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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.

Progressive myoclonic epilepsy (PME) is a progressive neurological disorder. Unfortunately, until now, no definitive curative treatment exists; however, it is of utmost importance to identify patients with PME. The underlying aetiology can be pinpointed if methodological clinical evaluation is performed, followed by subsequent genetic testing. We report a case of PME that was diagnosed as Lafora body disease. This case emphasises that, suspecting and identifying PME is important so as to start appropriate treatment and reduce the probability of morbidity and prognosticate the family.

BackgroundLafora disease (LD) is a rare fatal autosomal recessive form of progressive myoclonus epilepsy. It affects previously healthy children or adolescents, causing pharmacoresistant epilepsy, myoclonus and severe psychomotor deterioration. This work aims to describe the clinical course of LD and identify predictors of outcome by means of a prognostic systematic review with individual participant data meta-analysis.MethodsA search was conducted on MEDLINE and Embase with no restrictions on publication date. Only studies reporting genetically confirmed LD cases were included. Kaplan–Meier estimate was used to assess probability of death and loss of autonomy. Univariable and multivariable Cox regression models with mixed effects (clustered survival data) were performed to evaluate prognostic factors.ResultsSeventy-three papers describing 298 genetically confirmed LD cases were selected. Mean age at disease onset was 13.4 years (SD 3.7), with 9.1% aged ≥ 18 years. Overall survival rates in 272 cases were 93% [95% CI 89–96] at 5 years, 62% [95% CI 54–69] at 10 years and 57% [95% CI 49–65] at 15 years. Median survival time was 11 years. The probability of loss of autonomy in 110 cases was 45% [95% CI 36–55] at 5 years, 75% [95% CI 66–84] at 10 years, and 83% [95% CI 74–90] at 15 years. Median loss of autonomy time was 6 years. Asian origin and age at onset ConclusionsThis study documented that half of patients survived at least 11 years. The notion of actual survival rate and prognostic factors is crucial to design studies on the effectiveness of upcoming new disease-modifying therapies.

Aim: To develop novel cationic liposomes as a nonviral gene delivery vector for the treatment of rare diseases, such as Lafora disease – a neurodegenerative epilepsy. Materials & methods: DLinDMA and DOTAP liposomes were formulated and characterized for the delivery of gene encoding laforin and expression of functional protein in HEK293 and neuroblastoma cells. Results: Liposomes with cationic lipids DLinDMA and DOTAP showed good physicochemical characteristics. Nanosized DLinDMA liposomes demonstrated desired transfection efficiency, negligible hemolysis and minimal cytotoxicity. Western blotting confirmed successful expression and glucan phosphatase assay demonstrated the biological activity of laforin. Conclusion: Our study is a novel preclinical effort in formulating cationic lipoplexes containing plasmid DNA for the therapy of rare genetic diseases such as Lafora 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.

We report a long-lived patient with Lafora disease (LD). A 34-year-old woman experienced onset of seizures at the age of 11 years. She was bedridden in her early twenties due to frequent generalized tonic-clonic seizures, myoclonus, and progressive mental deterioration. Her seizures occurred all the time despite administration of multiple anticonvulsants at high doses. At the age of 31, she started perampanel, which resulted in reduction of anticonvulsants after her visible myoclonus and convulsions disappeared. Brain magnetic resonance imaging showed marked cerebral and cerebellar atrophy, and single-photon emission computed tomography using N-isopropyl-p-[123I] iodoamphetamine (IMP-SPECT) revealed significant hypoperfusion of the frontal lobe and cerebellum. We identified a W219R homozygous mutation in exon 1 of the NHLRC1 gene. Because perampanel may not only control seizures but also prevent mental deterioration in LD, we propose that perampanel should be administered from the early stage of LD.

Early signs of Lafora disease can mimic a benign genetic generalized epilepsy of adolescence.

Lafora disease is a genetic disease caused, in humans, by mutations in EPM2A and NHLRC1 genes, resulting in accumulation of polyglucosan bodies within neurons. Affected subjects present progressive neurological signs characterised primarily by myoclonic epilepsy. In dogs, Lafora disease has been described mainly in miniature wire-haired Dachshunds, where a dodecamer expansion in NHLRC1 gene has been identified. The same mutation has then been detected in the Basset Hound, Beagle, Chihuahua and Pembroke Welsh Corgi breeds. This is the first case of a Newfoundland dog with myoclonic epilepsy diagnosed with Lafora disease based on confirmed dodecamer expansion in the NHLRC1 gene. Lafora disease is being progressively recognised in different unrelated breeds suggesting a wider distribution in the canine population than previously thought.

Cortical atrophy has been identified using magnetic resonance imaging (MRI) in humans and dogs with Lafora disease (LD). In humans, proton magnetic resonance spectroscopy (1HMRS) of the brain indicates decreased N‐acetyl‐aspartate (NAA) relative to other brain metabolites. Brain 1HMRS findings in dogs with LD are lacking. A 6‐year‐old female Beagle was presented with a history of a single generalized tonic‐clonic seizure and episodic reflex myoclonus. Clinical, hematological, and neurological examination findings and 3‐Tesla MRI of the brain were unremarkable. Brain 1HMRS with voxel positioning in the thalamus was performed in the affected Beagle. It identified decreased amounts of NAA, glutamate‐glutamine complex, and increased total choline and phosphoethanolamine relative to water and total creatine compared with the reference range in healthy control Beagles. A subsequent genetic test confirmed LD. Abnormalities in 1HMRS despite lack of changes with conventional MRI were identified in a dog with LD.

