PTG protein depletion rescues malin-deficient Lafora disease in mouse.

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.

Deficiency of a glycogen synthase-associated protein, Epm2aip1, causes decreased glycogen synthesis and hepatic insulin resistance.

Glycogen synthesis is a major component of the insulin response, and defective glycogen synthesis is a major portion of insulin resistance. Insulin regulates glycogen synthase (GS) through incompletely defined pathways that activate the enzyme through dephosphorylation and, more potently, allosteric activation. We identify Epm2aip1 as a GS-associated protein. We show that the absence of Epm2aip1 in mice impairs allosteric activation of GS by glucose 6-phosphate, decreases hepatic glycogen synthesis, increases liver fat, causes hepatic insulin resistance, and protects against age-related obesity. Our work identifies a novel GS-associated GS activity-modulating component of insulin resistance.

Inhibiting glycogen synthesis prevents Lafora disease in a mouse model.

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.

Mild Lafora disease: clinical, neurophysiologic, and genetic findings.

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. NHLRC1 mutations were detected in 18 patients; EPM2A mutations were identified in 5. Patients who maintained >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 NHLRC1 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 NHLRC1 mutations in patients with mild LD should be taken into account in clinical practice for appropriate management and counseling.

PTG depletion removes Lafora bodies and rescues the fatal epilepsy of Lafora disease.

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.

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

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

Early-onset Lafora body disease.

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.

Glycogen hyperphosphorylation underlies lafora body formation.

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.

Mutations in CLN7/MFSD8 are a common cause of variant late-infantile neuronal ceroid lipofuscinosis.

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.

Laforin is a glycogen phosphatase, deficiency of which leads to elevated phosphorylation of glycogen in vivo.

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.

The autosomal recessively inherited progressive myoclonus epilepsies and their genes.

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.

Lafora disease as a cause of visually exacerbated myoclonic attacks in a dog.

An 8-year-old, castrated male, miniature wire-haired dachshund was presented with a 4-month history of intermittent facial twitching (myoclonus). The myoclonic episodes progressed over a 16-month period. Generalized seizure activity was infrequent. Clinical examination revealed visually stimulated myoclonus. Response to therapy with antiepileptic drugs was equivocal. Genetic testing identified the dog as being affected by Lafora disease.

Lafora progressive myoclonus epilepsy: disease course homogeneity in a genetic isolate.

Lafora epilepsy is characterized by starch formation in brain and skin and is diagnosed by skin biopsy or mutation detection. It has variable ages of onset (6-19 years) and death (18-32 years) even with the same mutation, likely due to extramutational factors. The authors identified 14 Lafora epilepsy patients in the genetic isolate of tribal Oman. The authors show that in this homogeneous environment and gene pool, the same mutation, EPM2B-c.468-469delAG, results in highly uniform ages of onset (14 years) and death (21 years). Biopsy, on the other hand, was not homogeneous (positive in 4/5 patients) and is, therefore, less sensitive than mutation testing.

Unverricht-Lundborg progressive myoclonus epilepsy in Oman.

We analyzed the clinical, electrophysiologic, and genetic features of Omani Arab patients suspected of manifesting the Unverricht-Lundborg form of progressive myoclonus epilepsy. Ten patients (five boys, five girls; mean age at onset, 10.2 years) were evaluated. Unverricht-Lundborg disease was confirmed in all by detection of dodecamer repeat expansion mutations in the EPM1 gene. There was no correlation between age at onset or severity of disease with sizes of dodecamer repeats. Myoclonic seizures were the presenting symptom in 70% of patients. Myoclonus was severe in adolescence, but remained stable or improved beyond 5-6 years of disease onset. No significant cognitive decline occurred. Nearly 75% of patients exhibited mild to moderate cerebellar dysfunction, which was nonprogressive after adulthood. Slowing of background activity, generalized spike-wave discharges, and photoparoxysmal responses were evident in all patients' electroencephalograms. Spike-wave discharges and photoparoxysmal responses tended to disappear in adulthood. This cluster of progressive myoclonus epilepsy patients manifested typical Unverricht-Lundborg disease. All cases had mutations in EPM1, the known gene for this disorder, and therefore do not contribute to identifying the gene in a second Unverricht-Lundborg disease locus recently mapped in Arab patients from Israel. Although Unverricht-Lundborg disease is very severe in adolescence, its clinical signs stabilize and improve somewhat in adulthood in this so-called "progressive epilepsy."

