Gene therapy for Lafora disease in the Epm2a-/- mouse model.

Lafora disease is a rare and fatal form of progressive myoclonic epilepsy typically occurring early in adolescence. The disease results from mutations in the EPM2A gene, encoding laforin, or the EPM2B gene, encoding malin. Laforin and malin work together in a complex to control glycogen synthesis and prevent the toxicity produced by misfolded proteins via the ubiquitin-proteasome system. Disruptions in either protein cause alterations in this complex, leading to the formation of Lafora bodies containing abnormal, insoluble, and hyperphosphorylated forms of glycogen. We used the Epm2a-/- knockout mouse model of Lafora disease to apply gene therapy by administering intracerebroventricular injections of a recombinant adeno-associated virus carrying the human EPM2A gene. We evaluated the effects of this treatment through neuropathological studies, behavioral tests, video-electroencephalography, electrophysiological recordings, and proteomic/phosphoproteomic analysis. Gene therapy ameliorated neurological and histopathological alterations, reduced epileptic activity and neuronal hyperexcitability, and decreased the formation of Lafora bodies. Moreover, differential quantitative proteomics and phosphoproteomics revealed beneficial changes in various molecular pathways altered in Lafora disease. Our results represent proof of principle for gene therapy with the coding region of the human EPM2A gene as a treatment for EPM2A-related Lafora disease.

Impaired malin expression and interaction with partner proteins in Lafora disease.

Lafora disease (LD) is an autosomal recessive myoclonus epilepsy with onset in the teenage years leading to death within a decade of onset. LD is characterized by the overaccumulation of hyperphosphorylated, poorly branched, insoluble, glycogen-like polymers called Lafora bodies. The disease is caused by mutations in either EPM2A, encoding laforin, a dual specificity phosphatase that dephosphorylates glycogen, or EMP2B, encoding malin, an E3-ubiquitin ligase. While glycogen is a widely accepted laforin substrate, substrates for malin have been difficult to identify partly due to the lack of malin antibodies able to detect malin in vivo. Here we describe a mouse model in which the malin gene is modified at the C-terminus to contain the c-myc tag sequence, making an expression of malin-myc readily detectable. Mass spectrometry analyses of immunoprecipitates using c-myc tag antibodies demonstrate that malin interacts with laforin and several glycogen-metabolizing enzymes. To investigate the role of laforin in these interactions we analyzed two additional mouse models: malin-myc/laforin knockout and malin-myc/LaforinCS, where laforin was either absent or the catalytic Cys was genomically mutated to Ser, respectively. The interaction of malin with partner proteins requires laforin but is not dependent on its catalytic activity or the presence of glycogen. Overall, the results demonstrate that laforin and malin form a complex in vivo, which stabilizes malin and enhances interaction with partner proteins to facilitate normal glycogen metabolism. They also provide insights into the development of LD and the rescue of the disease by the catalytically inactive phosphatase.

Induction of Translational Readthrough on Protein Tyrosine Phosphatases Targeted by Premature Termination Codon Mutations in Human Disease.

Nonsense mutations generating premature termination codons (PTCs) in various genes are frequently associated with somatic cancer and hereditary human diseases since PTCs commonly generate truncated proteins with defective or altered function. Induced translational readthrough during protein biosynthesis facilitates the incorporation of an amino acid at the position of a PTC, allowing the synthesis of a complete protein. This may evade the pathological effect of the PTC mutation and provide new therapeutic opportunities. Several protein tyrosine phosphatases (PTPs) genes are targeted by PTC in human disease, the tumor suppressor PTEN being the more prominent paradigm. Here, using PTEN and laforin as examples, two PTPs from the dual-specificity phosphatase subfamily, we describe methodologies to analyze in silico the distribution and frequency of pathogenic PTC in PTP genes. We also summarize laboratory protocols and technical notes to study the induced translational readthrough reconstitution of the synthesis of PTP targeted by PTC in association with disease in cellular models.

Lafora Disease: A Case Report and Evolving Treatment Advancements.