EPM2A has been certified as a causative gene in patients with Lafora disease (LD), which is a rare autosomal recessive and severe form of progressive myoclonus epilepsy. LD classically starts in adolescence, characterized by various types of seizure with myoclonic seizure as the main type. Typically within 10 years, intractable seizure attack, rapidly progressing dementia, and a vegetative state were present. LD is particularly frequently found in Mediterranean countries. Here, we report a Chinese family with a novel compound heterozygous mutation in the EPM2A gene, characterized by recurrent vomiting, intractable epilepsy, and progressive cognitive decline.

We report the atypical presentation of Lafora disease in a Senegalese girl carrying the homozygous variant, c.560A>C, in the NHLRC1 gene. At 13 years, the patient developed myoclonic and visual seizures, progressive psychomotor slowing, and cognitive decline. At 14 years, a neurological examination showed severe hypomimia, bradykinesia, rigidity and low-amplitude myoclonic jerks. Flash-visual and somatosensory evoked potentials showed an increased amplitude of the cortical components, while an electroretinogram showed attenuated responses. An EEG showed diffuse polyspikes associated with positive-negative jerks as well as posterior slow waves and irregular spikes. The electroclinical picture suggested the diagnosis of Lafora disease regarding the association of visual seizures, cognitive deterioration, and action myoclonus, together with the EEG and evoked potential findings. Two uncommon findings were the prominence of extrapyramidal signs in the early stage of disease (which are rarely reported) and attenuation of electroretinal responses. We consider that Lafora disease should be included in the diagnostic work-up for juvenile Parkinsonism, when associated with epilepsy.

The over-accumulation of glycogen appears as a hallmark in various glycogen storage diseases (GSDs), including Pompe, Cori, Andersen and Lafora disease. Accumulating evidence suggests that suppression of glycogen accumulation represents a potential therapeutic approach for treating these GSDs. Using a fluorescence polarization assay designed to screen for inhibitors of the key glycogen synthetic enzyme, glycogen synthase (GS), we identified a substituted imidazole, (rac)-2-methoxy-4-(1-(2-(1-methylpyrrolidin-2-yl)ethyl)-4-phenyl-1H-imidazol-5-yl)phenol (H23), as a first-in-class inhibitor for yeast glycogen synthase 2 (yGsy2p). Data from X-ray crystallography at 2.85 Å, as well as kinetic data, revealed that H23 bound within the UDP-glucose binding pocket of yGsy2p. The high conservation of residues between human and yeast GS in direct contact with H23 informed the development of around 500 H23 analogs. These analogs produced a structure-activity relationship (SAR) profile that led to the identification of a substituted pyrazole, 4-(4-(4-hydroxyphenyl)-3-(trifluoromethyl)-1H-pyrazol-5-yl)pyrogallol, with 300-fold improved potency against human GS. These substituted pyrazoles possess a promising scaffold for drug development efforts targeting GS activity in GSDs associated with excess glycogen accumulation.

Lafora disease is a hereditary, autosomal recessive progressive myoclonus epilepsy caused by mutations in the EPM2A (laforin) and EPM2B (malin) genes, with no substantial genotype-phenotype differences between the two. Clinical manifestations of the disease are determined by the accumulation of specific cytoplasmic “amyloid inclusions” consisting of polyglycosans (an abnormally branched glycogen molecule). Polyglycosans, or Lafora bodies, are typically found in the brain, hepatocytes of the liver, skeletal and cardiac muscles, in the ducts of sweat glands, and in the skin. The diagnosis is made following visual, generalized tonic-clonic and myoclonic seizures, progressing dementia, cerebellar ataxia, detection of specific Lafora bodies during sweat gland biopsy and data of genetic testing.The article describes a clinical case of Lafora disease in a patient with disease onset at 11 years old caused by the mutation in the EPM2A (laforine) gene with focal sensory visual seizures with subsequent generalized tonic-clonic seizures, progressive motor impairments in the form of ataxia and gait abnormality as well as behavioral and cognitive disorders. The presented clinical case demonstrates the need for additional research, such as biopsy and genetic testing, for diagnosing diseases proceeding with resistant epileptic seizures and progressive motor and cognitive impairments.

Backgrund/Aim. Lafora disease (LD) is progressive myoclonus epilepsy, characterized by intractable myoclonus and seizures, inevitable neurological deterioration, brutal cognitive decline and poor prognosis. The treatment still remains purely symptomatic. Recently, two single-case studies and one case series study reported the favourable effects of perampanel in LD. Our study aimed to test the benefits reported in three separate case studies. Methods. We performed an open label, prospective study of 4 patients aged between 22 and 34 years with mutation in NHLRC1 (EPM2B) gene, treated with perampanel (6?8 mg/day) as add-on therapy. Follow-up period comprised 14?26 months. Seizure frequency, myoclonus, functional disability and cognitive performance were analysed. Results. In 3 patients, both, seizures and myoclonus, showed remarkable improvement after the drug introduction (> 50% reduction). No significant effect was seen in one case. The functional and cognitive impairment maintained at the same level, though all patients were at the later stage of the disease. Psychiatric side effects were dose related. Conclusion. Our study supports the rare, previously reported observations that perampanel is beneficial in treating LD patients.

The use of antibodies as targeting molecules or cell-penetrating tools has emerged at the forefront of pharmaceutical research. Antibody-directed therapies in the form of antibody-drug conjugates, immune modulators, and antibody-directed enzyme prodrugs have been most extensively utilized as hematological, rheumatological, and oncological therapies, but recent developments are identifying additional applications of antibody-mediated delivery systems. A novel application of this technology is for the treatment of glycogen storage disorders (GSDs) via an antibody-enzyme fusion (AEF) platform to penetrate cells and deliver an enzyme to the cytoplasm, nucleus, and/or other organelles. Exciting developments are currently underway for AEFs in the treatment of the GSDs Pompe disease and Lafora disease (LD). Antibody-based therapies are quickly becoming an integral part of modern disease therapeutics.

