multiple samples to control for technical variation and enables the robust detection of CNVs. Using WGS and PopSV, we performed a comprehensive characterization of CNVs in 198 individuals affected with epilepsy and 301 controls. For both large and small variants, we found an enrichment of rare exonic events in epilepsy patients, especially in genes with predicted loss-of-function intolerance. Notably, this genome-wide survey also revealed an enrichment of rare non-coding CNVs near previously known epilepsy genes. This enrichment was strongest for non-coding CNVs located within 100 Kbp of an epilepsy gene and in regions associated with changes in the gene expression, such as expression QTLs or DNase I hypersensitive sites. Finally, we report on 21 potentially damaging events that could be associated with known or new candidate epilepsy genes. Our results suggest that comprehensive sequence-based profiling of CNVs could help explain a larger fraction of epilepsy cases.

Protein aggregation is a common cause of neuropathology. The protein aggregation myopathy Limb-Girdle Muscular Dystrophy 1D (LGMD1D) is caused by mutations of amino acids Phe89 or Phe93 of DNAJB6, a co-chaperone of the HSP70 anti-aggregation protein. Another DNAJB6 mutation, Pro96Arg, was found to cause a distal-onset myopathy in one family. We detail the mutational, neuropathological, neurophysiological, neurological and radiological features of five new DNAJB6-myopathy families. One has the known Phe93Leu mutation and classic late-onset slowly progressive LGMD1D. Two have different mutations of Phe91 causing a variant childhood-onset severe limb-girdle myopathy. One has a Phe100Val mutation and distal-onset myopathy, unique early bulbar involvement, and a gender-modified wide age-of-onset range. The last has childhood-onset severe distal-onset myopathy and the first non-missense DNAJB6 mutation, c.346 + 5G > A, causing a splicing defect that entirely eliminates DNAJB6’s G/F domain (ΔG/F), the domain that harbours all other mutations. Clinical and imaging examinations reveal that muscles considered uninvolved in DNAJB6-myopathy, e.g. lateral gastrocnemii, are affected in our patients with new mutations. Mutational modelling based on the known structure of the bacterial DNAJ2 protein indicates that all past and present mutated residues cluster within 15 Å in the G/F domain and all disturb the interface of this domain with the protein’s J domain that confers the interaction with HSP70. Our patients expand the phenotypic spectrum of DNAJB6-myopathy and allow tentative genotype-phenotype specifications. Combining with previous studies, the clinical severity spectrum is as follows: ΔG/F and Phe91 mutations, most severe; Phe100, Pro96, Phe89 mutations, intermediate; and Phe93, least severe. As it stands presently, proximal G/F domain mutations (Phe89, Phe91, Phe93) cause proximal limb-girdle myopathy, while distal G/F mutations (Pro96, Phe100) cause distal-onset myopathy. While all mutations affect the G/F–J interaction, each likely does so in different unknown extents or ways. One mutation, ΔG/F, causes its associated severe distal-onset myopathy phenotype in a clear way, through generation of a G/F domain-lacking DNAJB6 protein.

Platforms enabling targeted delivery of proteins into cells are needed to fully realize the potential of protein-based therapeutics with intracellular sites-of-action. Bacterial toxins are attractive systems to consider as templates for designing protein transduction systems as they naturally bind and enter specific cells with high efficiency. Here we investigated the capacity of diphtheria toxin to function as an intracellular protein delivery vector. We report that diphtheria toxin delivers an impressive array of passenger proteins spanning a range of sizes, structures, and stabilities into cells in a manner that indicates that they are "invisible" to the translocation machinery. Further, we show that α-amylase delivered into cells by a detoxified diphtheria toxin chimera digests intracellular glycogen in live cells, providing evidence that delivered cargo is folded, active, and abundant. The efficiency and versatility of diphtheria toxin over existing systems open numerous possibilities for intracellular delivery of bioactive proteins.

To expand the clinical phenotype associated with STXBP1 gene mutations and to understand the effect of STXBP1 mutations in the pathogenesis of focal cortical dysplasia (FCD).Patients with STXBP1 mutations were identified in various ways: as part of a retrospective cohort study of epileptic encephalopathy; through clinical referrals of individuals (10,619) with developmental delay (DD) for chromosomal microarray; and from a collection of 5,205 individuals with autism spectrum disorder (ASD) examined by whole-genome sequencing.Seven patients with heterozygous de novo mutations affecting the coding region of STXBP1 were newly identified. Three cases had radiologic evidence suggestive of FCD. One male patient with early infantile epileptic encephalopathy, DD, and ASD achieved complete seizure remission following resection of dysplastic brain tissue. Examination of excised brain tissue identified mosaicism for STXBP1, providing evidence for a somatic mechanism. Cell-type expression analysis suggested neuron-specific expression. A comprehensive analysis of the published data revealed that 3.1% of severe epilepsy cases carry a pathogenic de novo mutation within STXBP1. By contrast, ASD was rarely associated with mutations in this gene in our large cohorts.STXBP1 mutations are an important cause of epilepsy and are also rarely associated with ASD. In a case with histologically proven FCD, an STXBP1 somatic mutation was identified, suggesting a role in its etiology. Removing such tissue may be curative for STXBP1-related epilepsy.

Lafora disease (LD) and adult polyglucosan body disease (APBD) are glycogen storage diseases characterized by a pathogenic buildup of insoluble glycogen. Mechanisms causing glycogen insolubility are poorly understood. Here, in two mouse models of LD (Epm2a−/− and Epm2b−/−) and one of APBD (Gbe1ys/ys), the separation of soluble and insoluble muscle glycogen is described, enabling separate analysis of each fraction. Total glycogen is increased in LD and APBD mice, which, together with abnormal chain length and molecule size distributions, is largely if not fully attributed to insoluble glycogen. Soluble glycogen consists of molecules with distinct chain length distributions and differential corresponding solubility, providing a mechanistic link between soluble and insoluble glycogen in vivo. Phosphorylation states differ across glycogen fractions and mouse models, demonstrating that hyperphosphorylation is not a basic feature of insoluble glycogen. Lastly, model-specific variances in protein and activity levels of key glycogen synthesis enzymes suggest uninvestigated regulatory mechanisms.

Vacuolar H+-ATP complex (V-ATPase) is a multisubunit protein complex required for acidification of intracellular compartments. At least five different factors are known to be essential for its assembly in the endoplasmic reticulum (ER). Genetic defects in four of these V-ATPase assembly factors show overlapping clinical features, including steatotic liver disease and mild hypercholesterolemia. An exception is the assembly factor vacuolar ATPase assembly integral membrane protein (VMA21), whose X-linked mutations lead to autophagic myopathy.Here, we report pathogenic variants in VMA21 in male patients with abnormal protein glycosylation that result in mild cholestasis, chronic elevation of aminotransferases, elevation of (low-density lipoprotein) cholesterol and steatosis in hepatocytes. We also show that the VMA21 variants lead to V-ATPase misassembly and dysfunction. As a consequence, lysosomal acidification and degradation of phagocytosed materials are impaired, causing lipid droplet (LD) accumulation in autolysosomes. Moreover, VMA21 deficiency triggers ER stress and sequestration of unesterified cholesterol in lysosomes, thereby activating the sterol response element-binding protein-mediated cholesterol synthesis pathways.Together, our data suggest that impaired lipophagy, ER stress, and increased cholesterol synthesis lead to LD accumulation and hepatic steatosis. V-ATPase assembly defects are thus a form of hereditary liver disease with implications for the pathogenesis of nonalcoholic fatty liver disease.

Many adult and most childhood neurological diseases have a genetic basis. CRISPR/Cas9 biotechnology holds great promise in neurological therapy, pending the clearance of major delivery, efficiency, and specificity hurdles. We applied CRISPR/Cas9 genome editing in its simplest modality, namely inducing gene sequence disruption, to one adult and one pediatric disease. Adult polyglucosan body disease is a neurodegenerative disease resembling amyotrophic lateral sclerosis. Lafora disease is a severe late childhood onset progressive myoclonus epilepsy. The pathogenic insult in both is formation in the brain of glycogen with overlong branches, which precipitates and accumulates into polyglucosan bodies that drive neuroinflammation and neurodegeneration. We packaged Staphylococcus aureus Cas9 and a guide RNA targeting the glycogen synthase gene, Gys1, responsible for brain glycogen branch elongation in AAV9 virus, which we delivered by neonatal intracerebroventricular injection to one mouse model of adult polyglucosan body disease and two mouse models of Lafora disease. This resulted, in all three models, in editing of approximately 17% of Gys1 alleles and a similar extent of reduction of Gys1 mRNA across the brain. The latter led to approximately 50% reductions of GYS1 protein, abnormal glycogen accumulation, and polyglucosan bodies, as well as ameliorations of neuroinflammatory markers in all three models. Our work represents proof of principle for virally delivered CRISPR/Cas9 neurotherapeutics in an adult-onset (adult polyglucosan body) and a childhood-onset (Lafora) neurological diseases.

