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Berge A. Minassian - One of the best experts on this subject based on the ideXlab platform.
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Lafora Disease current biology and therapeutic approaches
Revue Neurologique, 2021Co-Authors: Sharmistha Mitra, Emrah Gumusgoz, Berge A. MinassianAbstract:Abstract The ubiquitin system impacts most cellular processes and is altered in numerous neurodegenerative Diseases. However, little is known about its role in neurodegenerative Diseases due to disturbances of glycogen metabolism such as Lafora Disease (LD). In LD, insufficiently branched and long-chained glycogen forms and precipitates into insoluble polyglucosan bodies (Lafora bodies), which drive neuroinflammation, neurodegeneration and epilepsy. LD is caused by mutations in the gene encoding the glycogen phosphatase laforin or the gene coding for the laforin interacting partner ubiquitin E3 ligase malin. The role of the malin-laforin complex in regulating glycogen structure remains with full of gaps. In this review we bring together the disparate body of data on these two proteins and propose a mechanistic hypothesis of the Disease in which malin-laforin's role to monitor and prevent over-elongation of glycogen branch chains, which drive glycogen molecules to precipitate and accumulate into Lafora bodies. We also review proposed connections between Lafora bodies and the ensuing neuroinflammation, neurodegeneration and intractable epilepsy. Finally, we review the exciting activities in developing therapies for Lafora Disease based on replacing the missing genes, slowing the enzyme – glycogen synthase – that over-elongates glycogen branches, and introducing enzymes that can digest Lafora bodies. Much more work is needed to fill the gaps in glycogen metabolism in which laforin and malin operate. However, knowledge appears already adequate to advance Disease course altering therapies for this catastrophic fatal Disease.
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targeting gys1 with aav sacas9 decreases pathogenic polyglucosan bodies and neuroinflammation in adult polyglucosan body and Lafora Disease mouse models
Neurotherapeutics, 2021Co-Authors: Emrah Gumusgoz, D R Guisso, S Kasiri, Felix Nitschke, Silvia Nitschke, Sharmistha Mitra, Brandy Verhalen, Matthew Dear, Berge A. MinassianAbstract:Many adult and most childhood neurological Diseases have a genetic basis. CRISPR/Cas9 biotechnology holds great promise in neurological therapy, pending the clearance of major delivery, efficiency, and specificity hurdles. We applied CRISPR/Cas9 genome editing in its simplest modality, namely inducing gene sequence disruption, to one adult and one pediatric Disease. Adult polyglucosan body Disease is a neurodegenerative Disease resembling amyotrophic lateral sclerosis. Lafora Disease is a severe late childhood onset progressive myoclonus epilepsy. The pathogenic insult in both is formation in the brain of glycogen with overlong branches, which precipitates and accumulates into polyglucosan bodies that drive neuroinflammation and neurodegeneration. We packaged Staphylococcus aureus Cas9 and a guide RNA targeting the glycogen synthase gene, Gys1, responsible for brain glycogen branch elongation in AAV9 virus, which we delivered by neonatal intracerebroventricular injection to one mouse model of adult polyglucosan body Disease and two mouse models of Lafora Disease. This resulted, in all three models, in editing of approximately 17% of Gys1 alleles and a similar extent of reduction of Gys1 mRNA across the brain. The latter led to approximately 50% reductions of GYS1 protein, abnormal glycogen accumulation, and polyglucosan bodies, as well as ameliorations of neuroinflammatory markers in all three models. Our work represents proof of principle for virally delivered CRISPR/Cas9 neurotherapeutics in an adult-onset (adult polyglucosan body) and a childhood-onset (Lafora) neurological Diseases.