Background Lafora progressive myoclonus epilepsy (Lafora disease) is a rare, usually childhood-onset, fatal neurodegenerative disease caused by biallelic mutations in EPM2A (Laforin) or EPM2B (NHLRC1; Malin). The epidemiology of Lafora disease in Germany is largely unknown. The objective of this retrospective case series is to characterize the genotypes and phenotypes of patients with Lafora disease living in Germany. Methods The patients described in this case series initially had the suspected clinical diagnosis of Lafora disease, or unclassified progressive myoclonus epilepsy. Molecular genetic diagnostics including next generation sequencing-based diagnostic panel analysis or whole exome sequencing was performed. Results The parents of four out of the 11 patients are nonconsanguineous and of German origin while the other patients had consanguineous parents. Various variants were found in EPM2A (six patients) and in EPM2B (five patients). Eight variants have not been reported in the literature so far. The patients bearing novel variants had typical disease onset during adolescence and show classical disease courses. Conclusions This is the first larger case series of Lafora patients in Germany. Our data enable an approximation of the prevalence of manifest Lafora disease in Germany to 1,69 per 10 million people. Broader application of gene panel or whole-exome diagnostics helps clarifying unclassified progressive myoclonus epilepsy and establish an early diagnosis, which will be even more important as causal therapy approaches have been developed and are soon to be tested in a phase I study.

Summary Lafora disease (LD) is a fatal childhood dementia characterized by progressive myoclonic epilepsy manifesting in the teenage years, rapid neurological decline, and death typically within ten years of onset. Mutations in either EPM2A, encoding the glycogen phosphatase laforin, or EPM2B, encoding the E3 ligase malin, cause LD. Whole exome sequencing has revealed many EPM2A variants associated with late-onset or slower disease progression. We established an empirical pipeline for characterizing the functional consequences of laforin missense mutations in vitro using complementary biochemical approaches. Analysis of 26 mutations revealed distinct functional classes associated with different outcomes that were supported by clinical cases. For example, F321C and G279C mutations have attenuated functional defects and are associated with slow progression. This pipeline enabled rapid characterization and classification of newly identified EPM2A mutations, providing clinicians and researchers genetic information to guide treatment of LD patients., Graphical abstract, Highlights • Lafora disease (LD) patients present with varying clinical progression • LD missense mutations differentially affect laforin function • An empirical in vitro pipeline is used to classify laforin missense mutations • Patient progression can be predicted based on mutation class, Disease; Biochemistry; Structural biology; Biophysics

AIMS Several neurodegenerative and neuromuscular disorders are characterised by storage of polyglucosan, consisting of proteins and amylopectin-like polysaccharides, which are less branched than in normal glycogen. Such diseases include Lafora disease, branching enzyme deficiency, glycogenin-1 deficiency, polyglucosan body myopathy type 1 (PGBM1) due to RBCK1 deficiency and others. The protein composition of polyglucosan bodies is largely unknown. METHODS We combined quantitative mass spectrometry, immunohistochemical and western blot analyses to identify the principal protein components of polyglucosan bodies in PGBM1. Histologically stained tissue sections of skeletal muscle from four patients were used to isolate polyglucosan deposits and control regions by laser microdissection. Prior to mass spectrometry, samples were labelled with tandem mass tags that enable quantitative comparison and multiplexed analysis of dissected samples. To study the distribution and expression of the accumulated proteins, immunohistochemical and western blot analyses were performed. RESULTS Accumulated proteins were mainly components of glycogen metabolism and protein quality control pathways. The majority of fibres showed depletion of glycogen and redistribution of key enzymes of glycogen metabolism to the polyglucosan bodies. The polyglucosan bodies also showed accumulation of proteins involved in the ubiquitin-proteasome and autophagocytosis systems and protein chaperones. CONCLUSIONS The sequestration of key enzymes of glycogen metabolism to the polyglucosan bodies may explain the glycogen depletion in the fibres and muscle function impairment. The accumulation of components of the protein quality control systems and other proteins frequently found in protein aggregate disorders indicates that protein aggregation may be an essential part of the pathobiology of polyglucosan storage.

Purpose Lafora disease is a genetic neurodegenerative metabolic disorder caused by insoluble polyglucosan aggregate accumulation throughout the central nervous system and body. The retina is an accessible neural tissue, which may offer alternative methods to assess neurological diseases quickly and noninvasively. In this way, noninvasive imaging may provide a means to characterize neurodegenerative disease, which enables earlier identification and diagnosis of disease and the ability to monitor disease progression. In this study, we sought to characterize the retina of individuals with Lafora disease using non-invasive retinal imaging. Methods One eye of three individuals with genetically confirmed Lafora disease were imaged with optical coherence tomography (OCT) and adaptive optics scanning light ophthalmoscopy (AOSLO). When possible, OCT volume and line scans were acquired to assess total retinal thickness, ganglion cell-inner plexiform layer thickness, and outer nuclear layer + Henle fiber layer thickness. OCT angiography (OCTA) scans were acquired in one subject at the macula and optic nerve head (ONH). AOSLO was used to characterize the photoreceptor mosaic and examine the retinal nerve fiber layer (RNFL). Results Two subjects with previous seizure activity demonstrated reduced retinal thickness, while one subject with no apparent symptoms had normal retinal thickness. All other clinical measures, as well as parafoveal cone density, were within normal range. Nummular reflectivity at the level of the RNFL was observed using AOSLO in the macula and near the ONH in all three subjects. Conclusions This multimodal retinal imaging approach allowed us to observe a number of retinal structural features in all three individuals. Most notably, AOSLO revealed nummular reflectivity within the inner retina of each subject. This phenotype has not been reported previously and may represent a characteristic change produced by the neurodegenerative process.