Malstructured glycogen accumulates over time in Lafora disease (LD) and precipitates into Lafora bodies (LBs), leading to neurodegeneration and intractable fatal epilepsy. Constitutive reduction of glycogen synthase-1 (GYS1) activity prevents murine LD, but the effect of GYS1 reduction later in disease course is unknown. Our goal was to knock out Gys1 in laforin (Epm2a)-deficient LD mice after disease onset to determine whether LD can be halted in midcourse, or even reversed. We generated Epm2a-deficient LD mice with tamoxifen-inducible Cre-mediated Gys1 knockout. Tamoxifen was administered at 4 months and disease progression assessed at 12 months. We verified successful knockout at mRNA and protein levels using droplet digital PCR and Western blots. Glycogen determination and periodic acid–Schiff–diastase staining were used to analyze glycogen and LB accumulation. Immunohistochemistry using astrocytic (glial fibrillary acidic protein) and microglial (ionized calcium-binding adapter molecule 1) markers was performed to investigate neuroinflammation. In the disease-relevant organ, the brain, Gys1 mRNA levels were reduced by 85% and GYS1 protein depleted. Glycogen accumulation was halted at the 4-month level, while LB formation and neuroinflammation were significantly, though incompletely, prevented. Skeletal muscle analysis confirmed that Gys1 knockout inhibits glycogen and LB accumulation. However, tamoxifen-independent Cre recombination precluded determination of disease halting or reversal in this tissue. Our study shows that Gys1 knockdown is a powerful means to prevent LD progression, but this approach did not reduce brain glycogen or LBs to levels below those at the time of intervention. These data suggest that endogenous mechanisms to clear brain LBs are absent or, possibly, compromised in laforin-deficient murine LD. Malstructured glycogen accumulates over time in Lafora disease (LD) and precipitates into Lafora bodies (LBs), leading to neurodegeneration and intractable fatal epilepsy. Constitutive reduction of glycogen synthase-1 (GYS1) activity prevents murine LD, but the effect of GYS1 reduction later in disease course is unknown. Our goal was to knock out Gys1 in laforin (Epm2a)-deficient LD mice after disease onset to determine whether LD can be halted in midcourse, or even reversed. We generated Epm2a-deficient LD mice with tamoxifen-inducible Cre-mediated Gys1 knockout. Tamoxifen was administered at 4 months and disease progression assessed at 12 months. We verified successful knockout at mRNA and protein levels using droplet digital PCR and Western blots. Glycogen determination and periodic acid–Schiff–diastase staining were used to analyze glycogen and LB accumulation. Immunohistochemistry using astrocytic (glial fibrillary acidic protein) and microglial (ionized calcium-binding adapter molecule 1) markers was performed to investigate neuroinflammation. In the disease-relevant organ, the brain, Gys1 mRNA levels were reduced by 85% and GYS1 protein depleted. Glycogen accumulation was halted at the 4-month level, while LB formation and neuroinflammation were significantly, though incompletely, prevented. Skeletal muscle analysis confirmed that Gys1 knockout inhibits glycogen and LB accumulation. However, tamoxifen-independent Cre recombination precluded determination of disease halting or reversal in this tissue. Our study shows that Gys1 knockdown is a powerful means to prevent LD progression, but this approach did not reduce brain glycogen or LBs to levels below those at the time of intervention. These data suggest that endogenous mechanisms to clear brain LBs are absent or, possibly, compromised in laforin-deficient murine LD. Lafora disease (LD) is teenage-onset progressive myoclonus epilepsy that is typically fatal within 10 years of symptom onset (1Delgado-Escueta A.V. Ganesh S. Yamakawa K. Advances in the genetics of progressive myoclonus epilepsy.Am. J. Med. Genet. 2001; 106: 129-138Crossref PubMed Scopus (67) Google Scholar, 2Minassian B.A. Lafora's disease: towards a clinical, pathologic, and molecular synthesis.Pediatr. Neurol. 2001; 25: 21-29Abstract Full Text Full Text PDF PubMed Scopus (138) Google Scholar). LD is caused by autosomal recessive mutations in either EPM2A or NHLRC1 (also known as EPM2B), encoding laforin, a glucan phosphatase, and malin, an E3 ubiquitin ligase, respectively (3Minassian B.A. Lee J.R. Herbrick J.A. Huizenga J. Soder S. Mungall A.J. Dunham I. Gardner R. Fong C.Y. Carpenter S. Jardim L. Satishchandra P. Andermann E. Snead 3rd, O.C. Lopes-Cendes I. et al.Mutations in a gene encoding a novel protein tyrosine phosphatase cause progressive myoclonus epilepsy.Nat. Genet. 1998; 20: 171-174Crossref PubMed Scopus (371) Google Scholar, 4Serratosa J.M. Gomez-Garre P. Gallardo M.E. Anta B. de Bernabe D.B. Lindhout D. Augustijn P.B. Tassinari C.A. Malafosse R.M. Topcu M. Grid D. Dravet C. Berkovic S.F. de Cordoba S.R. A novel protein tyrosine phosphatase gene is mutated in progressive myoclonus epilepsy of the Lafora type (EPM2).Hum. Mol. Genet. 1999; 8: 345-352Crossref PubMed Scopus (180) Google Scholar, 5Chan E.M. Young E.J. Ianzano L. Munteanu I. Zhao X. Christopoulos C.C. Avanzini G. Elia M. Ackerley C.A. Jovic N.J. Bohlega S. Andermann E. Rouleau G.A. Delgado-Escueta A.V. Minassian B.A. et al.Mutations in NHLRC1 cause progressive myoclonus epilepsy.Nat. Genet. 2003; 35: 125-127Crossref PubMed Scopus (224) Google Scholar). The exact roles of both enzymes are unclear. It is, however, established that laforin and malin form a functional complex that regulates glycogen metabolism, with absence of either protein causing formation of malstructured glycogen (6Gentry M.S. Worby C.A. Dixon J.E. Insights into Lafora disease: malin is an E3 ubiquitin ligase that ubiquitinates and promotes the degradation of laforin.Proc. Natl. Acad. Sci. U. S. A. 2005; 102: 8501-8506Crossref PubMed Scopus (167) Google Scholar, 7Sullivan M. Nitschke S. Steup M. Minassian B. Nitschke F. Pathogenesis of Lafora disease: transition of soluble glycogen to insoluble polyglucosan.Int. J. Mol. Sci. 2017; 18: 1743Crossref PubMed Scopus (29) Google Scholar). LD is widely considered a glycogen storage disease (8Nitschke F. Ahonen S.J. Nitschke S. Mitra S. Minassian B.A. Lafora disease - from pathogenesis to treatment strategies.Nat. Rev. Neurol. 2018; 14: 606-617Crossref PubMed Scopus (45) Google Scholar, 9Brewer M.K. Uittenbogaard A. Austin G.L. Segvich D.M. DePaoli-Roach A. Roach P.J. McCarthy J.J. Simmons Z.R. Brandon J.A. Zhou Z. Zeller J. Young L.E.A. Sun R.C. Pauly J.R. Aziz N.M. et al.Targeting pathogenic Lafora bodies in Lafora disease using an antibody-enzyme fusion.Cell Metab. 2019; 30: 689-705.e686Abstract Full Text Full Text PDF PubMed Scopus (26) Google Scholar, 10Tang B. Frasinyuk M.S. Chikwana V.M. Mahalingan K.K. Morgan C.A. Segvich D.M. Bondarenko S.P. Mrug G.P. Wyrebek P. Watt D.S. DePaoli-Roach A.A. Roach P.J. Hurley T.D. Discovery and development of small-molecule inhibitors of glycogen synthase.J. Med. Chem. 2020; 63: 3538-3551Crossref PubMed Scopus (13) Google Scholar, 11Gentry M.S. Afawi Z. Armstrong D.D. Delgado-Escueta A. Goldberg Y.P. Grossman T.R. Guinovart J.J. Harris F. Hurley T.D. Michelucci R. Minassian B.A. Sanz P. Worby C.A. Serratosa J.M. The 5th International Lafora Epilepsy Workshop: basic science elucidating therapeutic options and preparing for therapies in the clinic.Epilepsy Behav. 2020; 103: 106839Abstract Full Text Full Text PDF PubMed Scopus (9) Google Scholar). The abnormal glycogen molecules precipitate and aggregate to form Lafora bodies (LBs), which continually accumulate, leading to symptom onset and then inexorably worsening neurodegeneration, seizures, and cognitive decline, culminating in vegetative state and death in status epilepticus and related complications (8Nitschke F. Ahonen S.J. Nitschke S. Mitra S. Minassian B.A. Lafora disease - from pathogenesis to treatment strategies.Nat. Rev. Neurol. 2018; 14: 606-617Crossref PubMed Scopus (45) Google Scholar). Both laforin- and malin-deficient LD mouse models (12Ganesh S. Delgado-Escueta A.V. Sakamoto T. Avila M.R. Machado-Salas J. Hoshii Y. Akagi T. Gomi H. Suzuki T. Amano K. Agarwala K.L. Hasegawa Y. Bai D.-S. Ishihara T. Hashikawa T. et al.Targeted disruption of the Epm2a gene causes formation of Lafora inclusion bodies, neurodegeneration, ataxia, myoclonus epilepsy and impaired behavioral response in mice.Hum. Mol. Genet. 2002; 11: 1251-1262Crossref PubMed Scopus (158) Google Scholar, 13DePaoli-Roach A.A. Tagliabracci V.S. Segvich D.M. Meyer C.M. Irimia J.M. Roach P.J. Genetic depletion of the malin E3 ubiquitin ligase in mice leads to Lafora bodies and the accumulation of insoluble laforin.J. Biol. Chem. 2010; 285: 25372-25381Abstract Full Text Full Text PDF PubMed Scopus (87) Google Scholar, 14Turnbull J. Wang P. Girard J.M. Ruggieri A. Wang T.J. Draginov A.G. Kameka A.P. Pencea N. Zhao X. Ackerley C.A. Minassian B.A. Glycogen hyperphosphorylation underlies lafora body formation.Ann. Neurol. 2010; 68: 925-933Crossref PubMed Scopus (72) Google Scholar, 15Criado O. Aguado C. Gayarre J. Duran-Trio L. Garcia-Cabrero A.M. Vernia S. San Millan B. Heredia M. Roma-Mateo C. Mouron S. Juana-Lopez L. Dominguez M. Navarro C. Serratosa J.M. Sanchez M. et al.Lafora bodies and neurological defects in malin-deficient mice correlate with impaired autophagy.Hum. Mol. Genet. 2012; 21: 1521-1533Crossref PubMed Scopus (83) Google Scholar) recapitulate the hallmarks of LD: glycogen accumulation, LB formation, and neuroinflammation (16Turnbull J. DePaoli-Roach A.A. Zhao X. Cortez M.A. Pencea N. Tiberia E. Piliguian M. Roach P.J. Wang P. Ackerley C.A. Minassian B.A. PTG depletion removes Lafora bodies and rescues the fatal epilepsy of Lafora disease.PLoS Genet. 2011; 7e1002037Crossref PubMed Scopus (79) Google Scholar, 17Duran J. Gruart A. Garcia-Rocha M. Delgado-Garcia J.M. Guinovart J.J. Glycogen accumulation underlies neurodegeneration and autophagy impairment in Lafora disease.Hum. Mol. Genet. 2014; 23: 3147-3156Crossref PubMed Scopus (104) Google Scholar). In pursuit of a therapy for this intractable disease, one approach that emerged was to reduce glycogen synthase-1 (GYS1) activity, thereby interfering with chain elongation during glycogen synthesis, in order to prevent glycogen accumulation and LB formation. This approach proved successful with knockout of Gys1 or Ppp1r3c, encoding a protein (also known as PTG) involved in GYS1 activation. Both gene knockouts prevented LB formation, neurodegeneration, and other features of the disease in LD mice (16Turnbull J. DePaoli-Roach A.A. Zhao X. Cortez M.A. Pencea N. Tiberia E. Piliguian M. Roach P.J. Wang P. Ackerley C.A. Minassian B.A. PTG depletion removes Lafora bodies and rescues the fatal epilepsy of Lafora disease.PLoS Genet. 2011; 7e1002037Crossref PubMed Scopus (79) Google Scholar, 17Duran J. Gruart A. Garcia-Rocha M. Delgado-Garcia J.M. Guinovart J.J. Glycogen accumulation underlies neurodegeneration and autophagy impairment in Lafora disease.Hum. Mol. Genet. 2014; 23: 3147-3156Crossref PubMed Scopus (104) Google Scholar, 18Pederson B.A. Turnbull J. Epp J.R. Weaver S.A. Zhao X. Pencea N. Roach P.J. Frankland P.W. Ackerley C.A. Minassian B.A. Inhibiting glycogen synthesis prevents Lafora disease in a mouse model.Ann. Neurol. 2013; 74: 297-300PubMed Google Scholar, 19Turnbull J. Epp J.R. Goldsmith D. Zhao X. Pencea N. Wang P. Frankland P.W. Ackerley C.A. Minassian B.A. PTG protein depletion rescues malin-deficient Lafora disease in mouse.Ann. Neurol. 2014; 75: 442-446Crossref PubMed Scopus (40) Google Scholar). These results established proof of principle that targeting Gys1 for downregulation could be a promising therapeutic approach in LD. However, the mice in these studies had constitutively reduced GYS1. This left the question open as to how successful therapeutic targeting of Gys1 could be when LBs are already present, i.e., after disease onset and diagnosis. We generated laforin-deficient (Epm2a−/−) mice with tamoxifen (TAM)-inducible Gys1 knockout. We administered TAM at 4 months, after murine disease onset, and assessed subsequent disease progression at 12 months. We analyzed glycogen content, LB accumulation, and neuroinflammation to determine whether LD progression can be halted in midcourse, or even potentially reversed. We generated conditional Gys1 knockout mice to test whether genetic pharmacotherapy targeting Gys1 following disease onset would lead to a halt or even reversal of LD progression. We used mice carrying a Gys1-targeting cassette with loxP sites flanking exons 6 to 8 together with TAM-inducible Cre to generate conditional Gys1-knockout mice in wild-type (WT) and Epm2a−/− backgrounds (Fig. 1A). A subset of mice was harvested at 4 months, while the majority of mice were either TAM-treated or not treated and then aged until 12 months (Fig. 1B). Figure 1C gives an overview of all experimental mice, not only explaining their genotype and treatments but also introducing the names of each group used throughout the study. In addition to the WT and Epm2a−/− control (WT, LKO) and Gys1 knockout (WT-KO, LKO-KO) mice, we included LKO-L mice at 4 and 12 months. These LKO-L mice carried floxed Gys1 and expressed Cre but were not TAM-treated. They served to evaluate whether TAM-independent Cre-mediated recombination (referred to as “Cre leakage”) occurred. At 12 months, WT-KO mice exhibited GYS1 protein knockdown in the brain and muscle (Fig. 1D), which clearly indicated that Cre-mediated Gys1 recombination and knockout was successful. To assess the effect of TAM treatment in the brain of conditional Gys1-knockout mice (WT-KO, LKO-KO) and potential Cre-leakage mice (LKO-L), we determined the frequency of recombination events by quantifying Gys1 mRNA that still contained exons 6 to 8. Levels of intact Gys1 mRNA were decreased by approximately 85% in both WT-KO and LKO-KO mice, while LKO-L mice showed no reduction and hence no Cre leakage at 4 or 12 months (Fig. 2A, Fig. S1A). Like WT-KO mice (Fig. 1D), LKO-KO mice exhibited strong knockdown of GYS1 protein levels, while no decrease in GYS1 was detected in LKO-L compared with LKO mice (Fig. 2B). We then asked whether hallmarks of LD such as glycogen and LB accumulation in the brain were halted at the 4-month level, the time point of knockout induction, or perhaps even reversed. Glycogen determination confirmed an age-dependent increase in glycogen accumulation in LKO and LKO-L mice compared with WT in accordance with disease progression (Fig. 2C, Fig. S1B). There was a slight though not statistically significant trend toward less glycogen accumulation in LKO-L mice than in LKO control mice. Therefore, we directly compared LKO-KO mice with LKO-L mice in order to draw valid conclusions about disease halting or reversal. Doing so, we found clear stoppage in disease progression evidenced by LKO-L mice at 4 months having a very similar glycogen content as LKO-KO mice at 12 months (Fig. 2C, Fig. S1B). In WT-KO mice, glycogen levels were depleted, showing that Gys1 expression was sufficiently reduced to almost completely prevent synthesis of normal soluble glycogen. A similar reduction in soluble glycogen is to be expected in LKO-KO mice, and therefore, it can be concluded that the glycogen detected in these mice is mainly insoluble glycogen. Hippocampal LB quantification showed a small though significant amount of corpora amylacea–like (CAL) bodies (normal age-related astrocytic glucan inclusions (20Sinadinos C. Valles-Ortega J. Boulan L. Solsona E. Tevy M.F. Marquez M. Duran J. Lopez-Iglesias C. Calbo J. Blasco E. Pumarola M. Milan M. Guinovart J.J. Neuronal glycogen synthesis contributes to physiological aging.Aging Cell. 2014; 13: 935-945Crossref PubMed Scopus (52) Google Scholar, 21Augé E. Pelegrí C. Manich G. Cabezón I. Guinovart J.J. Duran J. Vilaplana J. Astrocytes and neurons produce distinct types of polyglucosan bodies in Lafora disease.Glia. 2018; 66: 2094-2107Crossref PubMed Scopus (25) Google Scholar)) in 12-month-old WT mice and age-dependent LB accumulation in LKO and LKO-L mice (Fig. 2D, Fig. S1C). CAL body formation was prevented by Gys1 knockout in WT-KO mice. LB accumulation was also significantly reduced in LKO-KO mice compared with LKO and LKO-L mice at 12 months, though slightly increased compared with LKO and LKO-L mice at 4 months. Therefore, Gys1 knockout at 4 months did not completely halt but significantly slowed LB formation in the hippocampus. In LD mice, the density of LBs is highest in the hippocampus, a structure whose pathologies commonly underlie epileptogenesis (22Chatzikonstantinou A. Epilepsy and the hippocampus.Front. Neurol. Neurosci. 