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targeting gys1 with aav sacas9 decreases pathogenic polyglucosan bodies and neuroinflammation in adult polyglucosan body and Lafora Disease mouse models
bioRxiv, 2021Co-Authors: Emrah Gumusgoz, D R Guisso, S Kasiri, Felix Nitschke, Silvia Nitschke, Sharmistha Mitra, Brandy Verhalen, Matthew Dear, Berge A. MinassianAbstract:Summary Many adult and most childhood neurological Diseases have a genetic basis. CRISPR/Cas9 biotechnology holds great promise in neurological therapy, pending the clearance of major delivery, efficiency and specificity hurdles. We apply CRISPR/Cas9 genome editing in its simplest modality, namely inducing gene sequence disruption, to one adult and one pediatric Disease. Adult polyglucosan body Disease is a neurodegenerative Disease resembling amyotrophic lateral sclerosis. Lafora Disease is a severe late childhood onset progressive myoclonus epilepsy. The pathogenic insult in both is formation in the brain of glycogen with overlong branches, which precipitates and accumulates into polyglucosan bodies that drive neuroinflammation and neurodegeneration. We packaged Staphylococcus aureus Cas9 and a guide RNA targeting the glycogen synthase gene Gys1 responsible for brain glycogen branch elongation in AAV9 virus, which we delivered by neonatal intracerebroventricular injection to one mouse model of adult polyglucosan body Disease and two mouse models of Lafora Disease. This resulted, in all three models, in editing of approximately 17% of Gys1 alleles and a similar extent of reduction of Gys1 mRNA across the brain. The latter led to approximately 50% reductions of GYS1 protein, of abnormal glycogen accumulation and of polyglucosan bodies, as well as corrections of neuroinflammatory markers in all three models. Our work represents proof of principle for virally-delivered CRISPR/Cas9 neurotherapeutics in an adult-onset (adult polyglucosan body) and a childhood-onset (Lafora) neurological Diseases.
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gys1 antisense therapy rescues neuropathological bases of murine Lafora Disease
bioRxiv, 2021Co-Authors: S Ahonen, Peixiang Wang, S Nitschke, Tamar R Grossman, Holly B Kordasiewicz, X Zhao, D R Guisso, S Kasiri, Felix Nitschke, Berge A. MinassianAbstract: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 the Disease's mouse models. 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 analyzed 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.
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ketogenic diet reduces Lafora bodies in murine Lafora Disease
Neurology Genetics, 2020Co-Authors: Lori Israelian, Xiaochu Zhao, Peixiang Wang, Shoghig Gabrielian, Berge A. MinassianAbstract:Lafora Disease (LD) is a teenage-onset fatal progressive myoclonus epilepsy caused by loss-of-function mutations in the EPM2A gene encoding the glycogen phosphatase laforin or EPM2B encoding the laforin-interacting ubiquitin E3 ligase malin. Concerted actions of glycogen synthase (GS) and branching enzyme generate normal short-branched soluble glycogen. In LD, some glycogen molecules develop long branches, precipitate, and accumulate into pathognomonic and pathogenic Lafora bodies (LBs). The precise mechanism by which the laforin-malin complex mitigates this is unknown, but thought to involve GS downregulation. In fact, transgenic GS downregulation in LD mouse models reduces LB formation and rescues the Disease.1,2
Felix Nitschke - One of the best experts on this subject based on the ideXlab platform.
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ppp1r3d deficiency preferentially inhibits neuronal and cardiac Lafora body formation in a mouse model of the fatal epilepsy Lafora Disease
Journal of Neurochemistry, 2021Co-Authors: Lori Israelian, Xiaochu Zhao, Peixiang Wang, Felix Nitschke, Silvia Nitschke, Ami M Perri, Jennifer P Y Lee, Brandy VerhalenAbstract: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.
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targeting gys1 with aav sacas9 decreases pathogenic polyglucosan bodies and neuroinflammation in adult polyglucosan body and Lafora Disease mouse models
Neurotherapeutics, 2021Co-Authors: Emrah Gumusgoz, D R Guisso, S Kasiri, Felix Nitschke, Silvia Nitschke, Sharmistha Mitra, Brandy Verhalen, Matthew Dear, Berge A. MinassianAbstract:Many adult and most childhood neurological Diseases have a genetic basis. CRISPR/Cas9 biotechnology holds great promise in neurological therapy, pending the clearance of major delivery, efficiency, and specificity hurdles. We applied CRISPR/Cas9 genome editing in its simplest modality, namely inducing gene sequence disruption, to one adult and one pediatric Disease. Adult polyglucosan body Disease is a neurodegenerative Disease resembling amyotrophic lateral sclerosis. Lafora Disease is a severe late childhood onset progressive myoclonus epilepsy. The pathogenic insult in both is formation in the brain of glycogen with overlong branches, which precipitates and accumulates into polyglucosan bodies that drive neuroinflammation and neurodegeneration. We packaged Staphylococcus aureus Cas9 and a guide RNA targeting the glycogen synthase gene, Gys1, responsible for brain glycogen branch elongation in AAV9 virus, which we delivered by neonatal intracerebroventricular injection to one mouse model of adult polyglucosan body Disease and two mouse models of Lafora Disease. This resulted, in all three models, in editing of approximately 17% of Gys1 alleles and a similar extent of reduction of Gys1 mRNA across the brain. The latter led to approximately 50% reductions of GYS1 protein, abnormal glycogen accumulation, and polyglucosan bodies, as well as ameliorations of neuroinflammatory markers in all three models. Our work represents proof of principle for virally delivered CRISPR/Cas9 neurotherapeutics in an adult-onset (adult polyglucosan body) and a childhood-onset (Lafora) neurological Diseases.