Lafora disease (LD) is a severe form of progressive myoclonus epilepsy inherited in an autosomal recessive fashion. It is associated with biallelic pathogenic variations in EPM2A or NHLRC1, which encode laforin and malin, respectively. The disease usually starts with adolescent onset seizures followed by progressive dementia, refractory status epilepticus and eventually death within 10 years of onset. LD is generally accepted as having a homogenous clinical course with no considerable differences between EPM2A or NHLRC1 associated forms. Nevertheless, late-onset and slow progressing forms of the disease have also been reported. Herein, we have performed clinical and genetic analyses of 14 LD patients from 12 different families and identified 8 distinct biallelic variations in these patients. Five of these variations were novel and/or associated with the LD phenotype for the first time. Interestingly, almost half of the cases were homozygous for the rare rs769301934 (NM_198586.3(NHLRC1): c.436 G > A; p.(Asp146Asn)) allele in NHLRC1. A less severe phenotype with an onset at a later age may be the reason for the biased inflation of this variant, which is already present in the human gene pool and can hence arise in the homozygous form in populations with increased parental consanguinity.

BackgroundLafora disease (LD) is a fatal childhood-onset dementia characterized by the extensive accumulation of glycogen aggregates—the so-called Lafora Bodies (LBs)—in several organs. The accumulation of LBs in the brain underlies the neurological phenotype of the disease. LBs are composed of abnormal glycogen and various associated proteins, including p62, an autophagy adaptor that participates in the aggregation and clearance of misfolded proteins.MethodsTo study the role of p62 in the formation of LBs and its participation in the pathology of LD, we generated a mouse model of the disease (malinKO) lacking p62.ResultsDeletion of p62 prevented LB accumulation in skeletal muscle and cardiac tissue. In the brain, the absence of p62 altered LB morphology and increased susceptibility to epilepsy.ConclusionsThese results demonstrate that p62 participates in the formation of LBs and suggest that the sequestration of abnormal glycogen into LBs is a protective mechanism through which to reduce the deleterious consequences of its accumulation in the brain.

17 páginas, 2 figuras, Metformin is a drug in the family of biguanide compounds that is widely used in the treatment of type 2 diabetes (T2D). Interestingly, the therapeutic potential of metformin expands its prescribed use as an anti-diabetic drug. In this sense, it has been described that metformin administration has beneficial effects on different neurological conditions. In this work, we review the beneficial effects of this drug as a neuroprotective agent in different neurological diseases, with a special focus on epileptic disorders and Lafora disease, a particular type of progressive myoclonus epilepsy. In addition, we review the different proposed mechanisms of action of metformin to understand its function at the neurological level., This work was supported by grants from the Spanish Ministry of Science and Innovation –SAF2017-83151-R to P.S. and Rti2018-095784b-100 SAF to M.P.S. and J.M.S.—and a grant from the National Institutes of Health (P01 NS097197), which established the Lafora Epilepsy Cure Initiative (LECI), to P.S., J.M.S. and M.P.S.

15 páginas, 6 figuras, 1 tablas, Lafora disease (LD; OMIM#274780) is a fatal rare neurodegenerative disorder characterized by generalized epileptic seizures and the presence of polyglucosan inclusions (PGs), called Lafora bodies (LBs), typically in the brain. LD is caused by mutations in two genes EPM2A or EPM2B, which encode respectively laforin, a glucan phosphatase, and malin, an E3-ubiquitin ligase. Much remains unknown about the molecular bases of LD and, unfortunately, appropriate treatment is still missing; therefore patients die within 10 years from the onset of the disease. Recently, we have identified neuroinflammation as one of the initial determinants in LD. In this work, we have investigated anti-inflammatory treatments as potential therapies in LD. With this aim, we have performed a preclinical study in an Epm2b-/- mouse model with propranolol, a β-adrenergic antagonist, and epigallocatechin gallate (EGCG), an antioxidant from green tea extract, both of which displaying additional anti-inflammatory properties. In vivo motor and cognitive behavioral tests and ex vivo histopathological brain analyses were used as parameters to assess the therapeutic potential of propranolol and EGCG. After 2 months of treatment, we observed an improvement not only in attention defects but also in neuronal disorganization, astrogliosis, and microgliosis present in the hippocampus of Epm2b-/- mice. In general, propranolol intervention was more effective than EGCG in preventing the appearance of astrocyte and microglia reactivity. In summary, our results confirm the potential therapeutic effectiveness of the modulators of inflammation as novel treatments in Lafora disease., This work was supported by a grant from the Spanish Ministry of Science and Innovation SAF2017-83151-R and a grant from the National Institutes of Health P01 NS097197, which established the Lafora Epilepsy Cure Initiative (LECI), to P.S.

An abnormal structural form of glycogen (with less branching points or amylopectin-like polysaccharide) called polyglucosan (PG) may accumulate in various tissues such as striated and smooth muscles, brain, nerve, liver and skin, and cause a group of nine different genetic disorders manifesting with a variety of clinical phenotypes that affect mainly the nervous system (Lafora disease, adult PG body disease), the heart (glycogen storage disease type XV, hypertrophic cardiomyopathy type 6, PG body myopathy type 1) and the skeletal muscle (glycogen storage disease type IV, glycogen storage disease type VII, PG body myopathy type 2), depending on the organs which are mostly affected by the PG aggregates. The pathological feature of PG storage in tissues is a hallmark of these disorders. Whole-genome sequencing has allowed to obtain a diagnosis in a large number of patients with a previously unrecognized disorder. We describe the clinical, pathological and molecular features of these genetic disorders, for many of which the pathological mechanisms underlying the corresponding mutant gene have been investigated and, at least in part, understood.