2014; 34: 121-142Crossref PubMed Scopus (28) Google Scholar). However, in order to show a more complete picture regarding the LB load in the whole brain, we also quantified LBs in two additional large brain regions, cerebellum and cortex (Fig. S2, A–B). The results are very similar to what we saw in the hippocampus with the exception that in the cerebellum, a complete halt, versus only slowing of LB accumulation, could be achieved in LKO-KO mice. Representative periodic acid–Schiff–diastase (PASD) images are shown for LB visualization in Figure 2E and Fig. S2C. Another hallmark of LD is glia-derived neuroinflammation (23Lahuerta M. Gonzalez D. Aguado C. Fathinajafabadi A. Garcia-Gimenez J.L. Moreno-Estelles M. Roma-Mateo C. Knecht E. Pallardo F.V. Sanz P. Reactive glia-derived neuroinflammation: a novel hallmark in Lafora progressive myoclonus epilepsy that progresses with age.Mol. Neurobiol. 2020; 57: 1607-1621Crossref PubMed Scopus (14) Google Scholar). By performing immunohistochemistry for glial fibrillary acidic protein (GFAP) and ionized calcium–binding adapter molecule 1 (IBA1), we, respectively, assessed whether astrogliosis and/or microgliosis can be halted or reversed by targeting Gys1. In the hippocampus, WT and WT-KO mice did not exhibit any significant differences irrespective of Gys1 genotype or age (Fig. 3, Fig. S3). LKO and LKO-L mice showed minimal (GFAP) or no (IBA1) neuroinflammation compared with WT mice at 4 months but significantly increased GFAP and IBA1 signals by 12 months, confirming increased hippocampal neuroinflammation with LD progression. Twelve-month-old LKO-KO mice showed partial rescue with GFAP and IBA1 signal levels between 4-month-old and 12-month-old LKO and LKO-L mice levels. GFAP and IBA1 signals were also quantified in the cerebellum (Fig. S4). However, astrogliosis was not significantly increased in LKO or LKO-L mice at 12 months, while microgliosis was significantly increased but not rescued in LKO-KO mice. In the cerebellum, LD-associated neuroinflammation was not as pronounced as in the hippocampus and consequently the timeframe to determine a halt in progression of neuroinflammation was too short to observe significant rescue in LKO-KO mice. While LD manifests as a neurological disease, LBs additionally accumulate in murine and patient skeletal muscle. We therefore analyzed the muscle to further assess the effect of Gys1 knockout on glycogen and LB accumulation post disease onset. Like in the brain, we determined the extent of recombination events by quantifying the reduction of Gys1 mRNA with exons 6 to 8 still present (Fig. 4A, Fig. S5A). Surprisingly, Gys1 mRNA levels were already approximately 90% reduced in LKO-L mice at 4 months, indicating major Cre leakage in the muscle, which was not the case in the brain (Fig. 2A). At 12 months, Gys1 mRNA levels were further reduced by 3.5-fold in LKO-KO and by approximately 2-fold in LKO-L mice, culminating in 97% and 94% knockdown, respectively, while LKO control mice remained unaffected. GYS1 protein levels in LKO-KO mice were as reduced as in WT-KO mice at 12 months (Fig. 1E, Fig. 4B). However, we detected a comparable depletion of GYS1 levels in LKO-L mice at 12 months. Even at 4 months, GYS1 levels were already reduced in LKO-L mice, confirming Cre leakage at the protein level in addition to that seen at the mRNA level. Further analyses focused on the effect of TAM-induced and TAM-independent Gys1 knockout on skeletal muscle total and insoluble glycogen content. For the latter, we used a recently established method that utilizes endogenously expressed metabolic enzymes to digest accessible soluble glycogen completely, leaving only the insoluble glycogen, which is not degradable (24Nitschke S. Petkovic S. Ahonen S. Minassian B.A. Nitschke F. Sensitive quantification of α-glucans in mouse tissues, cell cultures, and human cerebrospinal fluid.J. Biol. Chem. 2020; https://doi.org/10.1074/jbc.RA120.015061Abstract Full Text Full Text PDF Scopus (2) Google Scholar). This method allowed us to determine whether changes in total glycogen content were mostly due to changes in soluble glycogen, insoluble glycogen, or both. LKO mice showed an increase in total and insoluble glycogen at 12 months, while the amount of insoluble glycogen was only approximately 3% of the total glycogen at 4 months (Fig. 4, C–D, Fig. S5, B–C). These results are in line with the extensive LB accumulation in LKO mice at 12 months as well as the comparatively small LB load at 4 months, with a substantial number of PASD-positive cells but very small bodies (Fig. 4, E–F). Already at 4 months, LKO-L mice exhibited 50% reduced levels of total glycogen and an almost complete depletion of insoluble glycogen, indicating that not only was disease progression attenuated but also levels of soluble glycogen were significantly reduced. Accordingly, fewer cells were PASD-positive with less/smaller LBs. The extent of LB formation in 12-month-old LKO-KO mice looked very similar to 4-month-old LKO-L mice, in accordance with the comparable insoluble glycogen content. However, in the LKO-KO mice, even the soluble glycogen was reduced further, as total glycogen was reduced by approximately 95% compared with 4-month-old LKO mice. We detected an approximately 2-fold higher amount of total and insoluble glycogen in LKO-L mice at 12 months than in LKO-KO mice. The slightly higher amount, though not statistically significant, is in line with the PASD results that show a few cells with relatively many LBs, which are probably cells where Cre leakage did not occur or only affected one of the two alleles. Taken together, our results show that Gys1 knockout prevented the LD muscle phenotype; however, significant Cre leakage occluded assessment of disease reversal in this organ. Hallmarks of LD are glycogen accumulation and formation of LBs, which are insoluble, glycogen-like particles, characterized by reduced branching and long chains (25Nitschke F. Sullivan M.A. Wang P. Zhao X. Chown E.E. Perri A.M. Israelian L. Juana-Lopez L. Bovolenta P. Rodriguez de Cordoba S. Steup M. Minassian B.A. Abnormal glycogen chain length pattern, not hyperphosphorylation, is critical in Lafora disease.EMBO Mol. Med. 2017; 9: 906-917Crossref PubMed Scopus (27) Google Scholar). Therefore, reducing glycogen synthesis is an attractive avenue toward a therapy for the disease. From studying LD mouse models, we already know that reducing glycogen synthesis either by Gys1 knockout or indirectly by knockout of PTG prevents LB formation, neurodegeneration, and other features of the disease such as impaired autophagy (16Turnbull J. DePaoli-Roach A.A. Zhao X. Cortez M.A. Pencea N. Tiberia E. Piliguian M. Roach P.J. Wang P. Ackerley C.A. Minassian B.A. PTG depletion removes Lafora bodies and rescues the fatal epilepsy of Lafora disease.PLoS Genet. 2011; 7e1002037Crossref PubMed Scopus (79) Google Scholar, 17Duran J. Gruart A. Garcia-Rocha M. Delgado-Garcia J.M. Guinovart J.J. Glycogen accumulation underlies neurodegeneration and autophagy impairment in Lafora disease.Hum. Mol. Genet. 2014; 23: 3147-3156Crossref PubMed Scopus (104) Google Scholar, 18Pederson B.A. Turnbull J. Epp J.R. Weaver S.A. Zhao X. Pencea N. Roach P.J. Frankland P.W. Ackerley C.A. Minassian B.A. Inhibiting glycogen synthesis prevents Lafora disease in a mouse model.Ann. Neurol. 2013; 74: 297-300PubMed Google Scholar, 19Turnbull J. Epp J.R. Goldsmith D. Zhao X. Pencea N. Wang P. Frankland P.W. Ackerley C.A. Minassian B.A. PTG protein depletion rescues malin-deficient Lafora disease in mouse.Ann. Neurol. 2014; 75: 442-446Crossref PubMed Scopus (40) Google Scholar). Potential strategies to target brain glycogen synthesis are multiple, including antisense oligonucleotides, other forms of RNA interference, genome editing, and small-molecule inhibitors (8Nitschke F. Ahonen S.J. Nitschke S. Mitra S. Minassian B.A. Lafora disease - from pathogenesis to treatment strategies.Nat. Rev. Neurol. 2018; 14: 606-617Crossref PubMed Scopus (45) Google Scholar). Targeting glycogen synthesis as a therapy for LD has the advantage of applicability to patients with mutations in either EPM2A or NHLRC1, as opposed to gene replacement therapies, which would require separate development for each subgroup. A second major advantage is that downregulating a function is comparatively more practicable at the present time than replacing a missing function across the entire brain. Conventional knockout mouse models showed that it is possible to prevent LB formation and other disease-associated features when the capability to synthesize glycogen is blocked with the animal still in utero. Here, we asked the clinically more relevant question, namely, what would the effect of the intervention be after disease onset. We generated a conditional Gys1-knockout mouse, using TAM-induced Cre-mediated recombination, which enabled us to downregulate glycogen synthesis in laforin-deficient mice at 4 months when the LD neuropathological phenotype is already clearly detectable. We studied the hallmarks of LD, glycogen accumulation, LB formation, and consequent neuroinflammation. Interestingly, total brain glycogen in LKO-KO at 12 months was significantly lower than in LKO control mice at 4 months. Although we hoped to see a reversal, our results rather show a halt in disease progression for the following two reasons. Firstly, we expect the amount of soluble glycogen in LKO-KO mice to be reduced as strongly as in WT-KO mice, where the overall glycogen content was reduced by approximately 90%, because Gys1 mRNA and protein levels suggest a similar extent of Cre recombination in WT-KO and LKO-KO mice. This indicates that we detected mostly insoluble glycogen in the Gys1-knockout mice (LKO-KO). Secondly, we need to take into consideration that the amount of total glycogen in 4-month-old LKO control mice is a combination of soluble and insoluble glycogen. In order to estimate the amount of insoluble (LB) glycogen at 4 months, we can subtract the amount of WT glycogen from the amount in LKO mice because we have shown that the amount of soluble glycogen is not altered in LD mice, at least in the muscle (26Sullivan M.A. Nitschke S. Skwara E.P. Wang P. Zhao X. Pan X.S. Chown E.E. Wang T. Perri A.M. Lee J.P.Y. Vilaplana F. Minassian B.A. Nitschke F. Skeletal muscle glycogen chain length correlates with insolubility in mouse models of polyglucosan-associated neurodegenerative diseases.Cell Rep. 2019; 27: 1334-1344.e1336Abstract Full Text Full Text PDF PubMed Scopus (15) Google Scholar). This estimate gives approximately the same glycogen quantity as we see in LKO-KO mice at 12 months, further corroborating our conclusion of a halt in disease progression. Strikingly, although still present, the halt in disease progression was not as complete when we look at the LB quantification results in the hippocampus. In the cerebellum, however, we observed a complete halt in LB formation which is more in line with our findings on total glycogen level, measured in whole brain samples. It could be that Cre recombination was slightly less efficient in the hippocampus than in other parts of the brain such as the cerebellum. The Gys1 mRNA and protein levels are an average over the whole brain and do not reveal any potential disparities in recombination efficiency in different brain regions. An alternative explanation for differences seen in different parts of the brain could be cell type–specific differences in Cre recombination, being, for instance, less efficient in astrocytes than in other cell types (e.g., neurons or other glial cells). It has been shown that a great number of LBs in LD mice are found in astrocytes (21Augé E. Pelegrí C. Manich G. Cabezón I. Guinovart J.J. Duran J. Vilaplana J. Astrocytes and neurons produce distinct types of polyglucosan bodies in Lafora disease.Glia. 2018; 66: 2094-2107Crossref PubMed Scopus (25) Google Scholar, 27Rubio-Villena C. Viana R. Bonet J. Garcia-Gimeno M.A. Casado M. Heredia M. Sanz P. Astrocytes: new players in progressive myoclonus epilepsy of Lafora type.Hum. Mol. Genet. 2018; 27: 1290-1300Crossref PubMed Scopus (25) Google Scholar). Furthermore, it was found that the ratio between astrocytic and neuronal LBs can be different in different brain regions (21Augé E. Pelegrí C. Manich G. Cabezón I. Guinovart J.J. Duran J. Vilaplana J. Astrocytes and neurons produce distinct types of polyglucosan bodies in Lafora disease.Glia. 2018; 66: 2094-2107Crossref PubMed Scopus (25) Google Scholar). Assuming this ratio is different in the hippocampus and cerebellum, cell type–specific differences in Cre recombination could explain the different extent of rescue seen in these two brain regions. Furthermore, glycogen metabolism in astrocytes is very different from glycogen metabolism in neurons (28Saez I. Duran J. Sinadinos C. Beltran A. Yanes O. Tevy M.F. Martinez-Pons C. Milan M. Guinovart J.J. Neurons have an active glycogen metabolism that contributes to tolerance to hypoxia.J. Cereb. Blood Flow Metab. 2014; 34: 945-955Crossref PubMed Scopus (111) Google Scholar, 29Dienel G.A. The metabolic trinity, glucose-glycogen-lact

