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1.
Brain ; 145(7): 2361-2377, 2022 07 29.
Artigo em Inglês | MEDLINE | ID: mdl-35084461

RESUMO

Longer glucan chains tend to precipitate. Glycogen, by far the largest mammalian glucan and the largest molecule in the cytosol with up to 55 000 glucoses, does not, due to a highly regularly branched spherical structure that allows it to be perfused with cytosol. Aberrant construction of glycogen leads it to precipitate, accumulate into polyglucosan bodies that resemble plant starch amylopectin and cause disease. This pathology, amylopectinosis, is caused by mutations in a series of single genes whose functions are under active study toward understanding the mechanisms of proper glycogen construction. Concurrently, we are characterizing the physicochemical particularities of glycogen and polyglucosans associated with each gene. These genes include GBE1, EPM2A and EPM2B, which respectively encode the glycogen branching enzyme, the glycogen phosphatase laforin and the laforin-interacting E3 ubiquitin ligase malin, for which an unequivocal function is not yet known. Mutations in GBE1 cause a motor neuron disease (adult polyglucosan body disease), and mutations in EPM2A or EPM2B a fatal progressive myoclonus epilepsy (Lafora disease). RBCK1 deficiency causes an amylopectinosis with fatal skeletal and cardiac myopathy (polyglucosan body myopathy 1, OMIM# 615895). RBCK1 is a component of the linear ubiquitin chain assembly complex, with unique functions including generating linear ubiquitin chains and ubiquitinating hydroxyl (versus canonical amine) residues, including of glycogen. In a mouse model we now show (i) that the amylopectinosis of RBCK1 deficiency, like in adult polyglucosan body disease and Lafora disease, affects the brain; (ii) that RBCK1 deficiency glycogen, like in adult polyglucosan body disease and Lafora disease, has overlong branches; (iii) that unlike adult polyglucosan body disease but like Lafora disease, RBCK1 deficiency glycogen is hyperphosphorylated; and finally (iv) that unlike laforin-deficient Lafora disease but like malin-deficient Lafora disease, RBCK1 deficiency's glycogen hyperphosphorylation is limited to precipitated polyglucosans. In summary, the fundamental glycogen pathology of RBCK1 deficiency recapitulates that of malin-deficient Lafora disease. Additionally, we uncover sex and genetic background effects in RBCK1 deficiency on organ- and brain-region specific amylopectinoses, and in the brain on consequent neuroinflammation and behavioural deficits. Finally, we exploit the portion of the basic glycogen pathology that is common to adult polyglucosan body disease, both forms of Lafora disease and RBCK1 deficiency, namely overlong branches, to show that a unified approach based on downregulating glycogen synthase, the enzyme that elongates glycogen branches, can rescue all four diseases.


Assuntos
Doença de Depósito de Glicogênio Tipo IV , Doença de Lafora , Ubiquitina-Proteína Ligases , Animais , Regulação para Baixo , Glucanos/metabolismo , Glicogênio/metabolismo , Doença de Depósito de Glicogênio , Glicogênio Sintase/genética , Glicogênio Sintase/metabolismo , Doença de Lafora/genética , Doença de Lafora/patologia , Camundongos , Epilepsias Mioclônicas Progressivas , Doenças do Sistema Nervoso , Proteínas Tirosina Fosfatases não Receptoras/genética , Ubiquitina/genética , Ubiquitina-Proteína Ligases/genética , Ubiquitina-Proteína Ligases/metabolismo
2.
J Biol Chem ; 296: 100150, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33277363

