Lafora disease is a fatal form of progressive myoclonic epilepsy caused by mutations in the EPM2A or NHLRC1/EPM2B genes that usually appears during adolescence. The Epm2a-/- and Epm2b-/- knock-out mouse models of the disease develop behavioral and neurological alterations similar to those observed in patients. The aim of this work is to analyze whether early treatment with metformin (from conception to adulthood) ameliorates the formation of Lafora bodies and improves the behavioral and neurological outcomes observed with late treatment (during 2 months at 10 months of age). We also evaluated the benefits of metformin in patients with Lafora disease. To assess neurological improvements due to metformin administration in the two mouse models, we evaluated the effects on pentylenetetrazol sensitivity, posturing, motor coordination and activity, and memory. We also analyzed the effects on Lafora bodies, neurodegeneration, and astrogliosis. Furthermore, we conducted a follow-up study of an initial cohort of 18 patients with Lafora disease, 8 treated with metformin and 10 untreated. Our results indicate that early metformin was more effective than late metformin in Lafora disease mouse models improving neurological alterations of both models such as neuronal hyperexcitability, motor and memory alterations, neurodegeneration, and astrogliosis and decreasing the formation of Lafora bodies. Moreover, patients receiving metformin had a slower progression of the disease. Overall, early treatment improves the outcome seen with late metformin treatment in the two knock-out mouse models of Lafora disease. Metformin-treated patients exhibited an ameliorated course of the disease with slower deterioration of their daily living activities., This work was supported by grants from the Spanish Ministry of Economy (Rti2018-095784b-100SAF) to J.M.S. and M.P.S., from the Tatiana Perez de Guzman el Bueno Foundation (5258/002) to M.P.S. and J.M.S., from the CIBERER (ACCI 2020, 23—U744) to M.P.S., and a grant from the National Institute of Neurological Disorders and Stroke of the National Institutes of Health (P01NS097197), which established the Lafora Epilepsy Cure Initiative (LECI), to J.M.S. and M.P.S., Peer reviewed

Lafora disease is a rare and fatal form of progressive myoclonic epilepsy typically occurring early in adolescence. The disease results from mutations in the EPM2A gene, encoding laforin, or the EPM2B gene, encoding malin. Laforin and malin work together in a complex to control glycogen synthesis and prevent the toxicity produced by misfolded proteins via the ubiquitin-proteasome system. Disruptions in either protein cause alterations in this complex, leading to the formation of Lafora bodies containing abnormal, insoluble, and hyperphosphorylated forms of glycogen. We used the Epm2a-/- knockout mouse model of Lafora disease to apply gene therapy by administering intracerebroventricular injections of a recombinant adeno-associated virus carrying the human EPM2A gene. We evaluated the effects of this treatment through neuropathological studies, behavioral tests, video-electroencephalography, electrophysiological recordings, and proteomic/phosphoproteomic analysis. Gene therapy ameliorated neurological and histopathological alterations, reduced epileptic activity and neuronal hyperexcitability, and decreased the formation of Lafora bodies. Moreover, differential quantitative proteomics and phosphoproteomics revealed beneficial changes in various molecular pathways altered in Lafora disease. Our results represent proof of principle for gene therapy with the coding region of the human EPM2A gene as a treatment for EPM2A-related Lafora disease., This work was supported by grants from the Spanish Ministry of Economy (Rti2018-095784b-100SAF MCI/AEI/FEDER, UE) to J.M.S. and M.P.S.; from the Tatiana Perez de Guzman el Bueno Foundation to M.P.S. and J.M.S.; from the Centro de Investigación Biomédica en Red de Enfermedades Raras (CIBERER) (ACCI 2020, 23 - U744) to M.P.S.; from the Fondazione Malattie Rare Mauro Baschirotto BIRD Onlus to M.P.S., C.C., M.S., and L.Z.P.; and a grant from the National Institute of Neurological Disorders and Stroke of the National Institutes of Health (P01NS097197), which established the Lafora Epilepsy Cure Initiative (LECI), to J.M.S. and M.P.S. We thank Pascual Sanz Bigorra (Institute of Biomedicine of Valencia [IBV]) and Manuel Soto Catalán (Instituto de Investigación Sanitaria-Fundación Jiménez Díaz) for their generous gift of GS, BiP, and cleaved caspase-3 antibodies, and to Miguel Chillón Rodríguez (Viral Vector Production Unit or UPV-UAB-VHIR) for the generation of rAAVs and technical advice. We also thank Juan Antonio López del Olmo (CNIC Proteomics Unit), Ariadna Martín Blázquez (Instituto de Investigación Sanitaria-Fundación Jiménez Díaz), and the Animal Facility of Instituto de Investigación Sanitaria-Fundación Jiménez Díaz for their technical assistance., Peer reviewed

