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At the beginning of the 20th century, in view of the growing international recognition of Santiago Ramón y Cajal, the Spanish authorities took some important steps to support Cajal’s scientific work. This recognition peaked in 1906, when Camillo Golgi and Santiago Ramón y Cajal shared the Nobel Prize in Physiology or Medicine. The Spanish government provided Cajal a state-of-the-art laboratory in Madrid to allow him to continue with his research and they funded salaries to pay his first tenured collaborators, the number of which increased further after the creation of the Junta para Ampliación de Estudios (JAE). The JAE was an organism set up to help promising researchers develop their careers in different ways, thereby contributing to the development of science in Spain. Although largely forgotten or relatively unknown, there has been a recent revival in the recognition of the school that developed around Cajal, collectively referred to as the Spanish Neurological School (or colloquially, as the Cajal School or School of Madrid). Almost all Cajal’s collaborators were men, although a limited number of female scientists spent part of their careers at the heart of the Cajal School. Here we discuss these women and their work in the laboratory in Madrid. We have tracked the careers of Laura Forster (from Australia/United Kingdom), Manuela Serra, María Soledad Ruiz-Capillas and María Luisa Herreros (all Spanish), through their scientific publications, both in the journal founded by Cajal and elsewhere, and from other documentary sources. To complete the picture, we also outline the careers of other secondary figures that contributed to the production and running of Cajal’s laboratory in Madrid. We show here that the dawn of Spanish neuroscience included a number of contributions from female researchers who to date, have received little recognition., This work was supported by grants PIMCD2017-101 and PIMCD2018-168 from the Vicerrectorado de Calidad, Universidad Complutense de Madrid. Boston Scientific Ibérica has funded CN. The research group of FdC is currently supported by the Spanish Ministerio de Ciencia, Innovación y Universidades (Grants SAF2016-77575-R and RD16-0015-0019, partially financed by F.E.D.E.R.: European Union “Una manera de hacer Europa”), the Fundación Ramón Areces (Spain), the Fundación Inocente Inocente (Spain), the Comunidad de Madrid (Spain, Grant No. IND2018/BMD-9751), a supporting technological contract from AptaTargets, S.L. (Spain), and a grant from the Federation of European Neuroscience Societies (FENS) History Online Project Grants Call 2018.

Introduction: Lafora disease is a rare and severe form of progressive myoclonus epilepsy. It is an autosomal recessive disease, genetically heterogeneous. Aim: Our aims is to study the clinic-pathological features of this rare entitie We report a case of Lafora disease. Case Report: We report the case of a 16 year old girl, which shows from the age of 14 myoclonus epilepsy. Neurological examination showed cerebellar syndrome and intellectual deterioration. Skin biopsy was needed to guide the diagnosis. The Lafora disease has a constantly fatal prognosis. Histological examination confirms the diagnosis and molecular study may help to establish a genetic counseling. Conclusion: Lafora disease has significant clinical and evolutionary characteristics that should guide the clinician to achieve axillary skin biopsy to find Lafora bodies.