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targeting gys1 with aav sacas9 decreases pathogenic polyglucosan bodies and neuroinflammation in adult polyglucosan body and Lafora Disease mouse models
bioRxiv, 2021Co-Authors: Emrah Gumusgoz, D R Guisso, S Kasiri, Felix Nitschke, Silvia Nitschke, Sharmistha Mitra, Brandy Verhalen, Matthew Dear, Berge A. MinassianAbstract:Summary Many adult and most childhood neurological Diseases have a genetic basis. CRISPR/Cas9 biotechnology holds great promise in neurological therapy, pending the clearance of major delivery, efficiency and specificity hurdles. We apply CRISPR/Cas9 genome editing in its simplest modality, namely inducing gene sequence disruption, to one adult and one pediatric Disease. Adult polyglucosan body Disease is a neurodegenerative Disease resembling amyotrophic lateral sclerosis. Lafora Disease is a severe late childhood onset progressive myoclonus epilepsy. The pathogenic insult in both is formation in the brain of glycogen with overlong branches, which precipitates and accumulates into polyglucosan bodies that drive neuroinflammation and neurodegeneration. We packaged Staphylococcus aureus Cas9 and a guide RNA targeting the glycogen synthase gene Gys1 responsible for brain glycogen branch elongation in AAV9 virus, which we delivered by neonatal intracerebroventricular injection to one mouse model of adult polyglucosan body Disease and two mouse models of Lafora Disease. This resulted, in all three models, in editing of approximately 17% of Gys1 alleles and a similar extent of reduction of Gys1 mRNA across the brain. The latter led to approximately 50% reductions of GYS1 protein, of abnormal glycogen accumulation and of polyglucosan bodies, as well as corrections of neuroinflammatory markers in all three models. Our work represents proof of principle for virally-delivered CRISPR/Cas9 neurotherapeutics in an adult-onset (adult polyglucosan body) and a childhood-onset (Lafora) neurological Diseases.
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gys1 antisense therapy rescues neuropathological bases of murine Lafora Disease
bioRxiv, 2021Co-Authors: S Ahonen, Peixiang Wang, S Nitschke, Tamar R Grossman, Holly B Kordasiewicz, X Zhao, D R Guisso, S Kasiri, Felix Nitschke, Berge A. MinassianAbstract: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 the Disease's mouse models. 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 analyzed 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.
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Lafora Disease from pathogenesis to treatment strategies
Nature Reviews Neurology, 2018Co-Authors: Felix Nitschke, Berge A. Minassian, Saija J Ahonen, Silvia Nitschke, Sharmistha MitraAbstract:Lafora Disease is a severe, autosomal recessive, progressive myoclonus epilepsy. The Disease usually manifests in previously healthy adolescents, and death commonly occurs within 10 years of symptom onset. Lafora Disease is caused by loss-of-function mutations in EPM2A or NHLRC1, which encode laforin and malin, respectively. The absence of either protein results in poorly branched, hyperphosphorylated glycogen, which precipitates, aggregates and accumulates into Lafora bodies. Evidence from Lafora Disease genetic mouse models indicates that these intracellular inclusions are a principal driver of neurodegeneration and neurological Disease. The integration of current knowledge on the function of laforin-malin as an interacting complex suggests that laforin recruits malin to parts of glycogen molecules where overly long glucose chains are forming, so as to counteract further chain extension. In the absence of either laforin or malin function, long glucose chains in specific glycogen molecules extrude water, form double helices and drive precipitation of those molecules, which over time accumulate into Lafora bodies. In this article, we review the genetic, clinical, pathological and molecular aspects of Lafora Disease. We also discuss traditional antiseizure treatments for this condition, as well as exciting therapeutic advances based on the downregulation of brain glycogen synthesis and Disease gene replacement.