RESUMO

Malstructured glycogen accumulates over time in Lafora disease (LD) and precipitates into Lafora bodies (LBs), leading to neurodegeneration and intractable fatal epilepsy. Constitutive reduction of glycogen synthase-1 (GYS1) activity prevents murine LD, but the effect of GYS1 reduction later in disease course is unknown. Our goal was to knock out Gys1 in laforin (Epm2a)-deficient LD mice after disease onset to determine whether LD can be halted in midcourse, or even reversed. We generated Epm2a-deficient LD mice with tamoxifen-inducible Cre-mediated Gys1 knockout. Tamoxifen was administered at 4 months and disease progression assessed at 12 months. We verified successful knockout at mRNA and protein levels using droplet digital PCR and Western blots. Glycogen determination and periodic acid-Schiff-diastase staining were used to analyze glycogen and LB accumulation. Immunohistochemistry using astrocytic (glial fibrillary acidic protein) and microglial (ionized calcium-binding adapter molecule 1) markers was performed to investigate neuroinflammation. In the disease-relevant organ, the brain, Gys1 mRNA levels were reduced by 85% and GYS1 protein depleted. Glycogen accumulation was halted at the 4-month level, while LB formation and neuroinflammation were significantly, though incompletely, prevented. Skeletal muscle analysis confirmed that Gys1 knockout inhibits glycogen and LB accumulation. However, tamoxifen-independent Cre recombination precluded determination of disease halting or reversal in this tissue. Our study shows that Gys1 knockdown is a powerful means to prevent LD progression, but this approach did not reduce brain glycogen or LBs to levels below those at the time of intervention. These data suggest that endogenous mechanisms to clear brain LBs are absent or, possibly, compromised in laforin-deficient murine LD.


Assuntos
Gliose/prevenção & controle , Glicogênio Sintase/fisiologia , Inflamação/prevenção & controle , Doença de Lafora/patologia , Músculo Esquelético/metabolismo , Proteínas Tirosina Fosfatases não Receptoras/deficiência , Animais , Feminino , Gliose/metabolismo , Gliose/patologia , Inflamação/metabolismo , Inflamação/patologia , Doença de Lafora/tratamento farmacológico , Doença de Lafora/genética , Doença de Lafora/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Músculo Esquelético/patologia , Moduladores Seletivos de Receptor Estrogênico/administração & dosagem , Tamoxifeno/administração & dosagem
3.
Brain ; 144(10): 2985-2993, 2021 11 29.
Artigo em Inglês | MEDLINE | ID: mdl-33993268

RESUMO

Lafora disease is a fatal progressive myoclonus epilepsy. At root, it is due to constant acquisition of branches that are too long in a subgroup of glycogen molecules, leading them to precipitate and accumulate into Lafora bodies, which drive a neuroinflammatory response and neurodegeneration. As a potential therapy, we aimed to downregulate glycogen synthase, the enzyme responsible for glycogen branch elongation, in mouse models of the disease. We synthesized an antisense oligonucleotide (Gys1-ASO) that targets the mRNA of the brain-expressed glycogen synthase 1 gene (Gys1). We administered Gys1-ASO by intracerebroventricular injection and analysed the pathological hallmarks of Lafora disease, namely glycogen accumulation, Lafora body formation, and neuroinflammation. Gys1-ASO prevented Lafora body formation in young mice that had not yet formed them. In older mice that already exhibited Lafora bodies, Gys1-ASO inhibited further accumulation, markedly preventing large Lafora bodies characteristic of advanced disease. Inhibition of Lafora body formation was associated with prevention of astrogliosis and strong trends towards correction of dysregulated expression of disease immune and neuroinflammatory markers. Lafora disease manifests gradually in previously healthy teenagers. Our work provides proof of principle that an antisense oligonucleotide targeting the GYS1 mRNA could prevent, and halt progression of, this catastrophic epilepsy.


Assuntos
Glicogênio Sintase/administração & dosagem , Doença de Lafora/tratamento farmacológico , Doença de Lafora/patologia , Oligorribonucleotídeos Antissenso/administração & dosagem , Animais , Feminino , Injeções Intraventriculares , Doença de Lafora/genética , Masculino , Camundongos , Camundongos Knockout , RNA Mensageiro/antagonistas & inibidores , RNA Mensageiro/genética
4.
J Neurochem ; 157(6): 1897-1910, 2021 06.
Artigo em Inglês | MEDLINE | ID: mdl-32892347

