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11 páginas, 7 figuras. This is a pre-copyedited, author-produced version of an article accepted for publication in Human Molecular Genetics following peer review. The version of record Rubio-Villena C., Viana R., Bonet J., Garcia-Gimeno M.A., Casado M., Heredia M.,Sanz P. (2018). Astrocytes: new players in progressive myoclonus epilepsy of Lafora type. Hum Mol Genet 27(7): 1290-1300, is available online at: http://dx.doi.org/10.1093/hmg/ddy044, Lafora disease (LD) is a fatal form of progressive myoclonus epilepsy characterized by the accumulation of insoluble poorly branched glycogen-like inclusions named Lafora bodies (LBs) in the brain and peripheral tissues. In the brain, since its first discovery in 1911, it was assumed that these glycogen inclusions were only present in affected neurons. Mouse models of LD have been obtained recently, and we and others have been able to report the accumulation of glycogen inclusions in the brain of LD animals, what recapitulates the hallmark of the disease. In this work we present evidence indicating that, although in mouse models of LD glycogen inclusions co-localize with neurons, as originally established, most of them co-localize with astrocytic markers such as glial fibrillary acidic protein (GFAP) and glutamine synthase. In addition, we have observed that primary cultures of astrocytes from LD mouse models accumulate higher levels of glycogen than controls. These results suggest that astrocytes may play a crucial role in the pathophysiology of Lafora disease, as the accumulation of glycogen inclusions in these cells may affect their regular functionality leading them to a possible neuronal dysfunction., This work was supported by grants from the Spanish Ministry of Economy and Competitiveness SAF2014-54604-C3-1-R, a grant from Generalitat Valenciana (PrometeoII/2014/029), a grant form Fundación Ramón Areces (XVIII Concurso Nacional Ayudas Investigacion Ciencias Vida y Materia) and a grant from the National Institute of Health (NIH-NINDS) P01NS097197, which established the Lafora Epilepsy Cure Initiative (LECI), to PS and grants SAF2016-75004-R and Contribution to COST Action CA15203 MITOEAGLE to MC.

Content, composition and molecular weight distribution of the urinary glycosaminoglycans (GAG) were determined in five patients with progressive myoclonus epilepsy (PME). In one patient (Family B) this syndrome was associated with cerebral Lafora bodies and in four siblings of Family A, no Lafora bodies were present in brain biopsy. Only one of the five patients had a moderate increase of urinary GAG excretion as expressed by 24-h output or creatinine. The heparan sulfate component of the GAG was moderately increased in two other patients. The molecular weight distribution of the urinary GAG was normal. The results do not support the contention that urinary GAG excretion is abnormal in PME. Among nine lysosomal enzymes in leucocytes, only the activity of alpha-mannosidase was increased 3-fold in the four siblings.

We studied brainstem auditory-evoked potentials of 11 patients suffering from the Finnish type of progressive myoclonus epilepsy (without Lafora bodies). All patients were ambulatory and had regular antiepileptic medication. The responses of 5 patients were interpreted as totally normal. In 6 cases there were slight deformations of the wave forms or of the amplitudes of the responses. The central conduction time of all patients was within the +/- 2 S.D. limits of the normal values. Postauricular muscular responses were not more prominent in patients than in controls. The results indicate that there is not any significant dysfunction in the cholinergic auditory pathways of the brainstem of these patients.