Genetic sequencing technologies have led to an increase in the identification and characterization of monogenic epilepsy syndromes. This increase has, in turn, generated strong interest in developing "precision therapies" based on the unique molecular genetics of a given monogenic epilepsy syndrome. These therapies include diets, vitamins, cell-signaling regulators, ion channel modulators, repurposed medications, molecular chaperones, and gene therapies. In this review, we evaluate these therapies from the perspective of their clinical validity and discuss the future of these therapies for individual syndromes.

Adult polyglucosan body disease (APBD) and Lafora disease (LD) are autosomal recessive glycogen storage neurological disorders. APBD is caused by mutations in the glycogen branching enzyme (GBE1) gene and is characterized by progressive upper and lower motor neuron dysfunction and premature death. LD is a fatal progressive myoclonus epilepsy caused by loss of function mutations in the EPM2A or EPM2B gene. These clinically distinct neurogenetic diseases share a common pathology. This consists of time-dependent formation, precipitation, and accumulation of an abnormal form of glycogen (polyglucosan) into gradually enlarging inclusions, polyglucosan bodies (PBs) in ever-increasing numbers of neurons and astrocytes. The growth and spread of PBs are followed by astrogliosis, microgliosis, and neurodegeneration. The key defect in polyglucosans is that their glucan branches are longer than those of normal glycogen, which prevents them from remaining in solution. Since the lengths of glycogen branches are determined by the enzyme glycogen synthase, we hypothesized that downregulating this enzyme could prevent or hinder the generation of the pathogenic PBs. Here, we pursued an adeno-associated virus vector (AAV) mediated RNA-interference (RNAi) strategy. This approach resulted in approximately 15% reduction of glycogen synthase mRNA and an approximately 40% reduction of PBs across the brain in the APBD and both LD mouse models. This was accompanied by improvements in early neuroinflammatory markers of disease. This work represents proof of principle toward developing a single lifetime dose therapy for two fatal neurological diseases: APBD and LD. The approach is likely applicable to other severe and common diseases of glycogen storage.