RESUMO

Mammalian glycogen chain lengths are subject to complex regulation, including by seven proteins (protein phosphatase-1 regulatory subunit 3, PPP1R3A through PPP1R3G) that target protein phosphatase-1 (PP1) to glycogen to activate the glycogen chain-elongating enzyme glycogen synthase and inactivate the chain-shortening glycogen phosphorylase. Lafora disease is a fatal neurodegenerative epilepsy caused by aggregates of long-chained, and as a result insoluble, glycogen, termed Lafora bodies (LBs). We previously eliminated PPP1R3C from a Lafora disease mouse model and studied the effect on LB formation. In the present work, we eliminate and study the effect of absent PPP1R3D. In the interim, brain cell type levels of all PPP1R3 genes have been published, and brain cell type localization of LBs clarified. Integrating these data we find that PPP1R3C is the major isoform in most tissues including brain. In the brain, PPP1R3C is expressed at 15-fold higher levels than PPP1R3D in astrocytes, the cell type where most LBs form. PPP1R3C deficiency eliminates ~90% of brain LBs. PPP1R3D is quantitatively a minor isoform, but possesses unique MAPK, CaMK2 and 14-3-3 binding domains and appears to have an important functional niche in murine neurons and cardiomyocytes. In neurons, it is expressed equally to PPP1R3C, and its deficiency eliminates ~50% of neuronal LBs. In heart, it is expressed at 25% of PPP1R3C where its deficiency eliminates ~90% of LBs. This work studies the role of a second (PPP1R3D) of seven PP1 subunits that regulate the structure of glycogen, toward better understanding of brain glycogen metabolism generally, and in Lafora disease.


Assuntos
Modelos Animais de Doenças , Doença de Lafora/metabolismo , Miocárdio/metabolismo , Neurônios/metabolismo , Proteína Fosfatase 1/deficiência , Animais , Encéfalo/metabolismo , Encéfalo/patologia , Feminino , Glicogênio/metabolismo , Humanos , Doença de Lafora/genética , Doença de Lafora/patologia , Masculino , Camundongos , Camundongos da Linhagem 129 , Camundongos Endogâmicos C57BL , Camundongos Knockout , Miocárdio/patologia , Neurônios/patologia , Proteína Fosfatase 1/genética
5.
Ann Neurol ; 75(3): 442-6, 2014 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-24419970

RESUMO

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.


Assuntos
Peptídeos e Proteínas de Sinalização Intracelular/uso terapêutico , Doença de Lafora/enzimologia , Doença de Lafora/terapia , Ubiquitina-Proteína Ligases/deficiência , Animais , Encéfalo/metabolismo , Encéfalo/patologia , Condicionamento Psicológico , Regulação para Baixo , Medo/psicologia , Glicogênio/metabolismo , Glicogênio Sintase/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/genética , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Doença de Lafora/psicologia , Camundongos , Camundongos Knockout , Mioclonia/enzimologia , Mioclonia/genética , Mioclonia/terapia , Fármacos Neuroprotetores/metabolismo , Placa Amiloide , Convulsões/enzimologia , Convulsões/genética , Convulsões/terapia
6.
J Biol Chem ; 288(48): 34627-37, 2013 Nov 29.
Artigo em Inglês | MEDLINE | ID: mdl-24142699

RESUMO

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.


Assuntos
Fosfatases de Especificidade Dupla/genética , Glicogênio Sintase/metabolismo , Glicogênio/biossíntese , Resistência à Insulina/genética , Obesidade/patologia , Envelhecimento/genética , Animais , Fosfatases de Especificidade Dupla/metabolismo , Glucose-6-Fosfato/metabolismo , Glicogênio/genética , Glicogênio Sintase/genética , Humanos , Insulina/genética , Insulina/metabolismo , Fígado/enzimologia , Fígado/metabolismo , Fígado/patologia , Camundongos , Obesidade/etiologia , Obesidade/genética , Fosforilação , Proteínas Tirosina Fosfatases não Receptoras
7.
Ann Neurol ; 74(2): 297-300, 2013 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-23913475

RESUMO

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.


Assuntos
Glicogênio/antagonistas & inibidores , Glicogênio/biossíntese , Doença de Lafora/prevenção & controle , Animais , Modelos Animais de Doenças , Fosfatases de Especificidade Dupla/genética , Técnicas de Inativação de Genes , Glicogênio Sintase/genética , Glicogênio Sintase/metabolismo , Doença de Lafora/patologia , Doença de Lafora/fisiopatologia , Camundongos , Camundongos da Linhagem 129 , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteínas Tirosina Fosfatases não Receptoras
8.
PLoS Genet ; 7(4): e1002037, 2011 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-21552327

RESUMO

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.