Lafora disease is a rare genetic disorder characterized by a disruption in glycogen metabolism. It manifests as progressive myoclonus epilepsy and cognitive decline during adolescence. Pathognomonic is the presence of abnormal glycogen aggregates that, over time, produce large inclusions (Lafora bodies) in various tissues. This study aims to describe the clinical and histopathological aspects of a novel Lafora disease patient, and to provide an update on the therapeutical advancements for this disorder. A 20-year-old Libyan boy presented with generalized tonic-clonic seizures, sporadic muscular jerks, eyelid spasms, and mental impairment. Electroencephalography showed multiple discharges across both brain hemispheres. Brain magnetic resonance imaging was unremarkable. Muscle biopsy showed increased lipid content and a very mild increase of intermyofibrillar glycogen, without the polyglucosan accumulation typically observed in Lafora bodies. Despite undergoing three lines of antiepileptic treatment, the patient's condition showed minimal to no improvement. We identified the homozygous variant c.137G>A, p.(Cys46Tyr), in the EPM2B/NHLRC1 gene, confirming the diagnosis of Lafora disease. To our knowledge, the presence of lipid aggregates without Lafora bodies is atypical. Lafora disease should be considered during the differential diagnosis of progressive, myoclonic, and refractory epilepsies in both children and young adults, especially when accompanied by cognitive decline. Although there are no effective therapies yet, the development of promising new strategies prompts the need for an early and accurate diagnosis.

TgLaforin, a glucan phosphatase, reveals the dynamic role of storage polysaccharides in Toxoplasma gondii tachyzoites and bradyzoites.

The asexual stages of Toxoplasma gondii are defined by the rapidly growing tachyzoite during the acute infection and by the slow growing bradyzoite housed within tissue cysts during the chronic infection. These stages represent unique physiological states, each with distinct glucans reflecting differing metabolic needs. A defining feature of T. gondii bradyzoites is the presence of insoluble storage glucans known as amylopectin granules (AGs) that are believed to play a role in reactivation, but their functions during the chronic infection remain largely unexplored. More recently, the presence of storage glucans has been recognized in tachyzoites where their precise function and architecture have yet to be fully defined. Importantly, the T. gondii genome encodes activities needed for glucan turnover: a glucan phosphatase (TgLaforin; TGME49_205290) and a glucan kinase (TgGWD; TGME49_214260) that catalyze a cycle of reversible glucan phosphorylation required for glucan degradation by amylases. The expression of these enzymes in tachyzoites supports the existence of a storage glucan, evidence that is corroborated by specific labeling with the anti-glycogen antibody IV58B6. Disruption of reversible glucan phosphorylation via a CRISPR/Cas9 knockout (KO) of TgLaforin revealed no growth defects under nutrient-replete conditions in tachyzoites. However, the growth of TgLaforin-KO tachyzoites was severely stunted when starved of glutamine, even under glucose replete conditions. The loss of TgLaforin also resulted in the attenuation of acute virulence in mice accompanied by a lower cyst burden. Defective cyst formation due to profound changes in AG morphology was also observed in TgLaforin-KO parasites, both in vitro and in vivo. Together, these data demonstrate the importance of glucan turnover across the T. gondii asexual cycle. These findings, alongside our previously identified class of small molecules that inhibit TgLaforin, implicate reversible glucan phosphorylation as a legitimate target for the development of new drugs against chronic T. gondii infections.

Epm2aR240X knock-in mice present earlier cognitive decline and more epileptic activity than Epm2a-/- mice.

Lafora disease is a rare recessive form of progressive myoclonic epilepsy, usually diagnosed during adolescence. Patients present with myoclonus, neurological deterioration, and generalized tonic-clonic, myoclonic, or absence seizures. Symptoms worsen until death, usually within the first ten years of clinical onset. The primary histopathological hallmark is the formation of aberrant polyglucosan aggregates called Lafora bodies in the brain and other tissues. Lafora disease is caused by mutations in either the EPM2A gene, encoding laforin, or the EPM2B gene, coding for malin. The most frequent EPM2A mutation is R241X, which is also the most prevalent in Spain. The Epm2a-/- and Epm2b-/- mouse models of Lafora disease show neuropathological and behavioral abnormalities similar to those seen in patients, although with a milder phenotype. To obtain a more accurate animal model, we generated the Epm2aR240X knock-in mouse line with the R240X mutation in the Epm2a gene, using genetic engineering based on CRISPR-Cas9 technology. Epm2aR240X mice exhibit most of the alterations reported in patients, including the presence of LBs, neurodegeneration, neuroinflammation, interictal spikes, neuronal hyperexcitability, and cognitive decline, despite the absence of motor impairments. The Epm2aR240X knock-in mouse displays some symptoms that are more severe that those observed in the Epm2a-/- knock-out, including earlier and more pronounced memory loss, increased levels of neuroinflammation, more interictal spikes and increased neuronal hyperexcitability, symptoms that more precisely resemble those observed in patients. This new mouse model can therefore be specifically used to evaluate how new therapies affects these features with greater precision.