Lafora disease is a progressive myoclonus epilepsy with polyglucosan accumulations and a peculiar neurodegeneration with generalised organellar disintegration. It causes severe seizures, leading to dementia and eventually death in early adulthood.One Lafora disease gene, EPM2A, has been identified on chromosome 6q24. Locus heterogeneity led us to search for a second gene using a genome wide linkage scan in French-Canadian families.We mapped a second Lafora disease locus, EPM2B, to a 2.2 Mb region at 6p22, a region known to code for several proteins, including kinesins. Kinesins are microtubule dependent motor proteins that are involved in transporting cellular components. In neurones, they play a major role in axonal and dendritic transport.Analysis of the present locus in other non-EPM2A families will reveal whether there is further locus heterogeneity. Identification of the disease gene will be of major importance towards our understanding of the pathogenesis of Lafora disease.

Progressive myoclonus epilepsy of the Unverricht-Lundborg type (EPM1) is a recessively inherited neurodegenerative disease caused by loss-of-function mutations in the gene encoding cystatin B, a cysteine protease inhibitor. Mice with disruptions in this gene display myoclonic seizures, progressive ataxia, and cerebellar pathology closely paralleling EPMI in humans. To provide further insight into our understanding of EPM1, we report pathological findings in brains from cystatin B-deficient mice. In addition to confirming the loss of cerebellar granular cell neurons by apoptosis, we identified additional neuronal apoptosis in young mutant mice (3-4 months old) within the hippocampal formation and entorhinal cortex. In older mutant mice (16-18 months old), there was also gliosis most marked in the presubiculum and parasubiculum of the hippocampal formation, as well as the entorhinal cortex, neocortex, and striatum. Furthermore, widespread white matter gliosis was also noted, which may be a secondary phenomenon. Within the cerebral cortex, cellular atrophy was a prominent finding in the superficial neurons of the prosubiculum. Finally, we show that mutant mice in either a "seizure-prone" or "seizure-resistant" genetic background display similar neuropathological changes. These findings indicate that neuronal atrophy is an important consequence of cystatin-B deficiency independent of seizure events, suggesting a physiological role for this protein in the maintenance of normal neuronal structure.

EPM2B mutations have been found in a variable proportion of patients with Lafora disease (LD). Genotype-phenotype correlations suggested that EPM2B patients show a slower course of the disease, with delayed age at death, compared with EPM2A patients. We herein report clinical and genetic findings of 26 Italian LD patients.Disease progression was evaluated by means of a disability scale based on residual motor and cognitive functions and daily living and social abilities, at 4 years from the onset. Mutational analysis was performed by sequencing the coding regions of the EPM2A and EPM2B genes.Age at onset ranged from 8.5 to 18.5 years (mean, 13.7+/-2.6). The mean duration of follow-up was 7.1+/-3.9 years. Daily living activities and social interactions were preserved in five of 24 patients. The remaining patients showed moderate to extremely severe limitations of daily living and social abilities. Sixteen (72%) of 22 families showed mutations in the EPM2B gene, and five (22%), in the EPM2A gene. One family showed no mutations. A novel EPM2B mutation also was identified.In our series, EPM2B mutations occurred in 72% of families, thus indicating that EPM2B is the major gene for LD in the Italian population. Moreover, we found that six of 17 EPM2B patients preserved daily living activities and social interactions at 4 years from onset, suggesting a slow disease progression. Additional clinical and functional studies will clarify whether specific mutations may influence the course of the disease in LD patients.

X-linked myopathy with excessive autophagy (XMEA) is a childhood-onset disease characterized by progressive vacuolation and atrophy of skeletal muscle. We show that XMEA is caused by hypomorphic alleles of the VMA21 gene, that VMA21 is the diverged human ortholog of the yeast Vma21p protein, and that like Vma21p it is an essential assembly chaperone of the V-ATPase, the principal mammalian proton pump complex. Decreased VMA21 raises lysosomal pH, which reduces lysosomal degradative ability and blocks autophagy. This reduces cellular free amino acids, which upregulates the mTOR pathway and mTOR-dependent macroautophagy, resulting in proliferation of large and ineffective autolysosomes that engulf sections of cytoplasm, merge together, and vacuolate the cell. Our results uncover macroautophagic overcompensation leading to cell vacuolation and tissue atrophy as a mechanism of 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.

Synaptic function is central to brain function. Understanding the synapse is aided by studies of patients lacking individual synaptic proteins. Common neurological diseases are genetically complex. Their understanding is likewise simplified by studies of less common monogenic forms. We detail the disease caused by absence of the synaptic protein CNKSR2 in 8 patients ranging from 6 to 62 years old. The disease is characterized by intellectual disability, attention problems, and abrupt lifelong language loss following a brief early childhood epilepsy with continuous spike-waves in sleep. This study describes the phenotype of CNKSR2 deficiency and its involvement in systems underlying common neurological disorders.

Summary We report clinical, neurophysiologic, and genetic features of an Italian series of patients with Lafora disease ( LD ) to identify distinguishing features of those with a slowly progressive course. Twenty‐three patients with LD (17 female; 6 male) were recruited. Mean age (± SD) at the disease onset was 14.5 ± 3.9 years and mean follow‐up duration was 13.2 ± 8.0 years. NHLRC 1 mutations were detected in 18 patients; EPM 2A mutations were identified in 5. Patients who maintained &gt;10 years gait autonomy were labeled as “mild” and were compared with the remaining LD patients with a typical course. Six of 23 patients were mild and presented significantly delay in the age at onset, lower neurologic disability score at 4 years after the onset, less severe seizure phenotype, lower probability of showing both photoparoxysmal response on electroencephalography ( EEG ) and giant somatosensory evoked potentials, as compared to patients with typical LD . However, in both mild and typical LD patients, EEG showed disorganization of background activity and frequent epileptiform abnormalities. Mild LD patients had NHLRC 1 mutations and five of six carried homozygous or compound heterozygous D146N mutation. This mutation was found in none of the patients with typical LD . The occurrence of specific NHLRC 1 mutations in patients with mild LD should be taken into account in clinical practice for appropriate management and counseling.

We aimed to generate a review and description of the phenotypic and genotypic spectra of ARHGEF9 mutations.Patients with mutations or chromosomal disruptions affecting ARHGEF9 were identified through our clinics and review of the literature. Detailed medical history and examination findings were obtained via a standardized questionnaire, or if this was not possible by reviewing the published phenotypic features.A total of 18 patients (including 5 females) were identified. Six had de novo, 5 had maternally inherited mutations, and 7 had chromosomal disruptions. All females had strongly skewed X-inactivation in favor of the abnormal X-chromosome. Symptoms presented in early childhood with delayed motor development alone or in combination with seizures. Intellectual disability was severe in most and moderate in patients with milder mutations. Males with severe intellectual disability had severe, often intractable, epilepsy and exhibited a particular facial dysmorphism. Patients with mutations in exon 9 affecting the protein's PH domain did not develop epilepsy.ARHGEF9 encodes a crucial neuronal synaptic protein; loss of function of which results in severe intellectual disability, epilepsy, and a particular facial dysmorphism. Loss of only the protein's PH domain function is associated with the absence of epilepsy.

Voltage-gated sodium channels (Navs) are mainstays of neuronal function, and mutations in the genes encoding CNS Navs (Nav1.1 [SCN1A], Nav1.2 [SCN2A], Nav1.3 [SCN3A], and Nav1.6 [SCN8A]) are causes of some of the most common and severe genetic epilepsies and epileptic encephalopathies (EE).1 Fibroblast-growth-factor homologous factors (FHFs) compose a family of 4 proteins that interact with the C-terminal tails of Navs to modulate the channels' fast, and long-term, inactivations.2FHF2 mutation is a rare cause of generalized epilepsy with febrile seizures plus (GEFS+).3 Recently, a de novo FHF1 mutation (p.R52H) was reported in early-onset EE in 2 siblings.4 We report 3 patients from unrelated families with the same FHF1 p.R52H mutation. The 5 cases together frame the FHF1 R52H EE from infancy to adulthood. As discussed below, this gain-of-function disease may be amenable to personalized therapy.

Abstract The developmental and epileptic encephalopathies (DEE) are a group of rare, severe neurodevelopmental disorders, where even the most thorough sequencing studies leave 60–65% of patients without a molecular diagnosis. Here, we explore the incompleteness of transcript models used for exome and genome analysis as one potential explanation for a lack of current diagnoses. Therefore, we have updated the GENCODE gene annotation for 191 epilepsy-associated genes, using human brain-derived transcriptomic libraries and other data to build 3,550 putative transcript models. Our annotations increase the transcriptional ‘footprint’ of these genes by over 674 kb. Using SCN1A as a case study, due to its close phenotype/genotype correlation with Dravet syndrome, we screened 122 people with Dravet syndrome or a similar phenotype with a panel of exon sequences representing eight established genes and identified two de novo SCN1A variants that now - through improved gene annotation - are ascribed to residing among our exons. These two (from 122 screened people, 1.6%) molecular diagnoses carry significant clinical implications. Furthermore, we identified a previously classified SCN1A intronic Dravet syndrome-associated variant that now lies within a deeply conserved exon. Our findings illustrate the potential gains of thorough gene annotation in improving diagnostic yields for genetic disorders.