Summary A longitudinal clinicoelectrophysiologic study was undertaken of a 15-year 2-month-old girl with Lafora disease who was diagnosed by skin biopsy and an immunohistochemical method with antisera against Lafora bodies. From age 10 years 5 months, 4 months after onset, EEG disclosed progressive deterioration of background activity and incremental increase in epileptic discharges. Photosensitivity was unique: Occipital spikes and diffuse spike-wave discharges were provoked by low-frequency repetitive photic stimuli but without elicitation of myoclonic seizures. Photosensitivity completely disappeared after age 13 years 10 months. High-voltage somatosensory evoked potentials (SEPs) and high-voltage flash visual evoked potentials (F-VEPs) were seen before age 13. After age 13, progressive prolongation of I-III and I-V interpeak latencies of auditory brainstem responses (ABRs), progressive prolongation of latencies of photoevoked eyelid microvibrations, delayed latencies of pattern-reversal visual evoked potentials, and a decrease in the V/I amplitude ratio of ABRs and the previously high F-VEP amplitudes were observed. RESUME Les auteurs ont suivi une jeune fille âgee de 15 ans et 2 mois, Presentant une maladie de Lafora diagnostiquee par biopsie cutanee et etude immunohistochimique au moyen d'un serum reagissant sur les corps de Lafora. A partir de l'âge de 10 ans et 5 mois, 4 mois apres le debut de la maladie, l'EEG a mis en evidence une deterioration progressive de l'activite de fond et une augmentation progressive des decharges epileptiques. Une photosensibilite tres particuliere a ete mise en evidence: des Pointes occipitales et des pointes-ondes diffuses etaient provoquees par la stimulation lumineuse intermittente a basse frequence, sans provoquer toutefois de crises myocloniques, cette photosensibilite a disparu completement apres l'âge de 13 ans et 10 mois. Des potentiels evoques somato-sensitifs de haut voltage et des potentiels evoques visuels au flash de haut voltage ont ete enregistres avant l'âge de 13 ans. Apres cet âge, les auteurs ont constate un allongement progressif des latences interpics I-III et I-IV des potentiels evoques auditifs precoces, un allongement progressif des latences des microvibrations palpebrales evoquees par la lumiere, un allongement des latences des potentiels evoques visuels par inversion de pattern, et une diminution de l'amplitude du rapport V/I des potentiels evoques auditifs precoces et des amplitudes des potentiels evoques visuels au flash. RESUMEN Se ha realizado un estudio longitudinal clinico-electrofisiologico en una nina de 15 anos y 2 meses de edad que padecia la enfermedad de Lafora diagnosticada mediante una biopsia cutanea y un metodo histoquimico con antisuero anticuerpos de Lafora. Desde la edad de 10 anos y 5 meses, 4 meses despues del comienzo de la enfermedad, aaparecio un deterioro progresivo de la actividad de fondo con un incremento de las descargas epilepticas. La fotosensibilidad fue muy especial: puntas occipitales y descargas difusas punta-onda fueron provocadas mediante estimulacion fotica repetitiva efectiva, de baja frecuencia, pero sin que se observaran ataques mioclonicos. La fotosensibilidad desaparecio completamente despues de la edad de 13 anos y 10 meses. Antes de la edad de 13 anos se observaron potenciales evocados somatosensoriales de alto voltaje y potenciales evocados visuales (F-VEPs) tras flash de alto voltaje. Despues de la edad de 13 anos se observaron: una progresiva prolongacion de las latencias interpicos de I-III y I-V de las respuestas auditivas de tronco cerebral (APRs), una prolongacion progresiva de las latencias de las vibraciones de los parpados evocadas foticamente, un retraso de las latencias de los potenciales evocados visuales inducidos por patrones graficos y una reduccion de la relacion V-I de los ABRs y de las previas amplitudes de los F-VEP. ZUSAMMENFASSUNG Eine klinisch-elektrophysiologische Langzeitstudie wurde bei einem jetzt 15 Jahre alten Madchen mit Lafora-Erkrankung durch gefuhrt. Die Diagnose war durch Hautbiopsie un durch immunhistochemische Methoden mit Antiseren gegen Laforakorper gestellt worden. Im Alter von 10,5 Jahren, 4 Monate nach Auftreten der Erkrankung, zeigte das EEG einen progredienten Abbau der Grundaktivitat und allmahliche Zunahme epileptischer Entladungen. Die Fotosensibilitat war auffallig: durch langsame repetitive Stimuli wurden occipitale spikes und diffuse spike-waves Entladungen provoziert ohne Auslosung einer myoklonischen Reaktion. Im Alter von 13,10 Jahren verschwand die Fotosensibilitat vollstandig. Amplitudenhohe somatosensorische EP und hohe Blitz-VEP wurden vor dem Alter von 13 Jahren und danach beobachtet. Die AEP zeigten allmahliche Verlangerung der I-III und I-V Latenzen, ferner eine Verlangerung der Licht-evozierten Augenlid-Mikrovibrationen und verzogerte Latenzen der VEP bei Musterumkehr. Ein Abfall der Amplitudenkorrelation V/1 der AEP und der Amplituden der F-VEP wurde ebenfalls festgestellt.

Lafora disease (LD) is an inherited and fatal form of neurodegenerative disorder characterized by the presence of an abnormal form of glycogen inclusions, called Lafora bodies, in neurons and other tissues. While Lafora bodies have been thought to underlie the neuropathology in LD, the specific process by which these inclusions might affect the neuronal functions was not very well understood. Here we review one of our recent studies on the LD animal model, wherein we have shown that the Lafora bodies might contribute to the impairment in the endosomal-lysosomal and autophagy pathways.