[Lafora disease with a fatal outcome]. / Bolezn' Lafory s letal'nym iskhodom.

Lafora disease is a rare hereditary genetic pathology of the nervous system (a group of progressive myoclonic epilepsies). The distinctive morphological feature of this disease is the presence of specific abnormal structures - polyglucosane bodies («Lafora bodies¼) in the brain tissue, myocardium, liver, and epithelium of the sweat gland ducts. The article discusses the clinical data of the course of Lafora's disease in an 18-year-old patient with a fatal outcome and the results of a post-mortem examination. The diagnosis of Lafora disease was confirmed by genetic analysis data - the presence of a homozygous mutation in the 2nd exon of the EPM2A gene - laforin (chr6:146007412G>A, rs137852915). When analyzing literature, we did not find a description of Lafora's disease cases with a fatal outcome with the presentation of macroscopic examination data at autopsy, as well as the results of a pathohistological examination of altered organ tissues with the morphological manifestations specific for this pathology (Lafora bodies in the the brain, heart, sweat gland epithelium).

The Toxoplasma glucan phosphatase TgLaforin utilizes a distinct functional mechanism that can be exploited by therapeutic inhibitors.

Toxoplasma gondii is an intracellular parasite that generates amylopectin granules (AGs), a polysaccharide associated with bradyzoites that define chronic T. gondii infection. AGs are postulated to act as an essential energy storage molecule that enable bradyzoite persistence, transmission, and reactivation. Importantly, reactivation can result in the life-threatening symptoms of toxoplasmosis. T. gondii encodes glucan dikinase and glucan phosphatase enzymes that are homologous to the plant and animal enzymes involved in reversible glucan phosphorylation and which are required for efficient polysaccharide degradation and utilization. However, the structural determinants that regulate reversible glucan phosphorylation in T. gondii are unclear. Herein, we define key functional aspects of the T. gondii glucan phosphatase TgLaforin (TGME49_205290). We demonstrate that TgLaforin possesses an atypical split carbohydrate-binding-module domain. AlphaFold2 modeling combined with hydrogen-deuterium exchange mass spectrometry and differential scanning fluorimetry also demonstrate the unique structural dynamics of TgLaforin with regard to glucan binding. Moreover, we show that TgLaforin forms a dual specificity phosphatase domain-mediated dimer. Finally, the distinct properties of the glucan phosphatase catalytic domain were exploited to identify a small molecule inhibitor of TgLaforin catalytic activity. Together, these studies define a distinct mechanism of TgLaforin activity, opening up a new avenue of T. gondii bradyzoite biology as a therapeutic target.

Lafora disease: Current biology and therapeutic approaches.

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.

A novel gene therapy for neurodegenerative Lafora disease via EPM2A-loaded DLinDMA lipoplexes.

Aim: To develop novel cationic liposomes as a nonviral gene delivery vector for the treatment of rare diseases, such as Lafora disease - a neurodegenerative epilepsy. Materials & methods: DLinDMA and DOTAP liposomes were formulated and characterized for the delivery of gene encoding laforin and expression of functional protein in HEK293 and neuroblastoma cells. Results: Liposomes with cationic lipids DLinDMA and DOTAP showed good physicochemical characteristics. Nanosized DLinDMA liposomes demonstrated desired transfection efficiency, negligible hemolysis and minimal cytotoxicity. Western blotting confirmed successful expression and glucan phosphatase assay demonstrated the biological activity of laforin. Conclusion: Our study is a novel preclinical effort in formulating cationic lipoplexes containing plasmid DNA for the therapy of rare genetic diseases such as Lafora disease.

Generation and characterization of a laforin nanobody inhibitor.