Longer glucan chains tend to precipitate. Glycogen, by far the largest mammalian glucan and the largest molecule in the cytosol with up to 55 000 glucoses, does not, due to a highly regularly branched spherical structure that allows it to be perfused with cytosol. Aberrant construction of glycogen leads it to precipitate, accumulate into polyglucosan bodies that resemble plant starch amylopectin and cause disease. This pathology, amylopectinosis, is caused by mutations in a series of single genes whose functions are under active study toward understanding the mechanisms of proper glycogen construction. Concurrently, we are characterizing the physicochemical particularities of glycogen and polyglucosans associated with each gene. These genes include GBE1, EPM2A and EPM2B, which respectively encode the glycogen branching enzyme, the glycogen phosphatase laforin and the laforin-interacting E3 ubiquitin ligase malin, for which an unequivocal function is not yet known. Mutations in GBE1 cause a motor neuron disease (adult polyglucosan body disease), and mutations in EPM2A or EPM2B a fatal progressive myoclonus epilepsy (Lafora disease). RBCK1 deficiency causes an amylopectinosis with fatal skeletal and cardiac myopathy (polyglucosan body myopathy 1, OMIM# 615895). RBCK1 is a component of the linear ubiquitin chain assembly complex, with unique functions including generating linear ubiquitin chains and ubiquitinating hydroxyl (versus canonical amine) residues, including of glycogen. In a mouse model we now show (i) that the amylopectinosis of RBCK1 deficiency, like in adult polyglucosan body disease and Lafora disease, affects the brain; (ii) that RBCK1 deficiency glycogen, like in adult polyglucosan body disease and Lafora disease, has overlong branches; (iii) that unlike adult polyglucosan body disease but like Lafora disease, RBCK1 deficiency glycogen is hyperphosphorylated; and finally (iv) that unlike laforin-deficient Lafora disease but like malin-deficient Lafora disease, RBCK1 deficiency's glycogen hyperphosphorylation is limited to precipitated polyglucosans. In summary, the fundamental glycogen pathology of RBCK1 deficiency recapitulates that of malin-deficient Lafora disease. Additionally, we uncover sex and genetic background effects in RBCK1 deficiency on organ- and brain-region specific amylopectinoses, and in the brain on consequent neuroinflammation and behavioural deficits. Finally, we exploit the portion of the basic glycogen pathology that is common to adult polyglucosan body disease, both forms of Lafora disease and RBCK1 deficiency, namely overlong branches, to show that a unified approach based on downregulating glycogen synthase, the enzyme that elongates glycogen branches, can rescue all four diseases.

<h2>Summary</h2><h3>Background</h3> The developmental and epileptic encephalopathies (DEEs) are the most severe group of epilepsies which co-present with developmental delay and intellectual disability (ID). DEEs usually occur in people without a family history of epilepsy and have emerged as primarily monogenic, with damaging rare mutations found in 50% of patients. Little is known about the genetic architecture of patients with DEEs in whom no pathogenic variant is identified. Polygenic risk scoring (PRS) is a method that measures a person's common genetic burden for a trait or condition. Here, we used PRS to test whether genetic burden for epilepsy is relevant in individuals with DEEs, and other forms of epilepsy with ID. <h3>Methods</h3> Genetic data on 2,759 cases with DEEs, or epilepsy with ID presumed to have a monogenic basis, and 447,760 population-matched controls were analysed. We compared PRS for 'all epilepsy', 'focal epilepsy', and 'genetic generalised epilepsy' (GGE) between cases and controls. We performed pairwise comparisons between cases stratified for identifiable rare deleterious genetic variants and controls. <h3>Findings</h3> Cases of presumed monogenic severe epilepsy had an increased PRS for 'all epilepsy' (p<0.0001), 'focal epilepsy' (p<0.0001), and 'GGE' (p=0.0002) relative to controls, which explain between 0.08% and 3.3% of phenotypic variance. PRS was increased in cases both with and without an identified deleterious variant of major effect, and there was no significant difference in PRS between the two groups. <h3>Interpretation</h3> We provide evidence that common genetic variation contributes to the aetiology of DEEs and other forms of epilepsy with ID, even when there is a known pathogenic variant of major effect. These results provide insight into the genetic underpinnings of the severe epilepsies and warrant a shift in our understanding of the aetiology of the DEEs as complex, rather than monogenic, disorders. <h3>Funding</h3> Science foundation Ireland, Human Genome Research Institute; National Heart, Lung, and Blood Institute; German Research Foundation.

Lafora disease is characterized by pathognomonic inclusions, Lafora bodies (LB), in neurons and other cell types. In skin, LB have been reported in either eccrine sweat glands or in apocrine sweat glands. The disease is caused by mutations in either the EPM2A gene or in a second yet-unknown gene. Here the authors determine whether a genotype-phenotype correlation exists between the genetic form of the disease and the skin cell type affected by LB formation. Also is described an important source of false positivity in the use of axillary biopsies for disease diagnosis.

Progressive Myoclonus Epilepsy (PME) of the Lafora type is an autosomal recessive disease, which presents in teenage years with myoclonia and generalized seizures leading to death within a decade of onset. It is characterized by pathognomonic inclusions, Lafora bodies (LB), in neurons and other cell types. Two genes causing Lafora disease (LD), EPM2A on chromosome 6q24 and NHLRC1 (EPM2B) on chromosome 6p22.3 have been identified, and our recent results indicate there is at least one other gene causing the disease. The EPM2A gene product, laforin, is a protein tyrosine phosphatase (PTP) with a carbohydrate-binding domain (CBD) in the N-terminus. NHLRC1 encodes a protein named malin, containing a zinc finger of the RING type in the N-terminal half and 6 NHL-repeat domains in the C-terminal direction. To date 43 different variations in EPM2A and 23 in NHLRC1 are known, including missense, nonsense, frameshift, and deletions. We have developed a human LD mutation database using a new generic biological database cross-referencing platform. The database, which currently contains 66 entries is accessible on the World Wide Web (http://projects.tcag.ca/lafora). Entries can be submitted via the curator of the database or via a web-based form. © 2005 Wiley-Liss, Inc.

A 20-year-old woman presented to a specialist epilepsy center with a 3-year history of drug-resistant epileptic seizures, progressive myoclonus, ataxia, and cognitive decline.Neurological examination, neuropsychological testing, electrophysiological studies, skin biopsy, MRI, genetic testing, and autopsy.Lafora disease (EPM2), resulting from a homozygous missense mutation in EPM2B (NHLRC1; c205C>G; Pro69Ala).Symptomatic treatment with conventional antiepileptic and antimyoclonic drugs.

The progressive myoclonus epilepsies (PMEs) consist of a group of diseases with myoclonic seizures and progressive neurodegeneration, with onset in childhood and/or adolescence. Lafora disease is a neuronal glycogenosis in which normal glycogen is transformed into starch-like polyglucosans that accumulate in the neuronal somatodendritic compartment. It is caused by defects of two genes of yet unknown function, one encoding a glycogen phosphatase (laforin) and the other an ubiquitin E3 ligase (malin). Early cognitive deterioration, visual seizures affecting over half, and slowing down of EEG basic activity are three major diagnostic clues. Unverricht–Lundborg disease is presently thought to be due to damage to neurons by lysosomal cathepsins and reactive oxygen species due to absence of cystatin B, a small protein that inactivates cathepsins and, by ways yet unknown, quenches damaging redox compounds. Preserved cognition and background EEG activity, action myoclonus early morning and vertex spikes in REM sleep are the diagnostic clues. Sialidosis, with cherry-red spot, neuronopathic Gaucher disease, with paralysis of verticality, and ataxia-PME, with ataxia at onset in the middle of the first decade, are also lysosomal diseases. How the lysosomal defect culminates in myoclonus and epilepsy in these conditions remains unknown.

Alternating hemiplegia of childhood is a rare disorder caused by de novo mutations in the ATP1A3 gene, expressed in neurons and cardiomyocytes. As affected individuals may survive into adulthood, we use the term 'alternating hemiplegia'. The disorder is characterized by early-onset, recurrent, often alternating, hemiplegic episodes; seizures and non-paroxysmal neurological features also occur. Dysautonomia may occur during hemiplegia or in isolation. Premature mortality can occur in this patient group and is not fully explained. Preventable cardiorespiratory arrest from underlying cardiac dysrhythmia may be a cause. We analysed ECG recordings of 52 patients with alternating hemiplegia from nine countries: all had whole-exome, whole-genome, or direct Sanger sequencing of ATP1A3. Data on autonomic dysfunction, cardiac symptoms, medication, and family history of cardiac disease or sudden death were collected. All had 12-lead electrocardiogram recordings available for cardiac axis, cardiac interval, repolarization pattern, and J-point analysis. Where available, historical and prolonged single-lead electrocardiogram recordings during electrocardiogram-videotelemetry were analysed. Half the cohort (26/52) had resting 12-lead electrocardiogram abnormalities: 25/26 had repolarization (T wave) abnormalities. These abnormalities were significantly more common in people with alternating hemiplegia than in an age-matched disease control group of 52 people with epilepsy. The average corrected QT interval was significantly shorter in people with alternating hemiplegia than in the disease control group. J wave or J-point changes were seen in six people with alternating hemiplegia. Over half the affected cohort (28/52) had intraventricular conduction delay, or incomplete right bundle branch block, a much higher proportion than in the normal population or disease control cohort (P = 0.0164). Abnormalities in alternating hemiplegia were more common in those ≥16 years old, compared with those <16 (P = 0.0095), even with a specific mutation (p.D801N; P = 0.045). Dynamic, beat-to-beat or electrocardiogram-to-electrocardiogram, changes were noted, suggesting the prevalence of abnormalities was underestimated. Electrocardiogram changes occurred independently of seizures or plegic episodes. Electrocardiogram abnormalities are common in alternating hemiplegia, have characteristics reflecting those of inherited cardiac channelopathies and most likely amount to impaired repolarization reserve. The dynamic electrocardiogram and neurological features point to periodic systemic decompensation in ATP1A3-expressing organs. Cardiac dysfunction may account for some of the unexplained premature mortality of alternating hemiplegia. Systematic cardiac investigation is warranted in alternating hemiplegia of childhood, as cardiac arrhythmic morbidity and mortality are potentially preventable.