Peter J. Roach - One of the best experts on this subject based on the ideXlab platform.
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targeting pathogenic Lafora bodies in Lafora Disease using an antibody enzyme fusion
Cell Metabolism, 2019Co-Authors: Kathryn M Brewer, Peter J. Roach, Anna A Depaoliroach, Grant L Austin, Dyann M Segvich, Annette Uittenbogaard, John J Mccarthy, Zoe R SimmonsAbstract:Summary Lafora Disease (LD) is a fatal childhood epilepsy caused by recessive mutations in either the EPM2A or EPM2B gene. A hallmark of LD is the intracellular accumulation of insoluble polysaccharide deposits known as Lafora bodies (LBs) in the brain and other tissues. In LD mouse models, genetic reduction of glycogen synthesis eliminates LB formation and rescues the neurological phenotype. Therefore, LBs have become a therapeutic target for ameliorating LD. Herein, we demonstrate that human pancreatic α-amylase degrades LBs. We fused this amylase to a cell-penetrating antibody fragment, and this antibody-enzyme fusion (VAL-0417) degrades LBs in vitro and dramatically reduces LB loads in vivo in Epm2a−/− mice. Using metabolomics and multivariate analysis, we demonstrate that VAL-0417 treatment of Epm2a−/− mice reverses the metabolic phenotype to a wild-type profile. VAL-0417 is a promising drug for the treatment of LD and a putative precision therapy platform for intractable epilepsy.
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targeting pathogenic Lafora bodies in Lafora Disease using an antibody enzyme fusion
bioRxiv, 2019Co-Authors: Kathryn M Brewer, Peter J. Roach, Anna A Depaoliroach, Grant L Austin, Dyann M Segvich, Annette Uittenbogaard, John J MccarthyAbstract:Abstract Lafora Disease (LD) is a fatal childhood epilepsy and a non-classical glycogen storage disorder with no effective therapy or cure. LD is caused by recessive mutations in the EPM2A or EPM2B genes that encode the glycogen phosphatase laforin and an E3 ubiquitin ligase malin, respectively. A hallmark of LD is the intracellular accumulation of abnormal and insoluble α-linked polysaccharide deposits known as Lafora bodies (LBs) in several tissues, including most regions of the brain. In mouse models of LD, genetic reduction of glycogen synthesis eliminates LB formation and rescues the neurological phenotype. Since multiple groups have confirmed that neurodegeneration and epilepsy result from LB accumulation, a major focus in the field has shifted toward the development of therapies that reduce glycogen synthesis or target LBs for degradation with the goal of treating LD. Herein, we identify the optimal enzymes for degrading LBs, and we develop a novel therapeutic agent by fusing human pancreatic α-amylase to a cellpenetrating antibody fragment. This antibody-enzyme fusion (VAL-0417) degrades LBs in vitro, shows robust cellular uptake, and significantly reduces the LB load in vivo in Epm2a-/- mice. VAL-0417 is a promising therapeutic for the treatment of LD and a putative precision therapy for an intractable epilepsy. Antibody-enzyme fusions represent a new class of antibody-based drugs that could be utilized to treat glycogen storage disorders and other Diseases. One Sentence Summary An antibody-enzyme fusion delivering an amylase degrades the toxic polyglucosan bodies that cause Lafora Disease, a fatal childhood epilepsy.
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Lafora Disease offers a unique window into neuronal glycogen metabolism
Journal of Biological Chemistry, 2018Co-Authors: Matthew S Gentry, Joan J Guinovart, Peter J. Roach, Jose M Serratosa, Berge A. MinassianAbstract:Lafora Disease (LD) is a fatal, autosomal recessive, glycogen-storage disorder that manifests as severe epilepsy. LD results from mutations in the gene encoding either the glycogen phosphatase laforin or the E3 ubiquitin ligase malin. Individuals with LD develop cytoplasmic, aberrant glycogen inclusions in nearly all tissues that more closely resemble plant starch than human glycogen. This Minireview discusses the unique window into glycogen metabolism that LD research offers. It also highlights recent discoveries, including that glycogen contains covalently bound phosphate and that neurons synthesize glycogen and express both glycogen synthase and glycogen phosphorylase.