Background Lafora disease (LD) is a rare, lethal, progressive myoclonus epilepsy for which no targeted therapy is currently available. Studies on a mouse model of LD showed a good response to metformin, a drug with a well known neuroprotective effect. For this reason, in 2016, the European Medicines Agency granted orphan designation to metformin for the treatment of LD. However, no clinical data is available thus far. Methods We retrospectively collected data on LD patients treated with metformin referred to three Italian epilepsy centres. Results Twelve patients with genetically confirmed LD (6 EPM2A, 6 NHLRC1) at middle/late stages of disease were treated with add-on metformin for a mean period of 18 months (range: 6–36). Metformin was titrated to a mean maintenance dose of 1167 mg/day (range: 500–2000 mg). In four patients dosing was limited by gastrointestinal side-effects. No serious adverse events occurred. Three patients had a clinical response, which was temporary in two, characterized by a reduction of seizure frequency and global clinical improvement. Conclusions Metformin was overall safe in our small cohort of LD patients. Even though the clinical outcome was poor, this may be related to the advanced stage of disease in our cases and we cannot exclude a role of metformin in slowing down LD progression. Therefore, on the grounds of the preclinical data, we believe that treatment with metformin may be attempted as early as possible in the course of LD.

Summary: 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. : EPM2A, EPM2B, or GBE1 deficiency causes insoluble glycogen accumulation and neurodegenerative diseases. Sullivan et al. show that these defects do not impair the construction of WT-like soluble glycogen. Demonstrating varying chain length distributions and correlating precipitation propensity among WT-glycogen molecules, a mechanistic explanation emerges for the structural characteristics of insoluble glycogen. Keywords: Lafora disease, laforin, malin, APBD, glycogen branching enzyme, glycogen synthase, glycogen storage disease, polyglucosan bodies, phosphorylation, glycogen chain length distribution

Progressive myoclonic epilepsy (PME) is an autosomal recessive, apparently a rare complex epilepsy syndrome. Among different types of PME, lafora body disease is more quickly progressive usually fatal within 2nd and 3rd decade. They are characterized by childhood or adolescent onset difficult to control multiple type seizures including myoclonous, generalized tonic clonic, absences, psychomotor regression with ataxia, dementia, dysarthria, visual hallucinations, and other general features. Early suspicion is important that leads to the rational diagnostic workout. The electro-clinical criteria would help a lot to exclude the benign epilepsy syndrome such as juvenile myoclonic epilepsy (JME) and suspect PME at the early stage of the complex epilepsy syndrome. Diagnosis is further clarified and confirmed by finding lafora body in skin and genetic study. Genetic mutation found in more than 87% cases in EPM2A gene or the EPM2B also known as NHLRC1 gene and are inherited in an autosomal recessive manner. EMP2A gene is located on chromosome 6q24. They are reported from Mediterranean basin, central Asia, India, Pakistan, northern Africa and Middle East where consanguineous marriage is common. We report a diagnosed case for the first time in Bangladesh. With the detail clinical history, rational use of the available investigation tools and clinical suspicion, diagnosis of the disorder at its early stage is possible. The rapid progress in genetic therapy would be a great hope in near future. Bangladesh J Child Health 2018; VOL 42 (3) :138-147

We described a clinical case of Lafora disease, a rare genetically determined disease (mutations in the EPM2Aor EPM2Bgenes) from the group of progressive myoclonus-epilepsy forms with an autosomal recessive type of inheritance. The symptoms of the beginning of Laphora disease are similar to the symptoms of idiopathic generalized epilepsy. The absence of the effect of treatment with antiepileptic drugs, the manifestation of new types of seizures, the progression of cognitive impairments allowed us to exclude idiopathic generalized epilepsy with isolated convulsive seizures and juvenile myoclonic epilepsy. The case described in the article indicates the need for the neurologist to be aware of rare genetic syndromes and alertness to progressing myoclonus-epilepsies.

21 páginas, 7 figuras. Some elements used in the figures were adapted from Servier Medical Art (https://smart.servier.com/) (accessed on 12 February 2021)., licensed under a Creative Commons Attribution 3.0 Generic License. Supplementary Materials: The following are available online at https://www.mdpi.com/article/10.3390/cells10040820/s1, Table S1: Reported TRIM32 substrates/interactors listed alphabetically. Table S2: Reported malin substrates/interactors listed alphabetically, Tripartite motif (TRIM) proteins are RING E3 ubiquitin ligases defined by a shared domain structure. Several of them are implicated in rare genetic diseases, and mutations in TRIM32 and TRIM-like malin are associated with Limb-Girdle Muscular Dystrophy R8 and Lafora disease, respectively. These two proteins are evolutionary related, share a common ancestor, and both display NHL repeats at their C-terminus. Here, we revmniew the function of these two related E3 ubiquitin ligases discussing their intrinsic and possible common pathophysiological pathways., This work has received funding from the European Union’s Horizon 2020 research and innovation programme under the Marie Sklodowska-Curie grant agreement 813599 (TRIM-NET). We also want to acknowledge the support of the grant from the National Institutes of Health P01 NS097197, which established the Lafora Epilepsy Cure Initiative (LECI), to P.S. E.L. is supported by a post-doctoral grant issued by Fondazione Umberto Veronesi.