Assuntos
Glucanos/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Doença de Lafora/fisiopatologia , Animais , Encéfalo/fisiopatologia , Modelos Animais de Doenças , Glucanos/análise , Glicogênio Sintase/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/genética , Doença de Lafora/genética , Camundongos , Camundongos Knockout
9.
PLoS Genet ; 7(7): e1002194, 2011 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-21829378

RESUMO

One quadrillion synapses are laid in the first two years of postnatal construction of the human brain, which are then pruned until age 10 to 500 trillion synapses composing the final network. Genetic epilepsies are the most common neurological diseases with onset during pruning, affecting 0.5% of 2-10-year-old children, and these epilepsies are often characterized by spontaneous remission. We previously described a remitting epilepsy in the Lagotto romagnolo canine breed. Here, we identify the gene defect and affected neurochemical pathway. We reconstructed a large Lagotto pedigree of around 34 affected animals. Using genome-wide association in 11 discordant sib-pairs from this pedigree, we mapped the disease locus to a 1.7 Mb region of homozygosity in chromosome 3 where we identified a protein-truncating mutation in the Lgi2 gene, a homologue of the human epilepsy gene LGI1. We show that LGI2, like LGI1, is neuronally secreted and acts on metalloproteinase-lacking members of the ADAM family of neuronal receptors, which function in synapse remodeling, and that LGI2 truncation, like LGI1 truncations, prevents secretion and ADAM interaction. The resulting epilepsy onsets at around seven weeks (equivalent to human two years), and remits by four months (human eight years), versus onset after age eight in the majority of human patients with LGI1 mutations. Finally, we show that Lgi2 is expressed highly in the immediate post-natal period until halfway through pruning, unlike Lgi1, which is expressed in the latter part of pruning and beyond. LGI2 acts at least in part through the same ADAM receptors as LGI1, but earlier, ensuring electrical stability (absence of epilepsy) during pruning years, preceding this same function performed by LGI1 in later years. LGI2 should be considered a candidate gene for common remitting childhood epilepsies, and LGI2-to-LGI1 transition for mechanisms of childhood epilepsy remission.


Assuntos
Epilepsias Parciais/genética , Mutação/genética , Proteínas do Tecido Nervoso/genética , Proteínas ADAM/metabolismo , Animais , Encéfalo/metabolismo , Células COS , Chlorocebus aethiops , Cães , Epilepsias Parciais/metabolismo , Feminino , Regulação da Expressão Gênica no Desenvolvimento , Estudo de Associação Genômica Ampla , Células HEK293 , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Proteínas do Tecido Nervoso/metabolismo , Neurônios/metabolismo , Polimorfismo de Nucleotídeo Único/genética , Ligação Proteica/fisiologia , Ratos
10.
EBioMedicine ; 101: 104993, 2024 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-38324982

RESUMO

BACKGROUND: Macrophages are innate immune cells whose phagocytosis function is critical to the prognosis of stroke and peritonitis. cis-aconitic decarboxylase immune-responsive gene 1 (Irg1) and its metabolic product itaconate inhibit bacterial infection, intracellular viral replication, and inflammation in macrophages. Here we explore whether itaconate regulates phagocytosis. METHODS: Phagocytosis of macrophages was investigated by time-lapse video recording, flow cytometry, and immunofluorescence staining in macrophage/microglia cultures isolated from mouse tissue. Unbiased RNA-sequencing and ChIP-sequencing assays were used to explore the underlying mechanisms. The effects of Irg1/itaconate axis on the prognosis of intracerebral hemorrhagic stroke (ICH) and peritonitis was observed in transgenic (Irg1flox/flox; Cx3cr1creERT/+, cKO) mice or control mice in vivo. FINDINGS: In a mouse model of ICH, depletion of Irg1 in macrophage/microglia decreased its phagocytosis of erythrocytes, thereby exacerbating outcomes (n = 10 animals/group, p < 0.05). Administration of sodium itaconate/4-octyl itaconate (4-OI) promoted macrophage phagocytosis (n = 7 animals/group, p < 0.05). In addition, in a mouse model of peritonitis, Irg1 deficiency in macrophages also inhibited phagocytosis of Staphylococcus aureus (n = 5 animals/group, p < 0.05) and aggravated outcomes (n = 9 animals/group, p < 0.05). Mechanistically, 4-OI alkylated cysteine 155 on the Kelch-like ECH-associated protein 1 (Keap1), consequent in nuclear translocation of nuclear factor erythroid 2-related factor 2 (Nrf2) and transcriptional activation of Cd36 gene. Blocking the function of CD36 completely abolished the phagocytosis-promoting effects of Irg1/itaconate axis in vitro and in vivo. INTERPRETATION: Our findings provide a potential therapeutic target for phagocytosis-deficiency disorders, supporting further development towards clinical application for the benefit of stroke and peritonitis patients. FUNDING: The National Natural Science Foundation of China (32070735, 82371321 to Q. Li, 82271240 to F. Yang) and the Beijing Natural Science Foundation Program and Scientific Research Key Program of Beijing Municipal Commission of Education (KZ202010025033 to Q. Li).