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glycogen phosphorylation and Lafora Disease
PMC, 2015Co-Authors: Peter J. RoachAbstract:Covalent phosphorylation of glycogen, first described 35 years ago, was put on firm ground through the work of the Whelan laboratory in the 1990s. But glycogen phosphorylation lay fallow until interest was rekindled in the mid 2000s by the finding that it could be removed by a glycogen-binding phosphatase, laforin, and that mutations in laforin cause a fatal teenage-onset epilepsy, called Lafora Disease. Glycogen phosphorylation is due to phosphomonoesters at C2, C3 and C6 of glucose residues. Phosphate is rare, ranging from 1:500 to 1:5000 phosphates/glucose depending on the glycogen source. The mechanisms of glycogen phosphorylation remain under investigation but one hypothesis to explain C2 and perhaps C3 phosphate is that it results from a rare side reaction of the normal synthetic enzyme glycogen synthase. Lafora Disease is likely caused by over-accumulation of abnormal glycogen in insoluble deposits termed Lafora bodies in neurons. The abnormality in the glycogen correlates with elevated phosphorylation (at C2, C3 and C6), reduced branching, insolubility and an enhanced tendency to aggregate and become insoluble. Hyperphosphorylation of glycogen is emerging as an important feature of this deadly childhood Disease.
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glycogen phosphomonoester distribution in mouse models of the progressive myoclonic epilepsy Lafora Disease
Journal of Biological Chemistry, 2015Co-Authors: Anna A Depaoliroach, Christian Heiss, Christopher J Contreras, Mayumi Ishihara, Parastoo Azadi, Dyann M Segvich, Peter J. RoachAbstract:Glycogen is a branched polymer of glucose that acts as an energy reserve in many cell types. Glycogen contains trace amounts of covalent phosphate, in the range of 1 phosphate per 500-2000 glucose residues depending on the source. The function, if any, is unknown, but in at least one genetic Disease, the progressive myoclonic epilepsy Lafora Disease, excessive phosphorylation of glycogen has been implicated in the pathology by disturbing glycogen structure. Some 90% of Lafora cases are attributed to mutations of the EPM2A or EPM2B genes, and mice with either gene disrupted accumulate hyperphosphorylated glycogen. It is, therefore, of importance to understand the chemistry of glycogen phosphorylation. Rabbit skeletal muscle glycogen contained covalent phosphate as monoesters of C2, C3, and C6 carbons of glucose residues based on analyses of phospho-oligosaccharides by NMR. Furthermore, using a sensitive assay for glucose 6-P in hydrolysates of glycogen coupled with measurement of total phosphate, we determined the proportion of C6 phosphorylation in rabbit muscle glycogen to be ∼20%. C6 phosphorylation also accounted for ∼20% of the covalent phosphate in wild type mouse muscle glycogen. Glycogen phosphorylation in Epm2a(-/-) and Epm2b(-/-) mice was increased 8- and 4-fold compared with wild type mice, but the proportion of C6 phosphorylation remained unchanged at ∼20%. Therefore, our results suggest that C2, C3, and/or C6 phosphate could all contribute to abnormal glycogen structure or to Lafora Disease.
Peixiang Wang - One of the best experts on this subject based on the ideXlab platform.