Introduction: Progressive myoclonic epilepsies (PMEs) are a heterogenous group of genetic diseases presenting with epilepsy, cognitive impairment, and severe action myoclonus, which can severely affect daily life activities and independent walking ability. Perampanel is a recent commercially available antiseizure medication with high efficacy against generalized seizures. Some reports supported the role of perampanel in ameliorating action myoclonus in PMEs. Here, we aimed to describe a case series and provide a systematic literature review on perampanel effects on PMEs.Methods: We report the perampanel effectiveness on myoclonus, daily life activities, and seizures on an original Italian multicenter case series of 11 individuals with PMEs. Then, using the Preferred Reporting Items for Systematic Reviews and Meta-analyses (PRISMA) guidelines, we performed a systematic review on perampanel effect on myoclonus and disability in PMEs. We searched PubMed, Scopus, and Google Scholar articles on perampanel and PMEs up to June 2020. No prospective trials were found. We reviewed 11 case series manuscripts reporting 104 cases of different PMEs.Results: Here, we are reporting the effectiveness of perampanel in five individuals affected by Unverricht–Lundborg disease, three by Lafora disease, two by sialidosis, and one by an undetermined PME. Nine out of 11 individuals improved their disability related to the action myoclonus (two with Lafora disease did not). Among the 104 persons with PMEs collected by the systematic review, we found that more than half of the patients receiving perampanel exhibited an amelioration of action myoclonus and, consequently, of their independence in daily life activities. The Unverricht–Lundborg disease seemed to show the best clinical response to perampanel, in comparison with the other more severe PMEs. A significant seizure reduction was achieved by almost all persons with active epilepsy. Only 11% of PME patients dropped out due to inefficacy.Conclusions: Perampanel demonstrated a beneficial effect with regard to action myoclonus, disability, and seizures and was well-tolerated in people with PMEs, independently from their genetic diagnosis. Given the limited scientific evidence, broader prospective trials should be encouraged.

Objectives Mutations in the gene encoding the glycogen phosphatase laforin result in the fatal childhood dementia Lafora disease (LD). A cellular hallmark of LD is cytoplasmic, hyper-phosphorylated, glycogen-like aggregates called Lafora bodies (LBs) that form in nearly all tissues and drive disease progression. Additional tools are needed to define the cellular function of laforin, understand the pathological role of laforin in LD, and determine the role of glycogen phosphate in glycogen metabolism. In this work, we present the generation and characterization of laforin nanobodies, with one being a laforin inhibitor. Design and methods We identify multiple classes of specific laforin-binding nanobodies and determine their binding epitopes using hydrogen deuterium exchange (HDX) mass spectrometry. Using para-nitrophenyl phosphate (pNPP) and a malachite gold-based assay specific for glucan phosphatase activity, we assess the inhibitory effect of one nanobody on laforin’s catalytic activity. Results Six families of laforin nanobodies are characterized and their epitopes mapped. One nanobody is identified and characterized that serves as an inhibitor of laforin’s phosphatase activity. Conclusions The six generated and characterized laforin nanobodies, with one being a laforin inhibitor, are an important set of tools that open new avenues to define unresolved glycogen metabolism questions.

Lafora disease is a severe form of progressive myoclonic epilepsy with autosomal recessive inheritance diagnosed by inclusion body in biopsy. A 26-year-old woman was admitted due to complaints of frequent twitches and fainting. The 0.5x0.3x0.3 cm axillary skin punch biopsy was subjected to routine histopathological evaluation. Cytoplasmic PAS-positive inclusion bodies were observed at the basal side of the eccrine and apocrine glands. The diagnosis of Lafora disease can also be made by the observation of the polyglycosan cytoplasmic inclusion bodies in the brain, liver and skeletal muscle biopsies. Although we need more work to understand the etiopathogenesis of Lafora disease, we would like to draw attention to the importance of skin biopsy in the differential diagnosis of young patients with clinically refractory epilepsy, myoclonus, and cognitive decline.

We describe a cohort of 10 unrelated Greek patients (4 females, 6 males; median age 6.5 years, range 2–18 years) with heterogeneous epilepsy syndromes with a genetic basis. In these patients, causative genetic variants, including two novel ones, were identified in 9 known epilepsy-related genes through whole exome sequencing. A patient with glycine encephalopathy was a compound heterozygote for the p.Arg222Cys and the p.Ser77Leu AMT variant. A patient affected with Lafora disease carried the homozygous p.Arg171His EPM2A variant. A de novo heterozygous variant in the GABRG2 gene (p.Pro282Thr) was found in one patient and a pathogenic variant in the GRIN2B gene (p.Gly820Val) in another patient. Infantile-onset lactic acidosis with seizures was associated with the p.Arg446Ter PDHX gene variant in one patient. In two additional epilepsy patients, the p.Ala1662Val and the novel non-sense p.Phe1330Ter SCN1A gene variants were found. Finally, in 3 patients we observed a novel heterozygous missense variant in SCN2A (p.Ala1874Thr), a heterozygous splice site variant in SLC2A1 (c.517-2A>G), as a cause of Glut1 deficiency syndrome, and a pathogenic variant in STXBP1 (p.Arg292Leu), respectively. In half of our cases (patients with variants in the GRIN2B, SCN1A, SCN2A and SLC2A1 genes), a genetic cause with potential management implications was identified.

Lafora disease (LD) is a fatal adolescence-onset neurodegenerative condition. The hallmark of LD is the accumulation of aberrant glycogen aggregates called Lafora bodies (LBs) in the brain and other tissues. Impeding glycogen synthesis from early embryonic stages by genetic suppression of glycogen synthase (MGS) in an animal model of LD prevents LB formation and ultimately the pathological manifestations of LD thereby indicating that LBs are responsible for the pathophysiology of the disease. However, it is not clear whether eliminating glycogen synthesis in an adult animal after LBs have already formed would halt or reverse the progression of LD.Herein we generated a mouse model of LD with inducible MGS suppression. We evaluated the effect of MGS suppression at different time points on LB accumulation as well as on the appearance of neuroinflammation, a pathologic trait of LD models.In the skeletal muscle, MGS suppression in adult LD mice blocked the formation of new LBs and reduced the number of glycogen aggregates. In the brain, early but not late MGS suppression halted the accumulation of LBs. However, the neuroinflammatory response was still present, as shown by the levels of reactive astrocytes, microglia and inflammatory cytokines.Our results confirm that MGS as a promising therapeutic target for LD and highlight the importance of an early diagnosis for effective treatment of the disease.