Assuntos
Acidente Vascular Cerebral Hemorrágico , Peritonite , Succinatos , Humanos , Camundongos , Animais , Proteína 1 Associada a ECH Semelhante a Kelch , Acidente Vascular Cerebral Hemorrágico/metabolismo , Fator 2 Relacionado a NF-E2/genética , Fator 2 Relacionado a NF-E2/metabolismo , Macrófagos/metabolismo , Peritonite/tratamento farmacológico , Fagocitose , Prognóstico , Hidroliases/genética , Hidroliases/metabolismo , Hidroliases/farmacologia
11.
Cell Rep ; 43(1): 113653, 2024 01 23.
Artigo em Inglês | MEDLINE | ID: mdl-38175758

RESUMO

Omicron, as the emerging variant with enhanced vaccine tolerance, has sharply disrupted most therapeutic antibodies. Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) belongs to the subgenus Sarbecovirus, members of which share high sequence similarity. Herein, we report one sarbecovirus antibody, 5817, which has broad-spectrum neutralization capacity against SARS-CoV-2 variants of concern (VOCs) and SARS-CoV, as well as related bat and pangolin viruses. 5817 can hardly compete with six classes of receptor-binding-domain-targeted antibodies grouped by structural classifications. No obvious impairment in the potency is detected against SARS-CoV-2 Omicron and subvariants. The cryoelectron microscopy (cryo-EM) structure of neutralizing antibody 5817 in complex with Omicron spike reveals a highly conserved epitope, only existing at the receptor-binding domain (RBD) open state. Prophylactic and therapeutic administration of 5817 potently protects mice from SARS-CoV-2 Beta, Delta, Omicron, and SARS-CoV infection. This study reveals a highly conserved cryptic epitope targeted by a broad sarbecovirus neutralizing antibody, which would be beneficial to meet the potential threat of pre-emergent SARS-CoV-2 VOCs.


Assuntos
Coronavírus Relacionado à Síndrome Respiratória Aguda Grave , Animais , Camundongos , Anticorpos Amplamente Neutralizantes , Microscopia Crioeletrônica , Anticorpos Neutralizantes , Epitopos , Anticorpos Antivirais
12.
J Biol Chem ; 287(30): 25650-9, 2012 Jul 20.
Artigo em Inglês | MEDLINE | ID: mdl-22669944

RESUMO

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.


Assuntos
Fosfatases de Especificidade Dupla/metabolismo , Glicogênio/metabolismo , Doença de Lafora/enzimologia , Proteólise , Ubiquitina-Proteína Ligases/metabolismo , Adolescente , Animais , Células Cultivadas , Fosfatases de Especificidade Dupla/genética , Feminino , Glicogênio/genética , Humanos , Doença de Lafora/genética , Doença de Lafora/patologia , Masculino , Camundongos , Camundongos Knockout , Fosforilação/genética , Complexo de Endopeptidases do Proteassoma/genética , Complexo de Endopeptidases do Proteassoma/metabolismo , Ligação Proteica/genética , Proteínas Tirosina Fosfatases não Receptoras , Ubiquitina-Proteína Ligases/genética
13.
Brain ; 135(Pt 9): 2684-98, 2012 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-22961547

RESUMO

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.


Assuntos
Encéfalo/patologia , Proteínas de Transporte/genética , Doença de Lafora/genética , Músculo Esquelético/patologia , Proteínas Nucleares/genética , Adolescente , Adulto , Idade de Início , Atrofia , Criança , Pré-Escolar , Cromossomos Humanos Par 4 , Proteínas de Ligação a DNA , Progressão da Doença , Feminino , Histona Metiltransferases , Humanos , Doença de Lafora/patologia , Escore Lod , Masculino , Mutação , Pele/patologia
14.
Nat Genet ; 35(2): 125-7, 2003 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-12958597

RESUMO

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.