Summary: Since age 12 years, a 25-year-old woman had a syndrome with myoclonic epilepsy, cerebellar signs, and spontaneous myoclonus. Skin biopsy showed typical Lafora bodies (LB), but she lacked a progressive course and mental impairment, hallmarks of Lafora disease. Lysosomal enzyme assays showed low level arylsulfatase A (ASA) activity. DNA study disclosed a homozygous ASA Pd genotype. Both parents carried one Pd allele. The still-unknown relationship between the pathologic level of ASA activity and myoclonic epilepsies suggests introduction of ASA assays in patients with PME. RESUMEN Desde la edad de 12 anos una mujer de 25 anos padecio un sindrome con epilepsia mioclonica, trastornos cerebelosos y mioclonias espontaneas. La biopsia cutanea mostro tipicos cuerpos de Lafora pero no presento un cuadro progresivo ni alteraciones mentales, sintomas fundamentales de la enfermedad de Lafora. Los ensayos de enzima lisosomial mostraron niveles bajos de actividad ASA. El estudio de DNA revelo un genotipo homozigotico ASA Pd. Ambos familiares eran portadores del allelo Pd. Todavia permanece desconocida la relacion entre el nivel patologico de la actividad de la ASA y las epilepsias mioclonicas lo que sugiere la introduccion de los ensayos ASA en los pacientes con epilepsia mioclonica progresiva. ZUSAMMENFASSUNG Eine 25 jahrige Frau litt seit ihrem 12. Lebensjahr an einem Syndrom mit myoklonischer Epilepsie, zerebellaren Zeichen und spontanem Myoklonus. Die Hautbiopsie zeigte typische Laforakorper, die Patientin zeigte jedoch keinen progressiven Verlauf und mentalen Abbau, wie sie typisch fur dei Lafora-Erkrankungen sind. Die Bestimmung der lysosomalen Enzyme zeigte eins erniedrigts ASA-Aktivitat. DNA-Untersuchungen schlossen einen homozygoten ASA Pd Genotyp aus. Beide Eltern trugen sin Pd Allel. Die bislang unbekannte Beziehung zwischen pathologischen ASA Werten und myoklonischen Epilepsien legen nahe, ASA Assays bei Patienten mit PME durchzufuhren.

Massive occurrence of polyglucosan bodies (PBs) was found within the surgically removed temporal lobe of a 34 year old woman with complex partial seizures. This peculiar feature is very unusual in neuropathological examinations of epileptogenic foci. This patient could not be included in any of the classic diseases in which PBs are found. She exhibited a localised form of glycogen storage disease.

This report describes a sporadic late-onset myopathy in two unrelated adults which was marked by polyglucosan inclusions surrounded by abnormally structured mitochondria, the latter finding a localized, possibly reactive phenomenon. The polyglucosan material was characterized by a battery of histochemical and enzyme histochemical techniques; revealed common antigenicity with Lafora bodies, corpora amylacea and muscle fiber inclusions in types IV and VII glycogenoses; and contained ubiquitin. Additional lectin histochemical and associated digestion preparations disclosed the presence of alpha-glycosyl residues as apparently the sole carbohydrate component in polyglucosan bodies while the above mentioned common antigenicity with Lafora bodies and other inclusions suggests an additional, so far unidentified, protein component.

A cytochemical procedure for polysaccharides was carried out on a brain biopsy specimen, the thin-section study of which had shown excess glycogen granules and the corpora amylacea variety of polyglucosan bodies. Both granules and amyloid bodies were stained positively in contrast to the remaining structures of the brain tissue which remained unstained. This demonstrates that beta-granules as well as filamentous and amorphous components of amyloid bodies are just different aspects of the polysaccharide molecule. Up to now the same kind of cytochemical evidence has been supplied for Lafora bodies of human material and Lafora-like bodies of rat material. The present study on corpora amylacea of human material shows that amyloid, Lafora, and Lafora-like bodies all behave the same way when stained for polysaccharides.

Deposits similar to corpora amylacea were observed by electron microscopy and light microscopy within thin myelinated axons in the spinal gray matter in various diseases. By electron microscopy the deposits consisted of randomly interlacing short linear structures of various thickness which were different from tangles of neurofilaments. Sometimes they contained dense granules and electron-dense floccules. Though their incidence was relatively high in some cases of degenerative or metabolic disease of the central nervous system, they were not specific to any disease but seemed to be related to aging, indicating a peculiar aspect of chronic degeneration of the axons.