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canine Lafora Disease an unstable repeat expansion disorder
Life, 2021Co-Authors: Thilo Von Klopmann, Saija Ahonen, Travis Wang, Irene Espadassantiuste, Kaspar Matiasek, Daniel Sanchezmasian, Stefan Rupp, Helene Vandenberghe, Jeremy Rose, Peixiang WangAbstract:Canine Lafora Disease is a recessively inherited, rapidly progressing neurodegenerative Disease caused by the accumulation of abnormally constructed insoluble glycogen Lafora bodies in the brain and other tissues due to the loss of NHL repeat containing E3 ubiquitin protein ligase 1 (NHLRC1). Dogs have a dodecamer repeat sequence within the NHLRC1 gene, which is prone to unstable (dynamic) expansion and loss of function. Progressive signs of Lafora Disease include hypnic jerks, reflex and spontaneous myoclonus, seizures, vision loss, ataxia and decreased cognitive function. We studied five dogs (one Chihuahua, two French Bulldogs, one Griffon Bruxellois, one mixed breed) with clinical signs associated with canine Lafora Disease. Identification of polyglucosan bodies (Lafora bodies) in myocytes supported diagnosis in the French Bulldogs; muscle areas close to the myotendinous junction and the myofascial union segment had the highest yield of inclusions. Postmortem examination of one of the French Bulldogs revealed brain Lafora bodies. Genetic testing for the known canine NHLRC1 mutation confirmed the presence of a homozygous mutation associated with canine Lafora Disease. Our results show that Lafora Disease extends beyond previous known breeds to the French Bulldog, Griffon Bruxellois and even mixed-breed dogs, emphasizing the likely species-wide nature of this genetic problem. It also establishes these breeds as animal models for the devastating human Disease. Genetic testing should be used when designing breeding strategies to determine the frequency of the NHLRC1 mutation in affected breeds. Lafora Diseases should be suspected in any older dog presenting with myoclonus, hypnic jerks or photoconvulsions.
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ppp1r3d deficiency preferentially inhibits neuronal and cardiac Lafora body formation in a mouse model of the fatal epilepsy Lafora Disease
Journal of Neurochemistry, 2021Co-Authors: Lori Israelian, Xiaochu Zhao, Peixiang Wang, Felix Nitschke, Silvia Nitschke, Ami M Perri, Jennifer P Y Lee, Brandy VerhalenAbstract: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.
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gys1 antisense therapy rescues neuropathological bases of murine Lafora Disease
bioRxiv, 2021Co-Authors: S Ahonen, Peixiang Wang, S Nitschke, Tamar R Grossman, Holly B Kordasiewicz, X Zhao, D R Guisso, S Kasiri, Felix Nitschke, Berge A. MinassianAbstract: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 the Disease's mouse models. 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 analyzed 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.
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ketogenic diet reduces Lafora bodies in murine Lafora Disease
Neurology Genetics, 2020Co-Authors: Lori Israelian, Xiaochu Zhao, Peixiang Wang, Shoghig Gabrielian, Berge A. MinassianAbstract:Lafora Disease (LD) is a teenage-onset fatal progressive myoclonus epilepsy caused by loss-of-function mutations in the EPM2A gene encoding the glycogen phosphatase laforin or EPM2B encoding the laforin-interacting ubiquitin E3 ligase malin. Concerted actions of glycogen synthase (GS) and branching enzyme generate normal short-branched soluble glycogen. In LD, some glycogen molecules develop long branches, precipitate, and accumulate into pathognomonic and pathogenic Lafora bodies (LBs). The precise mechanism by which the laforin-malin complex mitigates this is unknown, but thought to involve GS downregulation. In fact, transgenic GS downregulation in LD mouse models reduces LB formation and rescues the Disease.1,2
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Nationwide genetic testing towards eliminating Lafora Disease from Miniature Wirehaired Dachshunds in the United Kingdom
Canine Genetics and Epidemiology, 2018Co-Authors: Saija Ahonen, Peixiang Wang, Ian Seath, Clare Rusbridge, Susan Holt, Gill Key, Travis Wang, Berge A. MinassianAbstract:Background Canine DNA-testing has become an important tool in purebred dog breeding and many breeders use genetic testing results when planning their breeding strategies. In addition, information obtained from testing of hundreds dogs in one breed gives valuable information about the breed-wide genotype frequency of Disease associated allele. Lafora Disease is a late onset, recessively inherited genetic Disease which is diagnosed in Miniature Wirehaired Dachshunds (MWHD). It is one of the most severe forms of canine epilepsy leading to neurodegeneration and, frequently euthanasia within a few years of diagnosis. Canine Lafora Disease is caused by a dodecamer repeat expansion mutation in the NHLRC1 gene and a DNA test is available to identify homozygous dogs at risk, carriers and dogs free of the mutation. Results Blood samples were collected from 733 MWHDs worldwide, mostly of UK origin, for canine Lafora Disease testing. Among the tested MWHD population 7.0% were homozygous for the mutation and at risk for Lafora Disease. In addition, 234 dogs were heterozygous, indicating a carrier frequency of 31.9% in the tested population. Among the tested MWHDs, the mutant allele frequency was 0.2. In addition, data from the tested dogs over 6 years (2012–2017) indicated that the frequency of the homozygous and carrier dogs has decreased from 10.4% to 2.7% and 41.5% to 25.7%, respectively among MWHDs tested. As a consequence, the frequency of dogs free of the mutation has increased from 48.1% to 71.6%. Conclusions This study provides valuable data for the MWHD community and shows that the DNA test is a useful tool for the breeders to prevent occurrence of Lafora Disease in MWHDs. DNA testing has, over 6 years, helped to decrease the frequency of carriers and dogs at risk. Additionally, the DNA test can continue to be used to slowly eradicate the Disease-causing mutation in the breed. However, this should be done carefully, over time, to avoid further compromising the genetic diversity of the breed. The DNA test also provides a diagnostic tool for veterinarians if they are presented with a dog that shows clinical signs associated with canine Lafora Disease.