Lafora disease is a rare and fatal form of progressive myoclonic epilepsy typically occurring early in adolescence. The disease results from mutations in the EPM2A gene, encoding laforin, or the EPM2B gene, encoding malin. Laforin and malin work together in a complex to control glycogen synthesis and prevent the toxicity produced by misfolded proteins via the ubiquitin-proteasome system. Disruptions in either protein cause alterations in this complex, leading to the formation of Lafora bodies containing abnormal, insoluble, and hyperphosphorylated forms of glycogen. We used the Epm2a-/- knockout mouse model of Lafora disease to apply gene therapy by administering intracerebroventricular injections of a recombinant adeno-associated virus carrying the human EPM2A gene. We evaluated the effects of this treatment through neuropathological studies, behavioral tests, video-electroencephalography, electrophysiological recordings, and proteomic/phosphoproteomic analysis. Gene therapy ameliorated neurological and histopathological alterations, reduced epileptic activity and neuronal hyperexcitability, and decreased the formation of Lafora bodies. Moreover, differential quantitative proteomics and phosphoproteomics revealed beneficial changes in various molecular pathways altered in Lafora disease. Our results represent proof of principle for gene therapy with the coding region of the human EPM2A gene as a treatment for EPM2A-related Lafora disease., This work was supported by grants from the Spanish Ministry of Economy (Rti2018-095784b-100SAF MCI/AEI/FEDER, UE) to J.M.S. and M.P.S.; from the Tatiana Perez de Guzman el Bueno Foundation to M.P.S. and J.M.S.; from the Centro de Investigación Biomédica en Red de Enfermedades Raras (CIBERER) (ACCI 2020, 23 - U744) to M.P.S.; from the Fondazione Malattie Rare Mauro Baschirotto BIRD Onlus to M.P.S., C.C., M.S., and L.Z.P.; and a grant from the National Institute of Neurological Disorders and Stroke of the National Institutes of Health (P01NS097197), which established the Lafora Epilepsy Cure Initiative (LECI), to J.M.S. and M.P.S. We thank Pascual Sanz Bigorra (Institute of Biomedicine of Valencia [IBV]) and Manuel Soto Catalán (Instituto de Investigación Sanitaria-Fundación Jiménez Díaz) for their generous gift of GS, BiP, and cleaved caspase-3 antibodies, and to Miguel Chillón Rodríguez (Viral Vector Production Unit or UPV-UAB-VHIR) for the generation of rAAVs and technical advice. We also thank Juan Antonio López del Olmo (CNIC Proteomics Unit), Ariadna Martín Blázquez (Instituto de Investigación Sanitaria-Fundación Jiménez Díaz), and the Animal Facility of Instituto de Investigación Sanitaria-Fundación Jiménez Díaz for their technical assistance., Peer reviewed

[EN] Lafora disease is a fatal neurodegenerative disorder caused by loss-of-function mutations in the EPM2A or EPM2B genes, which encode laforin and malin, respectively. These mutations lead to the accumulation of intracellular inclusions of abnormal glycogen, known as Lafora bodies, the hallmark of the disease. Symptoms typically begin in early adolescence with seizures and rapidly progress to cognitive and motor decline, ultimately resulting in dementia and death within a decade of onset. Disruption of Epm2a or Epm2b in mice causes neuronal degeneration and Lafora body accumulation in the brain and other tissues. Epm2a-/- and Epm2b-/- mice exhibit motor and memory impairments, epileptic activity, and molecular and histological abnormalities. We previously demonstrated that intracerebroventricular delivery of a recombinant adeno-associated virus carrying EPM2A significantly improved pathology in Epm2a-/- mice. In this study, we tested recombinant adeno-associated virus-mediated delivery of the human EPM2B gene in Epm2b-/- mice. The treatment partially improved neurological, molecular, and histopathological outcomes, although some pathological features persisted. Importantly, our findings reveal differences between EPM2A- and EPM2B-based gene therapies, highlighting the need to better understand their distinct mechanisms. Despite limitations, our study provides new insights into the complexity of targeting EPM2B mutations in Lafora disease., This research was funded by grants from the Spanish Ministry of Economy [Rti2018-095784b-100SAF MCI/AEI/FEDER, UE] to J.M.S. and M.P.S.; from the Tatiana Perez de Guzman el Bueno Foundation to M.P.S. and J.M.S.; from the Fondazione Malattie Rare Mauro Baschirotto BIRD Onlus to M.P.S. and L.Z.P; from the AEVEL Foundation to J.M.S. and L.Z.P.; from the Centro de Investigacion Biomedica en Red de Enfermedades Raras (CIBERER) [ACCI 2020, 23-U744 and-742] to J.M.S., P.S. and M.P.S.; and from the National Institute of Neurological Disorders and Stroke of the National Institutes of Health [P01NS097197], which established the Lafora Epilepsy Cure Initiative (LECI), to J.M.S, P.S. and M.P.S.