Summary Sixty-five patients with progressive myoclonus epilepsy without Lafora bodies were examined by the Wechsler Verbal or Terman-Merrill-Lehtovaara scale, and 25 were reexamined after a mean of 6 yr. Intelligence was relatively intact by contrast with the patients' poor physical condition. The estimated I.Q. at the onset was 92, in the low normal range, and it diminished by about 10 points in 10 yr. This diminution had begun before the appearance of clear clinical symptoms. Low test scores correlated significantly (p < 0.01) with duration of the disease and with age. On the Wechsler Verbal Scale, tests of similarities, information, and comprehension were least affected; scores on digit span and arithmetic tests were lowest. Speech was slow and dysarthric and became finally unintelligible. Personality was infantile and mood labile and often depressed. The surprisingly high intelligence differentiates these patients from those having the form of progressive myoclonus epilepsy that occurs with Lafora bodies. The two diseases seem to be distinct clinical and histopathologic entities. REsume 65 sujets avec une epilepsie myoclonique progressive sans corps de Lafora, ont ete examines avec l'echelle verbale Wechsler ou Terman-Merrill-Lehtovaara, et 25 ont ete examines a nouveau en moyenne 6 ans apres. L'intelligence etait relativement intacte en contraste avec les mauvaises conditions psychiques des sujets. Le Q.I. au debut etait environ G 92, dans les limites inferieures de la normale. Des pauvres performances etaient en correlation significative (p < 0.01) avec la duree de la maladie et l'âge. Dans l'echelle verbale de Wechsler, les tests de similitudes, l'information et la comprehension etaient moins atteints; les valeurs des tests, la memoire des chiffres et l'arithmetique, etaient les plus bas. Le langage etait ralenti et avec dysarthrie et devenait a la fin incomprehensible. La personnaliteetait infantile et l'heumeur labile et souvent depressive. Un niveau d'intelligence etonnament eleve differencie ces patients de ceux qui ont une forme de myoclono-epilepsie progressive avec corps de Lafora. Les deux maladies sont des entites distinctes du point de vue clinique et histo-pathologique. RESUMEN Utilizando la escala verbal de Wechsler o la de Terman-Merrill-Lehtovaara se han examinado 65 enfermos con epilepsyia mioclonica progresiva sin cuerpos de Lafora, 25 de los cuales fueron reexaminados despues de un promedio de 6 anos. La inteligencia estaba relativamente conservada, hecho que contrastaba con el considerable deterioro fisico de los enfermos. Al principio del estudio el Cociente Intelectual (IQ) fue de 92, normal bajo, disminuyendo 10 puntos al cabo de 10 anos. Se aprecio una significativa correlateon (p < 0.01) entre los IQ bajos, la duracion de la enfermedad y la edad de los enfermos. En la escala verbal de Wechsler, resultaron menos afectados los tests de semejanzas, information y comprension, mientras que los resultados mas bajos se obtuvieron en pruebas aritmeticas. El lenguaje era disartrico y lento llegando, finalmente, a ser incomprensible. La personalidad era infantil y el animo labil y frecuentemente deprimido. La inteligencia sorprendentemente alta diferencia a este tipo de enfermos con los que padecen la forma progresiva de epilepsyia mioclonica con cuerpos de Lafora. Estas dos enfermedades constituyen entidades clinicas e histopatologicas distintas. ZUSAMMEnfassung 65 Patienten mit progressiver Myoklonusepilepsie ohne Laforakorperchen wurden psychologisch untersucht mit dem verbalen Wechslertest oder der Terman-Merrill-Lehtovaara-Skala. 25 von ihnen wurden nach einer durchsehnittlichen Zeitdauer von 6 Jahren nachuntersucht. Im Gegensatz zu der schweren korperlichen Beeintrachtigung war die Intelligenz relativ intakt. Der durchschnittliche IQ lag im Beginn mit 92 im unteren Normbereich. Er verminderte sich um etwa 10 Punkte in 10 Jahren. Niedrige Testergebnisse korrelierten signifikant mit der Dauer der Erkrankung und mit dem Alter (p < 0.01). Im Wechslerverbaltest waren die Untertests fur Ahnlichkeiten, Informatione und Verstandnis am wenigsten beeintrachtigt. Die Scores fur Zahlennachsprechen und Rechnen lagen am niedrigsten. Die Sprache war langsam und dysarthrisch und wurde schliesslich unverstandlich. Die Personlichkeit war infantil, die Stimmung labil und haufig depressiv. Die uberraschenderweise hohe Intelligenz unterscheidet diese Patienten von solchen mit einer progressiven Myoklonusepilepsie, bei der Lafora-korperchen gefunden wurden. Beide Erkrankungen sind klinisch und histopathologisch zu unterscheidende Entitaten.

A patient with advanced progressive myoclonic epilepsy (Unverricht type) with Lafora bodies is presented. Although the clinical history and symptoms were classical, the regional distribution of the cerebral involvement differed from the classical picture: the corpora mamillaria, the nucleus subthalamicus, and the nucleus ruber, which are normally reported to be spared, contained multiple Lafora bodies, whereas the lateral geniculate body, which is usually involved, was intact. The number of inclusions per cell, up to 25, was extremely high and correlated with the marked cortical atrophy and the prolonged clinical course. Using electron microscopy, type I and type II Lafora bodies were found, but the latter lacked the typical filamentous ultrastructure in the peripheral zone. The lack of visceral Lafora bodies in this case suggests that liver, muscle, and skin biopsies, which are widely used for the diagnosis, may lead to false negative results and cannot always replace a stereotactic brain biopsy. The differential diagnosis on polyglucosan bodies is emphasized.