Silvia Nitschke - One of the best experts on this subject based on the ideXlab platform.
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ppp1r3d deficiency preferentially inhibits neuronal and cardiac Lafora body formation in a mouse model of the fatal epilepsy Lafora Disease
Journal of Neurochemistry, 2021Co-Authors: Lori Israelian, Xiaochu Zhao, Peixiang Wang, Felix Nitschke, Silvia Nitschke, Ami M Perri, Jennifer P Y Lee, Brandy VerhalenAbstract: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.
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targeting gys1 with aav sacas9 decreases pathogenic polyglucosan bodies and neuroinflammation in adult polyglucosan body and Lafora Disease mouse models
Neurotherapeutics, 2021Co-Authors: Emrah Gumusgoz, D R Guisso, S Kasiri, Felix Nitschke, Silvia Nitschke, Sharmistha Mitra, Brandy Verhalen, Matthew Dear, Berge A. MinassianAbstract:Many adult and most childhood neurological Diseases have a genetic basis. CRISPR/Cas9 biotechnology holds great promise in neurological therapy, pending the clearance of major delivery, efficiency, and specificity hurdles. We applied CRISPR/Cas9 genome editing in its simplest modality, namely inducing gene sequence disruption, to one adult and one pediatric Disease. Adult polyglucosan body Disease is a neurodegenerative Disease resembling amyotrophic lateral sclerosis. Lafora Disease is a severe late childhood onset progressive myoclonus epilepsy. The pathogenic insult in both is formation in the brain of glycogen with overlong branches, which precipitates and accumulates into polyglucosan bodies that drive neuroinflammation and neurodegeneration. We packaged Staphylococcus aureus Cas9 and a guide RNA targeting the glycogen synthase gene, Gys1, responsible for brain glycogen branch elongation in AAV9 virus, which we delivered by neonatal intracerebroventricular injection to one mouse model of adult polyglucosan body Disease and two mouse models of Lafora Disease. This resulted, in all three models, in editing of approximately 17% of Gys1 alleles and a similar extent of reduction of Gys1 mRNA across the brain. The latter led to approximately 50% reductions of GYS1 protein, abnormal glycogen accumulation, and polyglucosan bodies, as well as ameliorations of neuroinflammatory markers in all three models. Our work represents proof of principle for virally delivered CRISPR/Cas9 neurotherapeutics in an adult-onset (adult polyglucosan body) and a childhood-onset (Lafora) neurological Diseases.