Tesis doctoral, 192 páginas, figuras y tablas, [EN] Lafora progressive myoclonic epilepsy (LD) is a rare disease characterized by neurodegeneration, epilepsy and theaccumulation of polyglucosans in different tissues, known as Lafora bodies. At the moment, there is a limited knowledge on the molecular bases of the disease and there is no available treatment, which leads to the death of patients within 10 years after the onset of the first symptoms. Until now, mutations in two genes that could explain the basis of the disease in 92% of the cases: EPM2A, encoding phosphatase dual laforin, and EPM2B, encoding malin, an E3-ubiquitin ligase. Both proteins interact forming a complex able to ubiquitinate different substrates. Recently, the involvement of the complex in several pathways has been demonstrated. However, there has been little advance in the understanding of the molecular bases underlying one of the most important features of the disease: epilepsy. For this reason, the aim of this thesis was to attempt to understand this process. The most part of the studies analyzing the epilepsy in LD focused on the analysis of the neural systems. However, recent studies suggest that an impairment of the astrocytes due to existing neuroinflamation could contribute to the triggering and/or the development of epilepsy and other neurological diseases. For this reason, our efforts have been focused on the study of the main excitatory neurotransmitter homeostasis, glutamate, in cellular and animal models of LD., The mentioned homeostasis depends on the correct functioning of the astrocytic cells and their deregulation contributes significantly to an increase in neural hyperexcitability and death. In this work, an alteration of the main astrocytic glutamate transporter, GLT-1, has been identified. This transporter is responsible of 90% of extracellular glutamate uptake. Therefore, a decrease in the activity of GLT-1 leads to an increase in synaptic glutamate, contributing to the hyperexcitability. Primary astrocytes from cortex of mice deficient in Epm2a or Epm2b genes, models of LD, show a decrease in the amount of GLT-1 located at the cell surface, which is correlated to a lower capacity of these cells to take glutamate up. This change in the subcellular localization is regulated by changes in the ubiquitination of the transporter, which is increased in the presence of overexpressed laforin-malin complex. Finally, we demonstrated that mouse models for LD present a reduced capacity to uptake glutamate compared to the control group, using in vivo microdialysis. Hence this thesis presents evidences of a deregulation in the homeostasis of the glutamate transporter GLT-1 in animals and cells models for LD and suggests that this alteration could contribute to epilepsy in LD., [ES] La epilepsia mioclónica progresiva de tipo Lafora (LD) es una enfermedad rara caracterizada por neurodegeneración, epilepsia y acumulación de poliglucosanos en diferentes tejidos, denominados cuerpos de Lafora. Por el momento, existe un conocimiento limitado de las bases moleculares de la enfermedad y todavía no se dispone de un tratamiento adecuado, lo que lleva a la muerte de los pacientes 10 años después del debut de los primeros síntomas.Hasta la fecha se han identificado mutaciones en dos genes que explicarían la base de la enfermedad en un 92% de los casos: EPM2A, que codifica la fosfatasa dual laforina, y EPM2B, que codifica una E3-ubicuitina ligasa. Ambas proteínas interaccionan formando un complejo capaz de ubicuitinar diferentes sustratos.Recientemente se ha demostrado la afectación de varias vías en la enfermedad de Lafora. Sin embargo, se ha avanzado poco en la comprensión de las bases moleculares que subyacen a una de las características más importantes de la enfermedad: la epilepsia. Por eso, la finalidad de esta tesis ha sido tratar de comprender este proceso. Hasta la fecha, la mayor parte de estudios realizados analizando la epilepsia en la enfermedad de Lafora se han centrado en analizar los sistemas neurales. No obstante, estudios recientes sugieren que una afectación de los astrocitos debido a la neuroinflamación existente podría contribuir al desarrollo de la epilepsia. Por ese motivo, nuestros esfuerzos han ido dirigidos al estudio de la homeostasis del principal neurotransmisor excitatorio, el glutamato, en modelos celulares y animales de la enfermedad de Lafora. Dicha homeostasis depende del correcto funcionamiento de las células astrocitarias y su desregulación contribuye significativamente a un aumento de la hiperexcitabilidad y la muerte neuronal., En este trabajo se ha identificado una desregulación del principal transportador de glutamato astrocitario, GLT 1. Dicho transportador es el responsable de la captación del 90% del glutamato extracelular, por lo que una disminución de la actividad de GLT-1 provoca un aumento del glutamato sináptico, favoreciendo la hiperexcitabilidad. Astrocitos primarios procedentes de corteza de ratón deficientes en el gen Epm2a o Epm2b muestran una disminución de la cantidad de GLT-1 expresada en la superficie celular, que se correlaciona con una menor capacidad de estas células para captar glutamato. Este cambio en la localización subcelular está regulado por cambios en la ubicuitinación del transportador, que se encuentra aumentada en presencia de laforina-malina sobreexpresada. Por último, se ha demostrado que los ratones modelo para LD presentan una capacidad disminuida para captar glutamato respecto a los controles, utilizando microdiálisis in vivo.Por lo tanto, esta tesis presenta evidencias de una desregulación de la homeostasis del transportador de glutamato GLT-1 en células y animales modelo para LD y sugiere que esta alteración podría contribuir a la epilepsia presente en LD., Para la realización de esta tesis doctoral, María del Carmen Muñoz Ballester ha disfrutado de una ayuda para la Formación de Profesorado Universitario (FPU) (FPU12-00383), del Ministerio de Educación, Cultura y Deporte. El trabajo se ha llevado a cabo en el grupo U742 del CIBERER-Instituto de Salud Carlos III (IP, P. Sanz) enmarcado dentro de los siguientes proyectos de investigación: – Programa Nacional de Biomedicina. Proyecto SAF2011-27442. “Bases moleculares de la fisiopatología de la epilepsia mioclónica progresiva de tipo Lafora. Identificación de nuevos sustratos del complejo laforina-malina” Años 2012-2014. Investigador Principal: Dr. Pascual Sanz. Centro: Instituto de Biomedicina de Valencia (CSIC), Valencia. – Programa Nacional de Biomedicina. Proyecto SAF2014-54604-C3-1-R. “Buscando una terapia para la epilepsia mioclónica progresiva de Lafora” Años 01/01/2015-31/12/2017. Investigador Principal: Dr. Pascual Sanz. Centro: Instituto de Biomedicina de Valencia (CSIC), Valencia.

Tesis doctoral, 123 páginas., Para la realización de esta tesis doctoral, Pablo Sánchez Martín ha disfrutado de una ayuda para la Formación de Profesorado Universitario (FPU) (FPU13-00507), del Ministerio de Educación, Cultura y Deporte.El trabajo se ha llevado a cabo en el grupo U742 del CIBERER-Instituto de Salud Carlos III (IP, P. Sanz) enmarcado dentro de los siguientes proyectos de investigación: – Programa Nacional de Biomedicina. Proyecto SAF2011-27442. “Bases moleculares de la fisiopatología de la epilepsia mioclónica progresiva de tipo Lafora. Identificación de nuevos sustratos del complejo laforina-malina” Años 2012-2014. Investigador Principal: Dr. Pascual Sanz. Centro: Instituto de Biomedicina de Valencia (CSIC), Valencia. – Programa Nacional de Biomedicina. Proyecto SAF2014-54604-C3-1-R. “Buscando una terapia para la epilepsia mioclónica progresiva de Lafora” Años 01/01/2015-31/12/2017. Investigador Principal: Dr. Pascual Sanz. Centro: Instituto de Biomedicina de Valencia (CSIC), Valencia