Background Lafora disease (LD) is characterized by progressive myoclonus, refractory epilepsy, and cognitive deterioration. This complex neurodegenerative condition is caused by pathogenic variants in EPM2A/EPM2B genes, encoding two essential glycogen metabolism enzymes known as laforin and malin. Long-term follow-up data are lacking. We describe the clinical features and genetic findings of a cohort of 26 Italian patients with a long clinical follow-up. Methods Patients with EPM2A/EPM2B pathogenic variants were identified by direct gene sequencing or gene panels with targeted re-sequencing. Disease progression, motor functions, and mental performance were assessed by a simplified disability scale. Spontaneous/action myoclonus severity was scored by the Magaudda Scale. Results Age range was 12.2–46.2 years (mean:25.53 ± 9.14). Age at disease onset ranged from 10 to 22 years (mean:14.04 ± 2.62). The mean follow-up period was 11.48 ± 7.8 years. Twelve out of the 26 (46%) patients preserved walking ability and 13 (50%) maintained speech. A slower disease progression with preserved ambulation and speech after ≥4 years of follow-up was observed in 1 (11%) out of the 9 (35%) EPM2A patients and in 6 (35%) out of the 17 (65%) EPM2B patients. Follow-up was >10 years in 7 (41.2%) EPM2B individuals, including two harbouring the homozygous p.(D146N) pathogenic variant. Conclusions This study supports an overall worse disease outcome with severe deterioration of ambulation and speech in patients carrying EPM2A mutations. However, the delayed onset of disabling symptoms observed in the EPM2B subjects harbouring the p.(D146N) pathogenic variant suggests that the underlying causative variant may still influence LD severity.

Perampanel is an α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor antagonist that has been marked as an antiepileptic drug for partial-onset and primary generalized tonic-clonic seizures. There have been some recent reports of perampanel being effective against cortical myoclonus by Lafora disease and Unverricht-Lundborg disease. We herein report a 49-year-old man who presented with myoclonus due to Lance-Adams syndrome (LAS) after cardiopulmonary arrest caused by a severe bronchial asthma attack. Perampanel was very effective against myoclonus induced by LAS even in the chronic state, over 10 years after the remote onset. Perampanel should be considered for the treatment of extremely refractory myoclonus due to LAS.

Summary Glycosylation defects are a hallmark of many nervous system diseases. However, the molecular and metabolic basis for this pathology is not fully understood. In this study, we found that N-linked protein glycosylation in the brain is metabolically channeled to glucosamine metabolism through glycogenolysis. We discovered that glucosamine is an abundant constituent of brain glycogen, which functions as a glucosamine reservoir for multiple glycoconjugates. We demonstrated the enzymatic incorporation of glucosamine into glycogen by glycogen synthase, and the release by glycogen phosphorylase by biochemical and structural methodologies, in primary astrocytes, and in vivo by isotopic tracing and mass spectrometry. Using two mouse models of glycogen storage diseases, we showed that disruption of brain glycogen metabolism causes global decreases in free pools of UDP-N-acetylglucosamine and N-linked protein glycosylation. These findings revealed fundamental biological roles of brain glycogen in protein glycosylation with direct relevance to multiple human diseases of the central nervous system.

Lafora Disease (LD) is a rare, fatal, late-onset, progressive form of myoclonic epilepsy, occurring in humans and dogs. Clinical manifestations of LD usually include seizures, spontaneous and reflex myoclonus with contractions of the neck and limb muscles. We studied the electroencephalogram (EEG) patterns of two beagles in whom LD was subsequently confirmed by genetic testing. In both cases, the EEG recordings, accompanied by electromyography (EMG), have shown similar uncommon patterns. The hypovoltaged background rhythm was interrupted by waxing “crescendo” polyspikes-slow wave complexes appearing 80–250 ms after the start of intermittent photic stimulation, followed by myoclonic jerks after 80–150 ms. This study highlights the value of EEG in establishing a presumptive diagnosis of LD in dogs.

Lafora disease (LD) is a teenage-onset fatal progressive myoclonus epilepsy caused by loss-of-function mutations in the EPM2A gene encoding the glycogen phosphatase laforin or EPM2B encoding the laforin-interacting ubiquitin E3 ligase malin. Concerted actions of glycogen synthase (GS) and branching enzyme generate normal short-branched soluble glycogen. In LD, some glycogen molecules develop long branches, precipitate, and accumulate into pathognomonic and pathogenic Lafora bodies (LBs). The precise mechanism by which the laforin-malin complex mitigates this is unknown, but thought to involve GS downregulation. In fact, transgenic GS downregulation in LD mouse models reduces LB formation and rescues the disease.1,2

Heat shock response (HSR) is a conserved cytoprotective pathway controlled by the master transcriptional regulator, the heat shock factor 1 (HSF1), that activates the expression of heat shock proteins (HSPs). HSPs, as chaperones, play essential roles in minimizing stress-induced damages and restoring proteostasis. Therefore, compromised HSR is thought to contribute to neurodegenerative disorders. Lafora disease (LD) is a fatal form of neurodegenerative disorder characterized by the accumulation of abnormal glycogen as Lafora bodies in neurons and other tissues. The symptoms of LD include progressive myoclonus epilepsy, dementia, and cognitive deficits. LD is caused by the defects in the gene coding laforin phosphatase or the malin ubiquitin ligase. Laforin and malin are known to work upstream of HSF1 and are essential for the activation of HSR. Herein, we show that mice deficient for laforin or malin show reduced levels of HSF1 and their targets in their brain tissues, suggesting compromised HSR; this could contribute to the neuropathology in LD. Intriguingly, treatment of LD animals with dexamethasone, a synthetic glucocorticoid analogue, partially restored the levels of HSF1 and its targets. Dexamethasone treatment was also able to ameliorate the neuroinflammation and susceptibility to induced seizures in the LD animals. However, dexamethasone treatment did not show a significant effect on Lafora bodies or autophagy defects. Taken together, the present study establishes a role for HSR in seizure susceptibility and neuroinflammation and dexamethasone as a potential antiepileptic agent, suitable for further studies in LD.