Seven cases of progressive familial myoclonus epilepsy occurring in three families are presented. The patients were in different stages of the illness. The EEG was abnormal in all. It is suggested that these cases belong clinically to the Lafora bodies group. Nystagmus and optic atrophy, seen in one patient, have not been described previously. Myoclonic jerks did not respond to treatment with diazepam and ethosuximide.

We have studied four patients who had a clinical course compatible with Lafora disease. The diagnosis was confirmed in one by the presence of Lafora bodies in central nervous system neurons at autopsy and was supported in another by findings in the cerebral biopsy of a sibling. Our patients had no clinically apparent liver disease, but liver specimens in each instance showed a distinctive histological abnormality, with hepatocytes containing inclusions having a ground-glass appearance. The liver biopsy findings appear to be relatively specific for this disorder and can easily be differentiated from those in other liver diseases.

RECENT advances in the diagnosis and classifcxation of epileptic seizures and epileptic syndromes, together with improvemenss in anticonvulsant therapy, have enabled the great majority of patients ...

The Lafora type of progressive myoclonus epilepsy is a rare and fatal familial disease characterized by seizures, myoclonus, and dementia. This diagnosis was confirmed in 2 patients by demonstrating the presence of intracytoplasmic polygkicosan bodies, or Lafora bodies, in the peripheral portion of the eccrinc sweat gland duct. Exclusive use of the periodic acid-Schifl stain is recommended for demonstrating these diagnostic inclusions. Electron microscopy reveals fine pale-staining filaments, line clark-slaining granules, and dark-rimmed vacuoles within these non-inembranc-bound inclusions. Skin biopsy is the preferred method of confirming the diagnosis of Lafora disease.

Deposits similar to corpora amylacea were observed by electron microscopy within myelinated axons in the peripheral nerves in a case of familial spastic paralysis. Ultrastructurally the deposits consisted of randomly interlacing short filaments which were closely related to glycogen granules present in the periphery of the deposits. A possible relationship between the filamentous structures and glycogen granules is discussed. The significance of this inclusion and of related bodies is also discussed.

The authors present the case of a patient with Lafora disease proven by skin biopsy, who suffered two occipital seizures recorded on EEG and provoked by intermittent photic stimulation. On the basis of data in the literature and their present experience, the authors suggest the involvement of the occipital lobes as a whole in the epileptogenic activity.

Lafora bodies were studied in a parietal cortex biopsy of a patient suffering from progressive myoclonic epilepsy, both histochemically (with the optical microscope) and in the electron microscope. The bodies appear at the electron microscope as fibrillar and granular masses, not surrounded by a membrane. Most of them closely resemble corpora amylacea, but are located in neurons or their processes. The PAS positive “dust-like” particles, previously described in this disease with the light microscope, present, at the electron microscope, the same fundamental structure. Lafora bodies do not contain glycogen but mainly mucopolysaccharides. The hypothesis of their lysosomal nature is discarded.

Intraneuronal inclusions, consisting of polyglucosan and having histochemical and ultrastructural features identical to Lafora body of familial myoclonic epilepsy (Unverricht-Lafora disease), have been found restricted to the lateral pallidum in five patients. Two of these patients were also found to have status marmoratus of the basal ganglia. These lateral pallidal inclusions have been named after Bielschowsky, their original discoverer. We report two additional patients with status marmoratus and Bielschowsky bodies and suggest that these two conditions are frequent concomitant phenomena arising independently from a common cause.

This is the first report of an adult polysaccharide storage myopathy demonstrating polysaccharide deposits within the axons of the peripheral nerve. Histochemical and electron-microscopic studies revealed that the intra-axonal deposits were basophilic, PAS-positive, diastase-resistant, and composed of filamentous and granular material. The structural similarity among the polysaccharide deposits in our case, amylopectin-like materials in type IV glycogenosis, Lafora bodies, corpora amylacea, and polyglucosan bodies is discussed.

Cerebral biopsies of three patients aged 22, 18 and 16 years with myoclonic epilepsy contained Lafory bodies. Two were a brother and sister of consanguineous parents. The Lafora bodies occurred in most neurons but not in glial cells. The ultrastructure of these bodies showed a fibrillar and granular material in the perikaryon and neuropile. In some neurons small Lafora bodies were delimited by a double membrane, suggesting that in early phases they have a membrene which disappears when they become more developed. Other non-specific neuron alterations are described. One of the cases presented in the liver PAS positive bodies of a structure different from the Lafora bodies.

Thirty-one patients suffering from progressive myoclonus epilepsy, (also called Unverricht-Lundborg's disease) without Lafora bodies, were examined to check the findings reported in literature and to chart out the main abnormalities in routine laboratory findings. Many alterations could be pointed out, but a high proportion of them were due to factors which are secondary to the syndrome of progressive myoclonus epilepsy: continuous anticonvulsive medication; immobilization; frequent infections; and the patient's poor nutritional condition. The most remarkable finding, and the only one which supported the abnormality reported earlier, was the raised excretion of indican which could not be explained by fermentation in the bowels. The urinary 5-hydroxyindoleacetic acid excretion was a little low, but still within the normal range. The tryptophan and 5-hydroxytryptamine metabolism deserves further investigation in attempting to discover the aetiology of progressive myoclonus epilepsy.