Abstract Objective To investigate the genetic basis of the recessive form of primary familial brain calcification and study pathways linking a novel gene with known dominant genes that cause the disease. Methods Whole exome sequencing and Sanger‐based segregation analysis were used to identify possible disease causing mutations. Mutation pathogenicity was validated by structural protein modeling. Functional associations between the candidate gene, MYORG , and genes previously implicated in the disease were examined through phylogenetic profiling. Results We studied nine affected individuals from two unrelated families of Middle Eastern origin. The median age of symptom onset was 29.5 years (range 21–57 years) and dysarthria was the most common presenting symptom. We identified in the MYORG gene, a homozygous c.1233delC mutation in one family and c.1060_1062del GAC mutation in another. The first mutation results in protein truncation and the second in deletion of a highly conserved aspartic acid that is likely to disrupt binding of the protein with its substrate. Phylogenetic profiling analysis of the MYORG protein sequence suggests co‐evolution with a number of calcium channels as well as other proteins related to regulation of anion transmembrane transport (False Discovery Rate, FDR &lt; 10 −8 ) and with PDCD 6 IP , a protein interacting with PDGFR β which is known to be involved in the disease. Interpretation MYORG mutations are linked to a recessive form of primary familial brain calcification. This association was recently described in patients of Chinese ancestry. We suggest the possibility that MYORG mutations lead to calcification in a PDGFR β ‐related pathway.

Lafora disease (LD) is the only progressive myoclonus epilepsy with polyglucosan bodies. Among conditions with polyglucosan bodies, LD is unique for the subcellular location of its polyglucosans in neuronal perikarya and dendrites and not in axons. Here we report that the protein encoded by the EPM2A gene, which is mutated in LD, localizes at the plasma membrane and the endoplasmic reticulum and that it is a functional protein tyrosine phosphatase. The significance of these findings in the epilepsy of LD and in the origin and characteristic subcellular location of Lafora bodies is discussed.

The neuronal ceroid lipofuscinoses (NCLs) are a heterogeneous group of autosomal recessive neurodegenerative diseases comprising Batten and other related diseases plus numerous variants. They are characterized by progressive neuronal cell death. The CLN6 gene was recently identified, mutations in which cause one of the variant late infantile forms of NCL (vLINCL). We describe four novel mutations in the CLN6 gene. This brings the total number of CLN6 mutations known to 11 in 38 families. This suggests that the CLN6 gene may be highly mutable. An American patient of Irish/French/Native American origin was heterozygous for a 4-bp insertion (c.267_268insAACG) in exon 3. The other allele had a point mutation (c.898T>C) in exon 7 resulting in a W300R amino acid change. Two Trinidadian siblings of Indian origin were homozygous for a mutation at the 5' donor splice site of exon 4 (IVS4+1G>T), affecting the first base of the invariant GT at the beginning of intron 4. The fourth novel mutation, a double deletion of 4 bp and 1 bp in exon 7 (c.829_832delGTCG;c.837delG), was identified in a Portuguese patient heterozygous for the I154del Portuguese CLN6 mutation. Four of the 11 mutations identified are in exon 4. Three Portuguese patients with clinical profiles similar to CLN6 patients without defects in CLN6 or other known NCL genes are described. We conclude the following: 1) the CLN6 gene may be a highly mutable gene; 2) exon 4 must code for a segment of the protein crucial for function; 3) vLINCL disease in Portugal is genetically heterogeneous; 4) the I154del accounts for 81.25% of affected CLN6 Portuguese alleles; and 5) three vLINCL Portuguese patients may have defects in a new NCL gene.

Lafora disease (LD) can be diagnosed by skin biopsy, but this approach has both false negatives and false positives. Biopsies of other organs can also be diagnostic but are more invasive. Genetic diagnosis is also possible but can be inconclusive, for example, in patients with only one heterozygous <i>EPM2A</i> mutation and patients with apparently homozygous <i>EPM2B</i> mutations where one parent is not a carrier of the mutation. We sought to identify occult mutations and clarify the genotypes and confirm the diagnosis of LD in patients with apparent nonrecessive disease inheritance. We used single nucleotide polymorphism, quantitative PCR, and fluorescent in situ hybridization analyses. We identified large <i>EPM2A</i> and <i>EPM2B</i> deletions undetectable by PCR in the heterozygous state and describe simple methods for their routine detection. We report a coding sequence change in several patients and describe why the pathogenic role of this change remains unclear. We confirm that adult-onset LD is due to <i>EPM2B</i> mutations. Finally, we report major intrafamilial heterogeneity in age at onset in LD.

X-linked Myopathy with Excessive Autophagy (XMEA) affects proximal muscles of the lower extremities and follows a progressive course reminiscent of muscular dystrophy. It is caused by mutations in VMA21 whose protein product assembles lysosomes' proton pumps. All XMEA mutations to date have been single-nucleotide substitutions that reduce VMA21 expression, which leads to modest lysosomal pH increase, the first step in the disease's pathogenesis. We now report a new class of XMEA mutations. We identified two VMA21 non-coding microdeletions, one intronic (c.54-16_54-8del), the other in the 3'UTR (c.*13_*104del). Both resulted in a relatively more severe (early ambulation loss), diffuse (extra-ocular and upper extremity involvement), and early (neonatal) onset disease compared to previously reported patients. Our cases highlight the importance of including non-coding regions of VMA21 in genetic testing panels of dystrophies and myopathies. Specific diagnosis of XMEA will be particularly important as therapies aimed at correcting the modest rise in lysosomal pH at the root of this disease are developed.

Creatine transporter deficiency (CRTR-D) is an X-linked inherited disorder of creatine transport. All males and about 50% of females have intellectual disability or cognitive dysfunction. Creatine deficiency on brain proton magnetic resonance spectroscopy and elevated urinary creatine to creatinine ratio are important biomarkers. Mutations in the SLC6A8 gene occur de novo in 30% of males. Despite reports of high prevalence of CRTR-D in males with intellectual disability, there are no true prevalence studies in the general population. To determine carrier frequency of CRTR-D in the general population we studied the variants in the SLC6A8 gene reported in the Exome Variant Server database and performed functional characterization of missense variants. We also analyzed synonymous and intronic variants for their predicted pathogenicity using in silico analysis tools. Nine missense variants were functionally analyzed using transient transfection by site-directed mutagenesis with In-Fusion HD Cloning in HeLa cells. Creatine uptake was measured by liquid chromatography tandem mass spectrometry for creatine measurement. The c.1654G>T (p.Val552Leu) variant showed low residual creatine uptake activity of 35% of wild type transfected HeLa cells and was classified as pathogenic. Three variants (c.808G>A; p.Val270Met, c.942C>G; p.Phe314Leu and c.952G>A; p.Ala318Thr) were predicted to be pathogenic based on in silico analysis, but proved to be non-pathogenic by our functional analysis. The estimated carrier frequency of CRTR-D was 0.024% in females in the general population. We recommend functional studies for all novel missense variants by transient transfection followed by creatine uptake measurement by liquid chromatography tandem mass spectrometry as fast and cost effective method for the functional analysis of missense variants in the SLC6A8 gene.

Lafora disease is a fatal genetic disorder characterised by neurotoxic deposits of malformed insoluble glycogen. In humans it is caused by mutation in the EPM2A or NHLRC1 genes. There is a known mutation in miniature wirehaired dachshunds which has not been documented in other dog breeds, including beagles, in which the disease is relatively commonly reported. This case report describes the causative defect in two affected beagles, namely the same massive expansion as in miniature wirehaired dachshunds of a 12-nucleotide repeat sequence that is unique to the canine NHLRC1 gene. This is the first mutation described in beagles with Lafora disease, and so far the only Lafora disease genetic variant in dogs.

Background: Progressive myoclonus epilepsies (PMEs) are a group of rare inherited diseases featuring a combination of myoclonus, seizures and variable degree of cognitive impairment. Despite extensive investigations, a large number of PMEs remain undiagnosed. In this review, we focus on the current pharmacological approach to PMEs. Keywords: seizures, epilepsy, antiepileptic drugs, myoclonic jerks, therapy, photoparoxysmal response.

Lafora disease (LD) is an autosomal recessive late onset, progressive myoclonic epilepsy with a high prevalence in the miniature Wirehaired Dachshund. The disease is due to a mutation in the Epm2b gene which results in intracellular accumulation of abnormal glycogen (Lafora bodies). Recent breed-wide testing suggests that the carrier plus affected rate may be as high as 20%. A characteristic feature of the disease is spontaneous and reflex myoclonus; however clinical signs and disease progression are not well described. A survey was submitted to owners of MWHD which were homozygous for Epm2b mutation (breed club testing program) or had late onset reflex myoclonus and clinical diagnosis of LD. There were 27 dogs (11 male; 16 female) for analysis after young mutation-positive dogs that had yet to develop disease were excluded. Average age of onset of clinical signs was 6.94 years (3.5–12). The most common initial presenting sign was reflex and spontaneous myoclonus (77.8%). Other presenting signs included hypnic myoclonus (51.9%) and generalized seizures (40.7%). Less common presenting signs include focal seizures, "jaw smacking", "fly catching", "panic attacks", impaired vision, aggression and urinary incontinence. All these clinical signs may appear, and then increase in frequency and intensity over time. The myoclonus in particular becomes more severe and more refractory to treatment. Signs that developed later in the disease include dementia (51.9%), blindness (48.1%), aggression to people (25.9%) and dogs (33.3%), deafness (29.6%) and fecal (29.6%) and urinary (37.0%) incontinence as a result of loss of house training (disinhibited type behavior). Further prospective study is needed to further characterize the canine disease and to allow more specific therapeutic strategies and to tailor therapy as the disease progresses.