Assuntos
Proteínas de Transporte/genética , Mutação , Epilepsias Mioclônicas Progressivas/genética , Proteínas Tirosina Fosfatases/genética , Sequência de Bases , Estudos de Coortes , Feminino , Homozigoto , Humanos , Doença de Lafora/genética , Masculino , Dados de Sequência Molecular , Epilepsias Mioclônicas Progressivas/enzimologia , Linhagem , Proteínas Tirosina Fosfatases não Receptoras , Deleção de Sequência , Ubiquitina-Proteína Ligases
15.
Genes (Basel) ; 14(4)2023 03 31.
Artigo em Inglês | MEDLINE | ID: mdl-37107612

RESUMO

Lafora disease (LD) is a progressive neurologic disorder caused by biallelic pathogenic variants in EPM2A or EPM2B, leading to tissue accumulation of polyglucosan aggregates termed Lafora bodies (LBs). This study aimed to characterize the retinal phenotype in Epm2a-/- mice by examining knockout (KO; Epm2a-/-) and control (WT) littermates at two time points (10 and 14 months, respectively). In vivo exams included electroretinogram (ERG) testing, optical coherence tomography (OCT) and retinal photography. Ex vivo retinal testing included Periodic acid Schiff Diastase (PASD) staining, followed by imaging to assess and quantify LB deposition. There was no significant difference in any dark-adapted or light-adapted ERG parameters between KO and WT mice. The total retinal thickness was comparable between the groups and the retinal appearance was normal in both groups. On PASD staining, LBs were observed in KO mice within the inner and outer plexiform layers and in the inner nuclear layer. The average number of LBs within the inner plexiform layer in KO mice were 1743 ± 533 and 2615 ± 915 per mm2, at 10 and 14 months, respectively. This is the first study to characterize the retinal phenotype in an Epm2a-/- mouse model, demonstrating significant LB deposition in the bipolar cell nuclear layer and its synapses. This finding may be used to monitor the efficacy of experimental treatments in mouse models.


Assuntos
Doença de Lafora , Epilepsias Mioclônicas Progressivas , Camundongos , Animais , Doença de Lafora/genética , Doença de Lafora/patologia , Modelos Animais de Doenças , Retina/patologia , Epilepsias Mioclônicas Progressivas/patologia , Eletrorretinografia
16.
Ann Neurol ; 68(6): 925-33, 2010 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-21077101

RESUMO

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.


Assuntos
Glicogênio/metabolismo , Hiperfosfatemia/etiologia , Corpos de Inclusão/metabolismo , Doença de Lafora/complicações , Doença de Lafora/patologia , Músculo Esquelético/patologia , Animais , Encéfalo/metabolismo , Córtex Cerebelar/patologia , Córtex Cerebelar/ultraestrutura , Modelos Animais de Doenças , Fosfatases de Especificidade Dupla/metabolismo , Regulação da Expressão Gênica/genética , Glicogênio Sintase/genética , Glicogênio Sintase/metabolismo , Doença de Lafora/genética , Doença de Lafora/metabolismo , Camundongos , Camundongos Knockout , Músculo Esquelético/ultraestrutura , Fosfatos/metabolismo , Proteínas Tirosina Fosfatases não Receptoras/deficiência
17.
Neurol Asia ; 26(2): 427-433, 2021 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-34733372

RESUMO

Lafora body disease (MIM-254780), a glycogen storage disease, characterized by Lafora bodies (deformed glycogen molecules) accumulating in multiple organs, is a rare form of myoclonic epilepsy. It manifests in early adolescent years, initially with seizures and myoclonus, followed by dementia and progressive cognitive decline, ultimately culminating in death within 10 years. In Pakistan so far 5 cases have been reported. Here, we report a new case of Lafora body disease belonging to a consanguineous family from Pakistan. Histopathological analysis confirmed presence of lafora bodies in the patient`s skin. Sanger sequencing revealed novel homozygous 5bp deletion mutation (NM_005670.4; c.359_363delGTGTG) in exon 2 of the EPM2A gene, which was truly segregated in the family. These results will increase our understanding regarding the aetiology of this disorder and will further add to the mutation spectrum of EPM2A gene.

18.
Proc Natl Acad Sci U S A ; 104(49): 19262-6, 2007 Dec 04.
Artigo em Inglês | MEDLINE | ID: mdl-18040046

RESUMO

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.