Aim The aim of this report is to provide initial evidence that add-on treatment with perampanel might be highly effective in progressive myoclonic epilepsy such as Lafora disease. Case report We report on a 21-year-old woman suffering from persistent myoclonus and generalized tonic–clonic seizures for more than seven years. Additionally, ataxia, a disturbance in speech and gait, as well as a cognitive decline were rapidly progressing. Subsequently, the diagnosis of Lafora disease was confirmed by the identification of a novel homozygous missense mutation in exon 3 of the EPM2A gene (c.538C>G; p.L180V). Adjunctive therapy with perampanel was started in this patient with advanced Lafora disease and was titrated up to 8 mg/day. A sustained and reproducible remission of myoclonus and GTCS could be achieved for a follow-up of three months. After dosage reduction to 6 mg/day, seizures recurred; however, on increasing the daily dose to 10 mg, seizures stopped for another three months. The patient also regained her ability to walk with help and the aid of a walker. Conclusions Perampanel is a selective, noncompetitive antagonist of AMPA-type glutamate receptors and recently licensed as adjunctive therapy for the treatment of refractory focal onset seizures. There is evidence for its effectiveness in generalized epilepsies, and phase III studies for this indication are on the way. Our case illustrates the possibility that perampanel might be a valuable option for treatment in PME. Considering its impressive efficacy in this case, we suggest a prospective, multicenter study evaluating perampanel in PME.

The history of the progressive myoclonus epilepsies (PMEs) spans more than a century. However, the recent history of PMEs begins with a consensus statement published in the wake of the Marseille PME workshop in 1989 (Marseille Consensus Group, 1990). This consensus helped define the various types of PME known at the time and set the agenda for a new era of genetic research which soon lead to the discovery of many PME genes. Prior to the Marseille meeting, and before the molecular era, there had been much confusion and controversy. Because investigators had but limited and biased experience with these rare disorders due to the uneven, skewed distribution of PMEs around the world, opinions and nosologies were based on local expertise which did not match well with the experiences of other researchers and clinicians. The three major areas of focus included: (1) the nature and limits of the concept of PME in varying scopes, which was greatly debated; (2) the description of discrete clinical entities by clinicians; and (3) the description of markers (pathological, biological, neurophysiological, etc.) which could lead to a precise diagnosis of a given PME type, with, in the best cases, a reliable correlation with clinical findings. In this article, we shall also examine the breakthroughs achieved in the wake of the 1989 Marseille meeting and recent history in the field, following the identification of several PME genes. As in other domains, the molecular and genetic approach has challenged some established concepts and has led to the description of new PME types. However, as may already be noted, this approach has also confirmed the existence of the major, established types of PME, which can now be considered as true diseases.

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.

Lafora disease (LD) is caused by mutations in either the laforin or malin gene. The hallmark of the disease is the accumulation of polyglucosan inclusions called Lafora Bodies (LBs). Malin knockout (KO) mice present polyglucosan accumulations in several brain areas, as do patients of LD. These structures are abundant in the cerebellum and hippocampus. Here, we report a large increase in glycogen synthase (GS) in these mice, in which the enzyme accumulates in LBs. Our study focused on the hippocampus where, under physiological conditions, astrocytes and parvalbumin-positive (PV(+)) interneurons expressed GS and malin. Although LBs have been described only in neurons, we found this polyglucosan accumulation in the astrocytes of the KO mice. They also had LBs in the soma and some processes of PV(+) interneurons. This phenomenon was accompanied by the progressive loss of these neuronal cells and, importantly, neurophysiological alterations potentially related to impairment of hippocampal function. Our results emphasize the relevance of the laforin-malin complex in the control of glycogen metabolism and highlight altered glycogen accumulation as a key contributor to neurodegeneration in LD.

As epilepsias mioclônicas progressivas (EMP) são um grupo raro de epilepsias de evolução debilitante e prognóstico ruim. Seu desafio reside na dificuldade do diagnóstico etiológico e na ausência de um tratamento específico para cada entidade. Apesar disso, avançosrecentes na área de genética molecular vem possibilitando melhor compreensão da etiopatogenia e diagnóstico dessas doenças. Neste trabalho, revisamos os conhecimentos atuais a respeito das EMP com ênfase nos aspectos clínicos e genéticos. The progressive myoclonic epilepsies (PME) are a rare group of epilepsies of debilitating evolution and bad prognosis. The challenge concerning PME consists of the difficulty in establishing the etiology and the absence of a specific treatment for each entity. Nevertheless,recent advances in molecular genetics have enabled better understanding of the pathogenesis and diagnosis of these diseases. The present work is a review of the PME features, with emphasis oncurrent molecular and clinical findings.