Canine Lafora Disease: An Unstable Repeat Expansion Disorder

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.

Astrocytic glycogen accumulation drives the pathophysiology of neurodegeneration in Lafora disease

The hallmark of Lafora disease, a fatal neurodegenerative disorder, is the accumulation of intracellular glycogen aggregates, called Lafora bodies. Until recently, it was widely believed that brain Lafora bodies were present exclusively in neurons and thus that Lafora disease pathology derived from their accumulation in this cell population. However, recent evidence indicates that Lafora bodies are also present in astrocytes. To define the role of astrocytic Lafora bodies in Lafora disease pathology, we deleted glycogen synthase specifically from astrocytes in a mouse model of the disease (malinKO). Strikingly, blocking glycogen synthesis in astrocytes—thus impeding Lafora bodies accumulation in this cell type—prevented the increase in neurodegeneration markers, autophagy impairment, and metabolic changes characteristic of the malinKO model. Conversely, mice that overaccumulate glycogen in astrocytes showed an increase in these markers. These results unveil the deleterious consequences of the deregulation of glycogen metabolism in astrocytes and change the perspective that Lafora disease is caused solely by alterations in neurons.

Gys1 antisense therapy rescues neuropathological bases of murine Lafora disease

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.

Generation and characterization of a laforin nanobody inhibitor

Mutations in the gene encoding the glycogen phosphatase laforin result in the fatal childhood epilepsy Lafora disease (LD). A cellular hallmark of LD is cytoplasmic, hyper-phosphorylated, glycogen-like aggregates called Lafora bodies (LBs) that form in nearly all tissues and drive disease progression. Additional tools are needed to define the cellular function of laforin, understand the pathological role of laforin in LD, and determine the role of glycogen phosphate in glycogen metabolism. We present the generation and characterization of laforin nanobodies. We identify multiple classes of specific laforin-binding nanobodies and determine their binding epitopes using hydrogen deuterium exchange (HDX) mass spectrometry. Further, one family of nanobodies is identified that serves as an inhibitor of laforin catalytic activity. The laforin nanobodies are an important set of tools that open new avenues to define unresolved questions.

The possibility of using skin biopsy in the diagnosis of Lafora disease

Lafora disease is a hereditary, autosomal recessive progressive myoclonus epilepsy caused by mutations in the EPM2A (laforin) and EPM2B (malin) genes, with no substantial genotype-phenotype differences between the two. Clinical manifestations of the disease are determined by the accumulation of specific cytoplasmic “amyloid inclusions” consisting of polyglycosans (an abnormally branched glycogen molecule). Polyglycosans, or Lafora bodies, are typically found in the brain, hepatocytes of the liver, skeletal and cardiac muscles, in the ducts of sweat glands, and in the skin. The diagnosis is made following visual, generalized tonic-clonic and myoclonic seizures, progressing dementia, cerebellar ataxia, detection of specific Lafora bodies during sweat gland biopsy and data of genetic testing.The article describes a clinical case of Lafora disease in a patient with disease onset at 11 years old caused by the mutation in the EPM2A (laforine) gene with focal sensory visual seizures with subsequent generalized tonic-clonic seizures, progressive motor impairments in the form of ataxia and gait abnormality as well as behavioral and cognitive disorders. The presented clinical case demonstrates the need for additional research, such as biopsy and genetic testing, for diagnosing diseases proceeding with resistant epileptic seizures and progressive motor and cognitive impairments.

Targeting pathogenic Lafora bodies in Lafora disease using an antibody-enzyme fusion

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 theEPM2AorEPM2Bgenes 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 LBsin vitro, shows robust cellular uptake, and significantly reduces the LB loadin vivoinEpm2a-/- 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 SummaryAn antibody-enzyme fusion delivering an amylase degrades the toxic polyglucosan bodies that cause Lafora disease, a fatal childhood epilepsy.

Central Nervous System Delivery and Biodistribution Analysis of an Antibody-Enzyme Fusion for the Treatment of Lafora Disease

Lafora disease is a fatal juvenile epilepsy, characterized by the malignant accumulation of aberrant glucan inclusions called Lafora Bodies (LBs). Cerebral delivery of protein-based therapeutics for the clearance of Lafora Bodies remain a unique challenge in the field. Recently, a humanized antigen-binding fragment (hFab) derived from a murine systemic lupus erythematosus DNA autoantibody (3E10) has been shown to mediate cell penetration and been proposed as a broadly applicable carrier to mediate cellular targeting and uptake. We report studies on cerebral delivery of VAL-0417, an antibody-enzyme fusion composed of the 3E10 hFab and human pancreatic α-amylase for the clearance of LBs in a mouse model of lafora disease. Herein, we report development of an enzyme-linked immunosorbant-based bioassay to detect VAL-0417 post treatment as a measure of delivery efficacy. We demonstrate the robust and sensitive detection of the fusion protein in multiple tissue types. Using our method, we measured biodistribution in different methods of delivery. We found intracerebroventricular administration provided the most robust delivery, while intrathecal administration only showed modest biodistribution. These data define critical steps in the translational pipeline of VAL-0417for the treatment of Lafora disease.

Lafora Disease: A Review of Molecular Mechanisms and Pathology

Lafora's disease is a neurodegenerative disorder caused by recessive loss-of-function mutations in the EPM2A (laforin glycogen phosphatase) or EPM2B (malin E3 ubiquitin ligase) genes. Neuropathology is characterized by malformed precipitated glycogen aggregates termed Lafora bodies. Asymptomatic until adolescence, patients undergo first insidious then rapid progressive myoclonus epilepsy toward a vegetative state and death within a decade. Laforin and malin interact to regulate glycogen phosphorylation and chain length pattern, the latter critical to glycogen's solubility. Significant gaps remain in precise mechanistic understanding. However, demonstration that partial reduction in brain glycogen synthesis near-completely prevents the disease in its genetic animal models opens a direct present path to therapy.