Typical progression of myoclonic epilepsy of the Lafora type: a case report.

BACKGROUND: 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. INVESTIGATIONS: Neurological examination, neuropsychological testing, electrophysiological studies, skin biopsy, MRI, genetic testing, and autopsy. DIAGNOSIS: Lafora disease (EPM2), resulting from a homozygous missense mutation in EPM2B (NHLRC1; c205C>G; Pro69Ala). MANAGEMENT: Symptomatic treatment with conventional antiepileptic and antimyoclonic drugs.

Targeted deletion of Fgl-2/fibroleukin in the donor modulates immunologic response and acute vascular rejection in cardiac xenografts.

BACKGROUND: Xenografts ultimately fail as a result of acute vascular rejection (AVR), a process characterized by intravascular thrombosis, fibrin deposition, and endothelial cell activation. METHODS AND RESULTS: We studied whether targeted deletion of Fgl-2, an inducible endothelial cell procoagulant, (Fgl-2-/-) in the donor prevents AVR in a mouse-to-rat cardiac xenotransplantation model. By 3 days after transplant, Fgl-2+/+ grafts developed typical features of AVR associated with increased levels of donor Fgl-2 mRNA. Grafts from Fgl-2-/- mice had reduced fibrin deposition but developed cellular rejection. Treatment with a short course of cobra venom factor and maintenance cyclosporine resulted in long-term acceptance of both Fgl-2+/+ and Fgl-2-/- grafts. On withdrawal of cyclosporine, Fgl-2+/+ grafts developed features of AVR; in contrast, Fgl-2-/- grafts again developed acute cellular rejection. Rejecting Fgl-2+/+ hearts stained positively for IgG, IgM, C3, and C5b-9, whereas rejecting Fgl-2-/- hearts had minimal Ig and complement deposition despite xenoantibodies in the serum. Furthermore, serum containing xenoantibodies failed to stain Fgl-2-/- long-term treated hearts but did stain wild-type heart tissues. Treatment of Fgl-2-/- xenografts with mycophenolate mofetil and tacrolimus, a clinically relevant immune suppression protocol, led to long-term graft acceptance. CONCLUSIONS: Deletion of Fgl-2 ameliorates AVR by downregulation of xenoantigens and may facilitate successful clinical heart xenotransplantation.

Lafora disease.

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.

Deep intronic GBE1 mutation in manifesting heterozygous patients with adult polyglucosan body disease.

IMPORTANCE: We describe a deep intronic mutation in adult polyglucosan body disease. Similar mechanisms can also explain manifesting heterozygous cases in other inborn metabolic diseases. OBJECTIVE: To explain the genetic change consistently associated with manifesting heterozygous patients with adult polyglucosan body disease. DESIGN, SETTING, AND PARTICIPANTS: This retrospective study took place from November 8, 2012, to November 7, 2014. We studied 35 typical patients with adult polyglucosan body disease, of whom 16 were heterozygous for the well-known c.986A>C mutation in the glycogen branching enzyme gene (GBE1) but harbored no other known mutation in 16 exons. MAIN OUTCOMES AND MEASURES: All 16 manifesting heterozygous patients had lower glycogen branching activity compared with homozygous patients, which showed inactivation of the apparently normal allele. We studied the messenger ribonucleic acid (mRNA) structure and the genetic change due to the elusive second mutation. RESULTS: When we reverse transcribed and sequenced the mRNA of GBE1, we found that all manifesting heterozygous patients had the c.986A>C mutant mRNA and complete lack of mRNA encoded by the second allele. We identified a deep intronic mutation in this allele, GBE1-IVS15+5289_5297delGTGTGGTGGinsTGTTTTTTACATGACAGGT, which acts as a gene trap, creating an ectopic last exon. The mRNA transcript from this allele missed the exon 16 and 3'UTR and encoded abnormal GBE causing further decrease of enzyme activity from 18% to 8%. CONCLUSIONS AND RELEVANCE: We identified the deep intronic mutation, which acts as a gene trap. This second-most common adult polyglucosan body disease mutation explains another founder effect in all Ashkenazi-Jewish cases.

Progressive myoclonus epilepsy.

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.

Hyperphosphorylation of glucosyl C6 carbons and altered structure of glycogen in the neurodegenerative epilepsy Lafora disease.

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.