Lafora's disease is a neurometabolic disease characterized by progressive myoclonus epilepsy. It is thought to be transmitted by autosomal recessive inheritance. The diagnosis of Lafora's disease is based on both clinical symptomatology and histologic detection of intracytoplasmic periodic acid-Schiff-positive inclusions, termed Lafora bodies. Despite the absence of cutaneous clinical findings, typical inclusions are present in eccrine duct cells and peripheral nerve of skin biopsies. By electron microscopy, the inclusions are electron-lucent and contain glycogenlike granules and filamentous material. Skin biopsy is a convenient and the least invasive method of establishing the diagnosis of Lafora's disease.

We compared the ultrastructure of the intracytoplasmic inclusion substance present in Lafora bodies and the myocardium of patients with Lafora disease, with that in the hepatocytes and myocardium of patients with type IV glycogenosis. Fibril-like structures and associated electron-dense clumps (crumpled plates) are the main components of the deposits in these two diseases. Ultrastructurally, the inclusions in both diseases appeared quite similar. In Lafora bodies, the electron-dense materials formed their central cores, but were also scattered in their peripheral areas. In type IV glycogenosis, the materials tended to be localized in the centers of large storage masses. The fibril-like structures in Lafora bodies were somewhat thicker and more electron-dense than those in type IV glycogenosis. The fibril-like structures and the crumpled plates in the intracytoplasmic inclusions of both Lafora disease and type IV glycogenosis were intensely stained in sections subjected to Thiery staining. Despite previous considerations to the contrary, the findings of the present study suggested that the fibril-like structures are not true fibrils, but in fact plates.

A case is reported of an 18 year old man with progressive myoclonus epilepsy. Histopathological examination revealed the presence of numerous Lafora bodies in the cerebellar granular layer, without other significant changes in the central nervous system or in other organs.

Within the framework of an Early Postmortem Brain Sampling Program we had the opportunity to make some light and electron microscopic observations on the orbital cortex of two elderly subjects known to have been free of neurologic and psychiatric disease. Filamentous deposits with the ultrastructural appearance of corpora amylacea were seen inside astrocytic and, more rarely, inside neuronal processes. The findings and their interpretation are discussed in the light of current views on cerebral corpora amylacea and Lafora bodies. It is concluded that today's views on this matter do not account satisfactorily for the present observation or for observations we have quoted from the literature.

A rare case with pleomorphic intra-neuronal polyglucosan bodies is reported. A 45-year-old man died of gastric carcinoma with extensive visceral involvement. The autopsy showed numerous intra-neural pleomorphic PAs-positive deposits reminiscent of Bielschowsky bodies in the lateral segments of the pallidum, substantia nigra, and brain stem on both sides. Their histochemical properties were consistent with polyglucosan. The bodies were ultrastructurally composed of filamentous structures and seen in perikarya, axons, and dendrites. Occasionally, spotty electron-dense material was seen in the center of the bodies. The filaments were selectively stained with thiocarbohydrazide method after Thiery, indicating their chief component as glucose polymers. The bodies are compared with Bielschowsky bodies, Lafora bodies, corpora amylacea, and related bodies, and their nature and significance are briefly discussed.

A small number of intraneuronal polyglucosan bodies, the so-called Lafora bodies, is occasionally found in the tegmental areas of the midbrain in aged cases with no metabolic or degenerative diseases. The occasional appearance of peculiar bodies, which Kato et al.1982 reported in a case of presenile dementia, is also observed during the examination of the inclusions. The ultrastructure of the bodies reveals an amorphous, electron-dense central core and a peripheral zone of radially oriented 100 A wide filaments.

