Antonio V. Delgado-Escueta

Mutations in a gene encoding a novel protein tyrosine phosphatase cause progressive myoclonus epilepsy

Laforas disease (LD; OMIM 254780) is an autosomal recessive form of progressive myoclonus epilepsy characterized by seizures and cumulative neurological deterioration. Onset occurs during late childhood and usually results in death within ten years of the first symptoms1,2. With few exceptions, patients follow a homogeneous clinical course despite the existence of genetic heterogeneity 3. Biopsy of various tissues, including brain, revealed characteristic polyglucosan inclusions called Lafora bodies4–8, which suggested LD might be a generalized storage disease6,9. Using a positional cloning approach, we have identified at chromosome 6q24 a novel gene, EPM2A, that encodes a protein with consensus amino acid sequence indicative of a protein tyrosine phosphatase (PTP). mRNA transcripts representing alternatively spliced forms of EPM2A were found in every tissue examined, including brain. Six distinct DNA sequence variations in EPM2A in nine families, and one homozygous microdeletion in another family, have been found to cosegregate with LD. These mutations are predicted to cause deleterious effects in the putative protein product, named laforin, resulting in LD.

Rapid Preparation of Synaptosomes from Mammalian Brain Using Nontoxic Isoosmotic Gradient Material (Percoll)

Abstract: A new procedure is described for the isolation of synaptosomes from various parts of mammalian brain. This method utilizes an isoosmotic Percoll/sucrose discontinuous gradient and has some advantages over the traditionally used synaptosomal isolation techniques: (1) it is possible to prepare suitable gradients while retaining isoosmolarity; (2) the time of the preparation is remarkably short (approximately 1 h); (3) if necessary, the gradient material can be easily removed from the samples. Intact synaptosomes were recovered from the 10%/16% (vol/vol) Percoll interphase. The fractions were identified and characterized by electron microscopy and by several biochemical markers for synaptosomes and other subcellular organelles. The homogeneity of the preparations is comparable to or better than that of synaptosomes prepared by the conventional methods. This procedure has been successfully used for the isolation of synaptosomes from very small tissue samples of various experimental animals and human brain.

Mutations in EFHC1 cause juvenile myoclonic epilepsy

Juvenile myoclonic epilepsy (JME) is the most frequent cause of hereditary grand mal seizures. We previously mapped and narrowed a region associated with JME on chromosome 6p12–p11 (EJM1). Here, we describe a new gene in this region, EFHC1, which encodes a protein with an EF-hand motif. Mutation analyses identified five missense mutations in EFHC1 that cosegregated with epilepsy or EEG polyspike wave in affected members of six unrelated families with JME and did not occur in 382 control individuals. Overexpression of EFHC1 in mouse hippocampal primary culture neurons induced apoptosis that was significantly lowered by the mutations. Apoptosis was specifically suppressed by SNX-482, an antagonist of R-type voltage-dependent Ca2+ channel (Cav2.3). EFHC1 and Cav2.3 immunomaterials overlapped in mouse brain, and EFHC1 coimmunoprecipitated with the Cav2.3 C terminus. In patch-clamp analysis, EFHC1 specifically increased R-type Ca2+ currents that were reversed by the mutations associated with JME.

Mutations in NHLRC1 cause progressive myoclonus epilepsy

Lafora progressive myoclonus epilepsy is characterized by pathognomonic endoplasmic reticulum (ER)-associated polyglucosan accumulations. We previously discovered that mutations in EPM2A cause Lafora disease. Here, we identify a second gene associated with this disease, NHLRC1 (also called EPM2B), which encodes malin, a putative E3 ubiquitin ligase with a RING finger domain and six NHL motifs. Laforin and malin colocalize to the ER, suggesting they operate in a related pathway protecting against polyglucosan accumulation and epilepsy.

Calcific neurocysticercosis and epileptogenesis

Neurocysticercosis is responsible for increased rates of seizures and epilepsy in endemic regions. The most common form of the disease, chronic calcific neurocysticercosis, is the end result of the host’s inflammatory response to the larval cysticercus of Taenia solium. There is increasing evidence indicating that calcific cysticercosis is not clinically inactive but a cause of seizures or focal symptoms in this population. Perilesional edema is at times also present around implicated calcified foci. A better understanding of the natural history, frequency, epidemiology, and pathophysiology of calcific cysticercosis and associated disease manifestations is needed to define its importance, treatment, and prevention.

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 Km 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.