Health Impact Assessment (HIA) requires capacity building in technical, operational and strategic domains at individual and organisational levels. HIA capacity building frameworks also need to adapt to different types of HIA approaches and organisational, institutional and cultural contexts. To build capacity in these domains, many institutions around the world develop and provide training programs and technical support fit-for-purpose HIA in their respective local and regional institutional contexts. For example, in Australia, the Centre for Primary Health Care and Equity (CPHCE) has conducted Learning by Doing (LbD) programs in HIA since 2002 which aims at acquiring knowledge and skills through active involvement with practical, real-world experience. These training programs have supported building capacity to undertake HIAs in more than 30 countries and involved more than 2,000 participants. In South Korea, the Korea Institute of Health and Social Affairs (KIHASA) has provided technical and financial support for central and local governments to conduct HIAs since 2008. A total of 27 HIAs were conducted in diverse policy areas including urban planning, housing, transportation, education, welfare, and health services. In this session, we review the competencies for HIA in the multilevel domains and compare international HIA capacity building programs against these domains to discuss strengths, limitations, challenges and future directions.

Mammalian glycogen chain lengths are subject to complex regulation, including by seven proteins (protein phosphatase-1 regulatory subunit 3, PPP1R3A through PPP1R3G) that target protein phosphatase-1 (PP1) to glycogen to activate the glycogen chain-elongating enzyme glycogen synthase and inactivate the chain-shortening glycogen phosphorylase. Lafora disease is a fatal neurodegenerative epilepsy caused by aggregates of long-chained, and as a result insoluble, glycogen, termed Lafora bodies (LBs). We previously eliminated PPP1R3C from a Lafora disease mouse model and studied the effect on LB formation. In the present work, we eliminate and study the effect of absent PPP1R3D. In the interim, brain cell type levels of all PPP1R3 genes have been published, and brain cell type localization of LBs clarified. Integrating these data we find that PPP1R3C is the major isoform in most tissues including brain. In the brain, PPP1R3C is expressed at 15-fold higher levels than PPP1R3D in astrocytes, the cell type where most LBs form. PPP1R3C deficiency eliminates ~90% of brain LBs. PPP1R3D is quantitatively a minor isoform, but possesses unique MAPK, CaMK2 and 14-3-3 binding domains and appears to have an important functional niche in murine neurons and cardiomyocytes. In neurons, it is expressed equally to PPP1R3C, and its deficiency eliminates ~50% of neuronal LBs. In heart, it is expressed at 25% of PPP1R3C where its deficiency eliminates ~90% of LBs. This work studies the role of a second (PPP1R3D) of seven PP1 subunits that regulate the structure of glycogen, toward better understanding of brain glycogen metabolism generally, and in Lafora disease.

The hallmark of Lafora disease, a fatal neurodegenerative disorder, is the accumulation of intracellular glycogen aggregates called Lafora bodies. Until recently, it was widely believed that brain Lafora bodies were present exclusively in neurons and thus that Lafora disease pathology derived from their accumulation in this cell population. However, recent evidence indicates that Lafora bodies are also present in astrocytes. To define the role of astrocytic Lafora bodies in Lafora disease pathology, we deleted glycogen synthase specifically from astrocytes in a mouse model of the disease (malinKO). Strikingly, blocking glycogen synthesis in astrocytes—thus impeding Lafora bodies accumulation in this cell type—prevented the increase in neurodegeneration markers, autophagy impairment, and metabolic changes characteristic of the malinKO model. Conversely, mice that over-accumulate glycogen in astrocytes showed an increase in these markers. These results unveil the deleterious consequences of the deregulation of glycogen metabolism in astrocytes and change the perspective that Lafora disease is caused solely by alterations in neurons.

14 páginas, 8 figuras, 1 tabla, Introduction: Lafora disease (LD) is a rare form of progressive infantile epilepsy in which rapid neurological deterioration occurs as the disease advances, leading the patients to a vegetative state and then death, usually within the first decade of disease onset. Based on the capacity of the endogenous cannabinoid system (ECS) to modulate several cellular processes commonly altered in many neurodegenerative processes, as well as the antiepileptic properties of certain natural cannabinoids, the aim of this study was to evaluate the role of the ECS in LD progression. Materials and Methods: We tested whether a natural cannabis extract highly enriched in cannabidiol (CBD) might be effective in curbing the pathological phenotype of malin knockout (KO) mice as an animal model of LD. Results: Our results reveal for the first time that alterations in the ECS occur during the evolution of LD, mainly at the level of CB1, CB2, and G protein-coupled receptor 55 (GPR55) receptor expression, and that a CBD-enriched extract (CBDext) is able to reduce the cognitive impairment exhibited by malin KO mice. However, in contrast to what has previously been reported for other kinds of refractory epilepsy in childhood, the CBD-enriched extract does not reduce the severity of the epileptic seizures induced in this animal model of LD. Conclusions: In summary, this study reveals that the ECS might play a role in LD and that a CBD-enriched extract partially reduces the dementia-like phenotype, but not the increased vulnerability to epileptic seizures, exhibited by an animal model of such a life-threatening disease., This work was supported by CIBERNED (Institute of Health Carlos III, Spanish Ministry of Economy and Competitiveness), Unión Iberoamericana de Universidades (NL01/2017) and Hemp Solutions Group S.L. We wish to thank T. Yohannan for editorial assistance.