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targeting gys1 with aav sacas9 decreases pathogenic polyglucosan bodies and neuroinflammation in adult polyglucosan body and Lafora Disease mouse models
bioRxiv, 2021Co-Authors: Emrah Gumusgoz, D R Guisso, S Kasiri, Felix Nitschke, Silvia Nitschke, Sharmistha Mitra, Brandy Verhalen, Matthew Dear, Berge A. MinassianAbstract:Summary Many adult and most childhood neurological Diseases have a genetic basis. CRISPR/Cas9 biotechnology holds great promise in neurological therapy, pending the clearance of major delivery, efficiency and specificity hurdles. We apply CRISPR/Cas9 genome editing in its simplest modality, namely inducing gene sequence disruption, to one adult and one pediatric Disease. Adult polyglucosan body Disease is a neurodegenerative Disease resembling amyotrophic lateral sclerosis. Lafora Disease is a severe late childhood onset progressive myoclonus epilepsy. The pathogenic insult in both is formation in the brain of glycogen with overlong branches, which precipitates and accumulates into polyglucosan bodies that drive neuroinflammation and neurodegeneration. We packaged Staphylococcus aureus Cas9 and a guide RNA targeting the glycogen synthase gene Gys1 responsible for brain glycogen branch elongation in AAV9 virus, which we delivered by neonatal intracerebroventricular injection to one mouse model of adult polyglucosan body Disease and two mouse models of Lafora Disease. This resulted, in all three models, in editing of approximately 17% of Gys1 alleles and a similar extent of reduction of Gys1 mRNA across the brain. The latter led to approximately 50% reductions of GYS1 protein, of abnormal glycogen accumulation and of polyglucosan bodies, as well as corrections of neuroinflammatory markers in all three models. Our work represents proof of principle for virally-delivered CRISPR/Cas9 neurotherapeutics in an adult-onset (adult polyglucosan body) and a childhood-onset (Lafora) neurological Diseases.
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Lafora Disease from pathogenesis to treatment strategies
Nature Reviews Neurology, 2018Co-Authors: Felix Nitschke, Berge A. Minassian, Saija J Ahonen, Silvia Nitschke, Sharmistha MitraAbstract:Lafora Disease is a severe, autosomal recessive, progressive myoclonus epilepsy. The Disease usually manifests in previously healthy adolescents, and death commonly occurs within 10 years of symptom onset. Lafora Disease is caused by loss-of-function mutations in EPM2A or NHLRC1, which encode laforin and malin, respectively. The absence of either protein results in poorly branched, hyperphosphorylated glycogen, which precipitates, aggregates and accumulates into Lafora bodies. Evidence from Lafora Disease genetic mouse models indicates that these intracellular inclusions are a principal driver of neurodegeneration and neurological Disease. The integration of current knowledge on the function of laforin-malin as an interacting complex suggests that laforin recruits malin to parts of glycogen molecules where overly long glucose chains are forming, so as to counteract further chain extension. In the absence of either laforin or malin function, long glucose chains in specific glycogen molecules extrude water, form double helices and drive precipitation of those molecules, which over time accumulate into Lafora bodies. In this article, we review the genetic, clinical, pathological and molecular aspects of Lafora Disease. We also discuss traditional antiseizure treatments for this condition, as well as exciting therapeutic advances based on the downregulation of brain glycogen synthesis and Disease gene replacement.
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pathogenesis of Lafora Disease transition of soluble glycogen to insoluble polyglucosan
International Journal of Molecular Sciences, 2017Co-Authors: Mitchell A Sullivan, Berge A. Minassian, Silvia Nitschke, Martin Steup, Felix NitschkeAbstract:Lafora Disease (LD, OMIM #254780) is a rare, recessively inherited neurodegenerative Disease with adolescent onset, resulting in progressive myoclonus epilepsy which is fatal usually within ten years of symptom onset. The Disease is caused by loss-of-function mutations in either of the two genes EPM2A (laforin) or EPM2B (malin). It characteristically involves the accumulation of insoluble glycogen-derived particles, named Lafora bodies (LBs), which are considered neurotoxic and causative of the Disease. The pathogenesis of LD is therefore centred on the question of how insoluble LBs emerge from soluble glycogen. Recent data clearly show that an abnormal glycogen chain length distribution, but neither hyperphosphorylation nor impairment of general autophagy, strictly correlates with glycogen accumulation and the presence of LBs. This review summarizes results obtained with patients, mouse models, and cell lines and consolidates apparent paradoxes in the LD literature. Based on the growing body of evidence, it proposes that LD is predominantly caused by an impairment in chain-length regulation affecting only a small proportion of the cellular glycogen. A better grasp of LD pathogenesis will further develop our understanding of glycogen metabolism and structure. It will also facilitate the development of clinical interventions that appropriately target the underlying cause of LD.