Treatment of neurocysticercosis: Current status and future research needs

Here we put forward a roadmap that summarizes important questions that need to be answered to determine more effective and safer treatments. A key concept in management of neurocysticercosis is the understanding that infection and disease due to neurocysticercosis are variable and thus different clinical approaches and treatments are required. Despite recent advances, treatments remain either suboptimal or based on poorly controlled or anecdotal experience. A better understanding of basic pathophysiologic mechanisms including parasite survival and evolution, nature of the inflammatory response, and the genesis of seizures, epilepsy, and mechanisms of anthelmintic action should lead to improved therapies.

Neuroimaging in temporal lobe epilepsy : test sensitivity and relationships to pathology and postoperative outcome

Summary: We studied patients with documented temporal lobe seizures to evaluate the predictive value of computed tomography (CT), magnetic resonance imaging (MRI), and F‐18 fluorodeoxyglucose positron emission tomography (FDG‐PET) for surgical therapy and the relationships between these tests and the pathologic diagnoses. CT detected abnormalities in 32.5%, with an accuracy of 19% when accuracy was defined as congruence with electrophysiologic studies. MRI detected abnormalities in 81%, with an accuracy of 67%. FDG‐PET detected abnormalities in 85%, with an accuracy of 82%. Pathologic change was detected in 79% of the excised temporal neocortex, 65% of amygdalae, and 93% of hippocampi. After follow‐up periods of 20–71 months (mean 41 months), 67% of patients were free of seizures and 94% had at least a 90% reduction in seizure frequency. There was no relationship between the type of abnormality on MRI or the type of pathology and postoperative outcome. Better outcomes were associated with focal or regional ictal onsets as recorded by surface EEG. Worse out‐comes were associated with hypometabolism that extended outside the temporal lobe. Pathologic change in the temporal neocortex was associated with extension of hypometabolism outside the temporal lobe.

Neuroimaging in Patients with Seizures of Probable Frontal Lobe Origin

Twenty‐two patients whose electroclinical ictal characeristics suggested frontal lobe seizure foci were studied. Computed tomography (CT) scans showed abnormalities in only 32% of patients whereas magnetic resonance imaging was informative in 45%. 18FDG‐Positron emission tomography (PET) scanning revealed decreased metabolism in 64% of the group. The areas of hypometabolism were focal, regional, or hemispheric. Focal frontal hypometabolism was significantly correlated with the electroclinical (semiologic) ictal localization. Therefore, FDG‐PET scanning is a sensitive and specific technique for investigating patients with seizures of probable frontal lobe origins.

Hyperglycosylation and Reduced GABA Currents of Mutated GABRB3 Polypeptide in Remitting Childhood Absence Epilepsy

Childhood absence epilepsy (CAE) accounts for 10% to 12% of epilepsy in children under 16 years of age. We screened for mutations in the GABA(A) receptor (GABAR) beta 3 subunit gene (GABRB3) in 48 probands and families with remitting CAE. We found that four out of 48 families (8%) had mutations in GABRB3. One heterozygous missense mutation (P11S) in exon 1a segregated with four CAE-affected persons in one multiplex, two-generation Mexican family. P11S was also found in a singleton from Mexico. Another heterozygous missense mutation (S15F) was present in a singleton from Honduras. An exon 2 heterozygous missense mutation (G32R) was present in two CAE-affected persons and two persons affected with EEG-recorded spike and/or sharp wave in a two-generation Honduran family. All mutations were absent in 630 controls. We studied functions and possible pathogenicity by expressing mutations in HeLa cells with the use of Western blots and an in vitro translation and translocation system. Expression levels did not differ from those of controls, but all mutations showed hyperglycosylation in the in vitro translation and translocation system with canine microsomes. Functional analysis of human GABA(A) receptors (alpha 1 beta 3-v2 gamma 2S, alpha 1 beta 3-v2[P11S]gamma 2S, alpha 1 beta 3-v2[S15F]gamma 2S, and alpha 1 beta 3-v2[G32R]gamma 2S) transiently expressed in HEK293T cells with the use of rapid agonist application showed that each amino acid transversion in the beta 3-v2 subunit (P11S, S15F, and G32R) reduced GABA-evoked current density from whole cells. Mutated beta 3 subunit protein could thus cause absence seizures through a gain in glycosylation of mutated exon 1a and exon 2, affecting maturation and trafficking of GABAR from endoplasmic reticulum to cell surface and resulting in reduced GABA-evoked currents.