Summary: Lafora disease is an autosomal recessive and progressive degenerative disorder of the central nervous system (CNS). The pathogenic mechanism has been presumed to be an inborn error of carbohydrate metabolism, although this has never been proved. In a case of proven Lafora disease, pyruvate metabolism, which has a central position in carbohydrate metabolism, was studied in body fluids under various conditions and in brain biopsy material. No abnormalities in this metabolic pathway were found. This finding plus earlier reports in the literature exclude a defect in glycolysis; thus, a disturbance of carbohydrate metabolism as the pathogenic mechanism of Lafora disease is unlikely RESUME La maladie de Lafora est une affection autosomique recessive degenerative et progressive du systeme nerveux central. Le mecanisme pathogenique est vraisemblablement represente par une erreur innee du metabolisme qui n'a jamais ete prouvee. Dans un cas de Lafora confirme, les auteurs ont etudie le meta-bolisme du pyruvate, qui occupe une position centrale dans le metabolisme des glucides, dans les liquides biologiques et a par-tir une biopsie cerebrale. lis n'ont pas constate anomalie dans le meltabolisme du pyruvate. Ces resultats, joints a des travaux anterieurs de la Litterature, excluent hypothese un RESUMEN La enfermedad de Lafora consiste en un proceso degenerativo del Sistema Nervioso Central de naturaleza progresiva y debido a mecanismos hereditarios autosdmicos recesivos. Los mecanismos patogenicos parecen corresponder a un defecto congenita del metabolismo de los carbohidratos a pesar de que nunca nan sido probados. En un caso de enfermedad de Lafora compro-bado patologicamente, se ha estudiado el metabolismo del piru-vato, que ocupa un lugar central en el metabolismo de los carbohidratos, en diversos fluidos corporales bajo condiciones di-versas y tambten en material cerebral de biopsia sin que se encontraran anomalfas de las vias metaolicas. Estos hallazgos unidos a informes previos de la literature excluyen un defecto en la glicolisis por lo que un trastorno en el mecanismo de los carbohidratos, como mecanismo patogenico en la enfermedad de Lafora parece improbable. ZUSAMMENFASSUNG Die Lafora-Erkrankung ist eine autosomal rezessive und progressiv-degenerative Erkrankung des ZNS. Als pathogenetischer Mechanismus wird bislang unbewiesen eine angeborene Stoning des Kohlehydratstoffwechsels vermutet. Bei einem erwiesenen Fall einer Lafora-Erkrankung wurde der Pyruvat-Metabolismus, der eine zentrale Stelle im Kohlehydrat-Metabolismus einnimmt, sowohl in Korperflussigkeiten unter unterschiedlichen Bedin-gungen als auch im bioptischen Hirngewebe untersucht. Es wur-den keine Abnormalitaten dieses Stoffwechselweges gefunden. Dies last zusammen mit alteren Berichten der Literatur einen Glycolysedefekt ausschliesen. Somit scheint eine Stoning des Kohlehydrat-Stoflwechsels als pathogenetischer Mechanismus fur die Lafora-Erkrankung unwahrscheinlich

We studied CSF and serum samples from 16 patients with progressive myoclonus epilepsy (PME). These patients had juvenile-onset PME with evidence of autosomal recessive inheritance and no Lafora bodies. Twelve of the 16 patients with PME had immunologic abnormalities. Oligoclonal gamma bands were seen in six of the eight patients from whom sufficient CSF was available. The CSF albumin and serum/CSF albumin ratios were normal in all 16 patients, indicating the presence of intact blood-brain barriers. Six of the 16 patients showed increased CSF IgG levels and five had an increased CNS IgG synthesis. All patients had normal serum and CSF IgM and IgA levels. Three patients, all with bands, had reduced measles and/or vaccinia serum/CSF antibody ratios. The findings suggest altered immune response of the CNS of some patients with PME apparently caused by nonspecific immunostimulation.

SUMMARY 1 In the brains of two siblings suffering from progressive myoclonus epilepsy, cells were observed which suggested the presence of a storage process. A fragment of the brainstem of a four-day-old nephew of these patients also showed “storage cells”. The neuropathological aspects are briefly described and found to correspond with classical descriptions of progressive myoclonus epilepsy with Lafora bodies in the nervous system. 2 Complete anatomical investigation of the visceral organs was possible in only one of the siblings. The heart contained deposits which were strongly PAS-positive and remained so after treatment of the specimen with saliva. In the liver of this patient, two different types of PAS-positive products could be found: (a) products which remain PAS-positive after treatment of the specimens with saliva and (b) products resembling glycogen in being saliva- and acid formaldehyde-labile. The latter type of product was found in cells arranged in insular patterns. 3 The hexosamine content of the brains of the two siblings and of the heart of one of them was normal. The liver of the last named patient contained an excess of nonlipid hexosamine. 4 It is tentatively suggested that a pathological alteration of glycogen metabolism existed in one of the patients described. This hypothesis could not be verified in the other two patients from this family who came to autopsy, because the preservation of the post mortem material was not appropriate for histochemical or biochemical studies. RESUME 1 Des cellules temoignant de la presence d'une thesaurismose ont ete observees dans le cerveau de deux malades (frere et soeur) atteints d'epilepsie myoclonique et le meme type de cellules a ete retrouve dans le tronc cerebral d'un cousin de ces malades, âge de quatre jours. L'aspect neuro-pathologique est resume en bref. II correspond a la description classique de l'epilepsie myoclonique progressive avec corpuscules de Lafora dans le systeme nerveux. 2 Un examen anatomique complet des organes visceraux n'a ete possible que chez Tun des malades adultes. Le coeur contenait des depots fortement PAS-positifs, resistant au traitement de la coupe par de la salive. On a trouve dans le foie de ce malade deux types diffeArents de produits PAS-positifs: (a) des produits restant PAS-positifs apres le traitement des coupes par la salive et (b) des produits ressemblant au glycogene en ce sens qu'ils disparaissent apres le traitement par la salive et par le formaldehyde acide. Ce dernier type de produit est present dans des cellules rangees selon un type insulaire. 3 Le taux d'hexosamine du cerveau de chacun des deux malades adultes, ainsi que celui du coeur de l'un d'eux etaient normaux. Le taux d'hexosamine non-lipidique etait eleve dans le foie de ce malade. 4 L'auteur tend a suggerer, comme hypothese de travail, la presence d'un trouble du metabolisme de glycogene chez l'un des malades decrits. Cette hypothese n'a pu etre confirmee a l'autopsie chez les deux autres malades de cette famille parce que la conservation du materiel post-mortem ne permettait point des etudes histochimiques et biochimiques.