Assuntos
Fosfatases de Especificidade Dupla/deficiência , Glicogênio/metabolismo , Doença de Lafora/enzimologia , Animais , Modelos Animais de Doenças , Fosfatases de Especificidade Dupla/genética , Glicogênio Sintase/análise , Glicogênio Sintase/metabolismo , Masculino , Camundongos , Camundongos Knockout , Mutação , Fosforilação , Proteínas Tirosina Fosfatases não Receptoras , Coelhos , Proteínas Recombinantes/farmacologia
19.
Sci Adv ; 6(50)2020 12.
Artigo em Inglês | MEDLINE | ID: mdl-33310843

RESUMO

Enzyme replacement therapy, in which a functional copy of an enzyme is injected either systemically or directly into the brain of affected individuals, has proven to be an effective strategy for treating certain lysosomal storage diseases. The inefficient uptake of recombinant enzymes via the mannose-6-phosphate receptor, however, prohibits the broad utility of replacement therapy. Here, to improve the efficiency and efficacy of lysosomal enzyme uptake, we exploited the strategy used by diphtheria toxin to enter into the endolysosomal network of cells by creating a chimera between the receptor-binding fragment of diphtheria toxin and the lysosomal hydrolase TPP1. We show that chimeric TPP1 binds with high affinity to target cells and is efficiently delivered into lysosomes. Further, we show superior uptake of chimeric TPP1 over TPP1 alone in brain tissue following intracerebroventricular injection in mice lacking TPP1, demonstrating the potential of this strategy for enhancing lysosomal storage disease therapy.


Assuntos
Toxina Diftérica , Terapia de Reposição de Enzimas , Animais , Encéfalo/metabolismo , Toxina Diftérica/metabolismo , Toxina Diftérica/farmacologia , Lisossomos/metabolismo , Camundongos , Receptor IGF Tipo 2/genética , Receptor IGF Tipo 2/metabolismo , Proteínas Recombinantes/metabolismo
20.
Ann Clin Transl Neurol ; 7(11): 2186-2198, 2020 11.
Artigo em Inglês | MEDLINE | ID: mdl-33034425

RESUMO

OBJECTIVE: Adult polyglucosan body disease (APBD) is an adult-onset neurological variant of glycogen storage disease type IV. APBD is caused by recessive mutations in the glycogen branching enzyme gene, and the consequent accumulation of poorly branched glycogen aggregates called polyglucosan bodies in the nervous system. There are presently no treatments for APBD. Here, we test whether downregulation of glycogen synthesis is therapeutic in a mouse model of the disease. METHODS: We characterized the effects of knocking out two pro-glycogenic proteins in an APBD mouse model. APBD mice were crossed with mice deficient in glycogen synthase (GYS1), or mice deficient in protein phosphatase 1 regulatory subunit 3C (PPP1R3C), a protein involved in the activation of GYS1. Phenotypic and histological parameters were analyzed and glycogen was quantified. RESULTS: APBD mice deficient in GYS1 or PPP1R3C demonstrated improvements in life span, morphology, and behavioral assays of neuromuscular function. Histological analysis revealed a reduction in polyglucosan body accumulation and of astro- and micro-gliosis in the brains of GYS1- and PPP1R3C-deficient APBD mice. Brain glycogen quantification confirmed the reduction in abnormal glycogen accumulation. Analysis of skeletal muscle, heart, and liver found that GYS1 deficiency reduced polyglucosan body accumulation in all three tissues and PPP1R3C knockout reduced skeletal muscle polyglucosan bodies. INTERPRETATION: GYS1 and PPP1R3C are effective therapeutic targets in the APBD mouse model. These findings represent a critical step toward the development of a treatment for APBD and potentially other glycogen storage disease type IV patients.


Assuntos
Doença de Depósito de Glicogênio/metabolismo , Glicogênio Sintase/deficiência , Peptídeos e Proteínas de Sinalização Intracelular/deficiência , Doenças do Sistema Nervoso/metabolismo , Animais , Comportamento Animal/fisiologia , Modelos Animais de Doenças , Doença de Depósito de Glicogênio/fisiopatologia , Doença de Depósito de Glicogênio/terapia , Camundongos , Camundongos Knockout , Doenças do Sistema Nervoso/fisiopatologia , Doenças do Sistema Nervoso/terapia
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