Abstract The history of the progressive myoclonus epilepsies (PMEs) spans more than a century. However, the recent history of PMEs begins with a consensus statement published in the wake of the Marseille PME workshop in 1989 (Marseille Consensus Group, ). This consensus helped define the various types of PME known at the time and set the agenda for a new era of genetic research which soon lead to the discovery of many PME genes. Prior to the Marseille meeting, and before the molecular era, there had been much confusion and controversy. Because investigators had but limited and biased experience with these rare disorders due to the uneven, skewed distribution of PMEs around the world, opinions and nosologies were based on local expertise which did not match well with the experiences of other researchers and clinicians. The three major areas of focus included: (1) the nature and limits of the concept of PME in varying scopes, which was greatly debated; (2) the description of discrete clinical entities by clinicians; and (3) the description of markers (pathological, biological, neurophysiological, etc .) which could lead to a precise diagnosis of a given PME type, with, in the best cases, a reliable correlation with clinical findings. In this article, we shall also examine the breakthroughs achieved in the wake of the 1989 Marseille meeting and recent history in the field, following the identification of several PME genes. As in other domains, the molecular and genetic approach has challenged some established concepts and has led to the description of new PME types. However, as may already be noted, this approach has also confirmed the existence of the major, established types of PME, which can now be considered as true diseases.

This work employs adult polyglucosan body disease (APBD) models to explore the efficacy and mechanism of action of the polyglucosan-reducing compound 144DG11. APBD is a glycogen storage disorder (GSD) caused by glycogen branching enzyme (GBE) deficiency causing accumulation of poorly branched glycogen inclusions called polyglucosans. 144DG11 improved survival and motor parameters in a GBE knockin (Gbeys/ys ) APBD mouse model. 144DG11 reduced polyglucosan and glycogen in brain, liver, heart, and peripheral nerve. Indirect calorimetry experiments revealed that 144DG11 increases carbohydrate burn at the expense of fat burn, suggesting metabolic mobilization of pathogenic polyglucosan. At the cellular level, 144DG11 increased glycolytic, mitochondrial, and total ATP production. The molecular target of 144DG11 is the lysosomal membrane protein LAMP1, whose interaction with the compound, similar to LAMP1 knockdown, enhanced autolysosomal degradation of glycogen and lysosomal acidification. 144DG11 also enhanced mitochondrial activity and modulated lysosomal features as revealed by bioenergetic, image-based phenotyping and proteomics analyses. As an effective lysosomal targeting therapy in a GSD model, 144DG11 could be developed into a safe and efficacious glycogen and lysosomal storage disease therapy.

We have identified an interacting partner protein (encoded by the human EPM2AIP1 gene (approved symbol)) for laforin, the product of the EPM2A gene, which is mutated in an autosomal recessive form of adolescent progressive myoclonus epilepsy. The EPM2AIP1 gene was identified in a screen for laforin-interacting proteins with a human brain cDNA library using the yeast two-hybrid system. The specificity of the interaction was confirmed by coimmunoprecipitation of in vivo-transfected protein and by using EPM2A deletion constructs. Subcellular colocalization of laforin and EPM2AIP1 protein was also demonstrated. The human EPM2AIP1 gene, corresponding to the KIAA0766 cDNA clone in the databases, was characterized and shown, like EPM2A, to be ubiquitously expressed. The gene, which comprises one large exon 1824 nucleotides in length and has alternative 3′ untranslated regions, maps to human chromosome 3p22.1. The function is currently not known and extensive analyses do not reveal any homology to other proteins or any obvious structural motifs. Because genetic heterogeneity in Lafora disease has been described, mutational analysis of the EPM2AIP1 gene was performed on non-EPM2A patients, but no mutations were found. The identification of this first binding partner for laforin promises to be an important step toward unraveling the underlying pathogenesis of this severest form of teenage-onset epilepsy.

Lafora disease is the most severe teenage-onset progressive epilepsy, a unique form of glycogenosis with perikaryal accumulation of an abnormal form of glycogen, and a neurodegenerative disorder exhibiting an unusual generalized organellar disintegration. The disease is caused by mutations of the EPM2A gene, which encodes two isoforms of the laforin protein tyrosine phosphatase, having alternate carboxyl termini, one localized in the cytoplasm (endoplasmic reticulum) and the other in the nucleus. To date, all documented disease mutations, including the knockout mouse model deletion, have been in the segment of the protein common to both isoforms. It is therefore not known whether dysfunction of the cytoplasmic, nuclear, or both isoforms leads to the disease. In the present work, we identify six novel mutations, one of which, c.950insT (Q319fs), is the first mutation specific to the cytoplasmic laforin isoform, implicating this isoform in disease pathogenesis. To confirm this mutation's deleterious effect on laforin, we studied the resultant protein's subcellular localization and function and show a drastic reduction in its phosphatase activity, despite maintenance of its location at the endoplasmic reticulum. Hum Mutat 23:170–176, 2004. © 2003 Wiley-Liss, Inc.

Laforin, encoded by the EPM2A gene, by sequence is a member of the dual specificity protein phosphatase family. 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, muscle and other tissues. Glycogen metabolizing enzymes were analyzed in a transgenic mouse over-expressing a dominant negative form of laforin that accumulates Lafora bodies in several tissues. Skeletal muscle glycogen was increased 2-fold as was the total glycogen synthase protein. However, the −/+glucose-6-P activity of glycogen synthase was decreased from 0.29 to 0.16. Branching enzyme activity was increased by 30%. Glycogen phosphorylase activity was unchanged. In whole brain, no differences in glycogen synthase or branching enzyme activities were found. Although there were significant differences in enzyme activities in muscle, the results do not support the hypothesis that Lafora body formation is caused by a major change in the balance between glycogen elongation and branching activities.

Progressive myoclonus epilepsies are severe, intractable, and neurodegenerative. They afflict patients of all ages, but more commonly adolescents, and comprise the main differential diagnosis of common juvenile myoclonic epilepsy. Genetic or minimally invasive pathologic diagnoses are available for many but not all teenage-onset progressive myoclonus epilepsies. We describe a multiplex family with autosomal recessive teenage-onset progressive myoclonus epilepsy that had remained undiagnosed despite extensive genetic and pathologic testing. We describe whole exome sequencing combined with homozygosity mapping to identify the disease gene directly and diagnose the family. The affected gene is CLN6, previously known to underlie variant late-infantile and adult-onset neuronal ceroid lipofuscinoses. Combined with other recent work, our results add CLN6 to the genetic mutations causing teenage-onset progressive myoclonus epilepsy, expand the group of teenage-onset progressive myoclonus epilepsy patients who can be diagnosed by genetic testing, and extend the clinical spectrum of CLN6 mutations to include teenage-onset progressive myoclonus epilepsy. This work also exemplifies the potentiality of next-generation sequencing in the genetic identification and diagnosis of patients with neurologic diseases of unknown cause.

Abstract The overwhelming majority of Rett syndrome cases are caused by mutations in the gene MECP2 . MECP2 has two isoforms, termed MECP2_e1 and MECP2_e2, which differ in their N‐terminal amino acid sequences. A growing body of evidence has indicated that MECP2_e1 may be the etiologically relevant isoform in Rett Syndrome based on its expression profile in the brain and because, strikingly, no mutations have been discovered that affect MECP2_e2 exclusively. In this study we sought to characterize four classical Rett patients with mutations that putatively affect only the MECP2_e1 isoform. Our hypothesis was that the classical Rett phenotype seen here is the result of disrupted MECP2_e1 expression, but with MECP2_e2 expression unaltered. We used quantitative reverse transcriptase PCR to assay mRNA expression for each isoform independently, and used cytospinning methods to assay total MECP2 in peripheral blood lymphocytes (PBL). In the two Rett patients with identical 11 bp deletions within the coding portion of exon 1, MECP2_e2 levels were unaffected, whilst a significant reduction of MECP2_e1 levels was detected. In two Rett patients harboring mutations in the exon 1 start codon, MECP2_e1 and MECP2_e2 mRNA amounts were unaffected. In summary, we have shown that patients with exon 1 mutations transcribe normal levels of MECP2_e2 mRNA, and most PBL are positive for MeCP2 protein, despite them theoretically being unable to produce the MECP2_e1 isoform, and yet still exhibit the classical RTT phenotype. Altogether, our work further supports our hypothesis that MECP2_e1 is the predominant isoform involved in the neuropathology of Rett syndrome. © 2011 Wiley Periodicals, Inc.

Alternating hemiplegia of childhood and rapid-onset dystonia parkinsonism are two separate movement disorders with different dominant mutations in the same sodium-potassium transporter ATPase subunit gene, ATP1A3.We present a child with topiramate-responsive alternating hemiplegia of childhood who was tested for an ATP1A3 gene mutation.Gene sequencing revealed an identical ATP1A3 mutation as in three typical adult-onset rapid-onset dystonia parkinsonism cases but never previously described in an alternating hemiplegia of childhood case.The discordance of these phenotypes suggests that there are other undiscovered environmental, genetic, or epigenetic factors influencing the development of alternating hemiplegia of childhood or rapid-onset dystonia parkinsonism.

X-linked myopathy with excessive autophagy (XMEA), caused by mutations of the VMA21 gene, is a strictly skeletal muscle disease. Extensive studies in yeast established VMA21 as the master assembly chaperone of V-ATPase, the complex multisubunit proton pump that acidifies organelles and that is vital to all mammalian tissues. As such, skeletal muscle disease exclusivity in XMEA is highly surprising. We now show that the severest VMA21 mutation, c.164-6t>g, does result in XMEA-typical pathology with autophagic vacuolar changes outside skeletal muscle, namely in the heart. However, even patients with this mutation do not exhibit clinical extramuscular disease, including cardiac disease, despite extreme skeletal muscle wasting to the extent of ventilation dependence. Uncovering the unique skeletal muscle vulnerability to defective organellar acidification, and resultant tissue-destructive excessive autophagy, will be informative to the understanding of muscle physiology. Alternatively, understanding extramuscular resistance to VMA21 mutation might disclose heretofore unknown mammalian V-ATPase assembly chaperones other than VMA21.

<h2>Abstract</h2> Lafora disease (LD) is both a fatal childhood epilepsy and a glycogen storage disease caused by recessive mutations in either the <i>Epilepsy progressive myoclonus 2A</i> (<i>EPM2A</i>) or <i>EPM2B</i> genes. Hallmarks of LD are aberrant, cytoplasmic carbohydrate aggregates called Lafora bodies (LBs) that are a disease driver. The 5th International Lafora Epilepsy Workshop was recently held in Alcala de Henares, Spain. The workshop brought together nearly 100 clinicians, academic and industry scientists, trainees, National Institutes of Health (NIH) representation, and friends and family members of patients with LD. The workshop covered aspects of LD ranging from defining basic scientific mechanisms to elucidating a LD therapy or cure and a recently launched LD natural history study.