SUMMARY A sister and brother, children of consanguineous parents, were suffering from progressive myoclonus epilepsy. In the boy, the myoclonic jerks of the Unverricht type remained very discrete. In both cases there were numerous Lafora bodies in the brain; one patient in addition showed cardiac and hepatic changes in the form of accumulations of unidentified material. Patients with progressive myoclonus epilepsy who belong to the Lafora group, have similar clinical symptoms and similar microscopic abnormalities in the brain. The clinical picture is characterized by the onset of generalized seizures at age 10–17, myoclonus of the Unverricht type, early dementia, normal ocular fundi and a duration of illness not exceeding 10 years; EEG findings are to some extent characteristic, and remain practically constant during the course of the illness; the disease often is a familial condition; the parents are free of the affection, but often consanguineous. Numerous Lafora bodies in the substantia nigra, dentate nucleus, the entire cerebral cortex and the thalamus are considered by us to be pathognomonic for a particular subgroup of the clinical syndrome progressive myoclonus epilepsy; this probably also applies to the changes in the heart and liver. RESUME Un frere et une soeur, issus de parents consanguins, sont atteints d'une epilepsie myoclonique progressive dont les secousses du type Unverricht sont tres discretes chez le garcon. L'examen anatomique montre de nombreux corpuscules de Lafora dans les deux cerveaux et, chez l'un des malades seulement, des alterations cardiaques et hepatiques representees par l'accumulation d'une substance non identifyee. Ces deux observations confirment le fait que les malades souffrant d'une epilepsie myoclonique progressive du type Lafora presentent une semeiologie clinique et des anomalies microscopiques cerebrales semblables. L'aspect clinique est caracterise par l'apparition de crises generalisees entre 10 et 17 ans, par des myoclonies du type Unverricht, une demence relativement precoce et un fond d'oeil normal; l'evolution n'excede jamais dix ans. Les donnees de L'E.E.G. sont relativement caracteristiques et restent pratiquement constantes pendant le cours de la maladie. L'affection est souvent familiale, mais elle respecte les parents qui sont frequemment consanguins. L'existence de nombreux corpuscules de Lafora dans la substance noire, dans le noyau dentele, dans le thalamus et dans tout le cortex cerebral est, a notre avis, pathognomonique d'un subgroupe du syndrome clinique d'epilepsie myoclonique progressive; il en va probablement de meme en ce qui concerne les alterations cardiaques et hepatiques.

Three cases of olivopontocerebellar atrophy, one of which included a cerebellar biopsy, have been examined for ultrastructural changes. Degenerative changes were identified within the afferent cerebellar fibers and within the cerebellar neurons, especially the Purkinje cells. Although the majority of the degenerative alterations have been previously described in a variety of disease states, there were some findings seen in our material that have not been previously reported, including unusual striped bodies and Lafora body inclusions within Purkinje dendrites. The relationship between OPCA and other diseases is discussed and a possible slow-viral etiology is considered.

Skin biopsies of three patients with Lafora disease (clinically typical and proven by liver biopsy) showed PAS-positive inclusions in numerous peripheral cells of eccrine sweat ducts. By electronmicroscopy, the inclusions consisted of polyglucosan-type material; they were not bounded by a membrane. Skin biopsy should be a convenient method of diagnosing Lafora disease.

A 17-year-old patient had myoclonic epilepsy caused by Lafora's disease. Biopsy showed polysaccharide accumulations within membrane-bound spaces in skeletal muscle cells. Some of the accumulations were morphologically similar to Lafora bodies as they have been seen in the brain. The histochemical reactions of these membrane-bound spaces suggested that they were peroxisomes. Polysaccharide accumulations also were demonstrated in hepatic cells, where they probably were located in the endoplasmic reticulum. Lafora's disease can be diagnosed by histochemical and electron microscopic study of skeletal muscle.