OBJECTIVES: Mutations in the gene encoding the glycogen phosphatase laforin result in the fatal childhood dementia 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. In this work, we present the generation and characterization of laforin nanobodies, with one being a laforin inhibitor. DESIGN AND METHODS: We identify multiple classes of specific laforin-binding nanobodies and determine their binding epitopes using hydrogen deuterium exchange (HDX) mass spectrometry. Using para-nitrophenyl phosphate (pNPP) and a malachite gold-based assay specific for glucan phosphatase activity, we assess the inhibitory effect of one nanobody on laforin's catalytic activity. RESULTS: Six families of laforin nanobodies are characterized and their epitopes mapped. One nanobody is identified and characterized that serves as an inhibitor of laforin's phosphatase activity. CONCLUSIONS: The six generated and characterized laforin nanobodies, with one being a laforin inhibitor, are an important set of tools that open new avenues to define unresolved glycogen metabolism questions.

Ppp1r3d deficiency preferentially inhibits neuronal and cardiac Lafora body formation in a mouse model of the fatal epilepsy Lafora disease.

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.

Accurate and sensitive quantitation of glucose and glucose phosphates derived from storage carbohydrates by mass spectrometry.

The addition of phosphate groups into glycogen modulates its branching pattern and solubility which all impact its accessibility to glycogen interacting enzymes. As glycogen architecture modulates its metabolism, it is essential to accurately evaluate and quantify its phosphate content. Simultaneous direct quantitation of glucose and its phosphate esters requires an assay with high sensitivity and a robust dynamic range. Herein, we describe a highly-sensitive method for the accurate detection of both glycogen-derived glucose and glucose-phosphate esters utilizing gas-chromatography coupled mass spectrometry. Using this method, we observed higher glycogen levels in the liver compared to skeletal muscle, but skeletal muscle contained many more phosphate esters. Importantly, this method can detect femtomole levels of glucose and glucose phosphate esters within an extremely robust dynamic range with excellent accuracy and reproducibility. The method can also be easily adapted for the quantification of plant starch, amylopectin or other biopolymers.

Regulation of the autophagic PI3KC3 complex by laforin/malin E3-ubiquitin ligase, two proteins involved in Lafora disease.

Lafora progressive myoclonus epilepsy is a fatal rare neurodegenerative disorder characterized by the accumulation of insoluble abnormal glycogen deposits in the brain and peripheral tissues. Mutations in at least two genes are responsible for the disease: EPM2A, encoding the glucan phosphatase laforin, and EPM2B, encoding the RING-type E3-ubiquitin ligase malin. Both laforin and malin form a functional complex in which laforin recruits the substrates to be ubiquitinated by malin. We and others have described that, in cellular and animal models of this disease, there is an autophagy impairment which leads to the accumulation of dysfunctional mitochondria. In addition, we established that the autophagic defect occurred at the initial steps of autophagosome formation. In this work, we present evidence that in cellular models of the disease there is a decrease in the amount of phosphatidylinositol-3P. This is probably due to defective regulation of the autophagic PI3KC3 complex, in the absence of a functional laforin/malin complex. In fact, we demonstrate that the laforin/malin complex interacts physically and co-localizes intracellularly with core components of the PI3KC3 complex (Beclin1, Vps34 and Vps15), and that this interaction is specific and results in the polyubiquitination of these proteins. In addition, the laforin/malin complex is also able to polyubiquitinate ATG14L and UVRAG. Finally, we show that overexpression of the laforin/malin complex increases PI3KC3 activity. All these results suggest a new role of the laforin/malin complex in the activation of autophagy via regulation of the PI3KC3 complex and explain the defect in autophagy described in Lafora disease.

Skeletal Muscle Glycogen Chain Length Correlates with Insolubility in Mouse Models of Polyglucosan-Associated Neurodegenerative Diseases.

Lafora disease (LD) and adult polyglucosan body disease (APBD) are glycogen storage diseases characterized by a pathogenic buildup of insoluble glycogen. Mechanisms causing glycogen insolubility are poorly understood. Here, in two mouse models of LD (Epm2a-/- and Epm2b-/-) and one of APBD (Gbe1ys/ys), the separation of soluble and insoluble muscle glycogen is described, enabling separate analysis of each fraction. Total glycogen is increased in LD and APBD mice, which, together with abnormal chain length and molecule size distributions, is largely if not fully attributed to insoluble glycogen. Soluble glycogen consists of molecules with distinct chain length distributions and differential corresponding solubility, providing a mechanistic link between soluble and insoluble glycogen in vivo. Phosphorylation states differ across glycogen fractions and mouse models, demonstrating that hyperphosphorylation is not a basic feature of insoluble glycogen. Lastly, model-specific variances in protein and activity levels of key glycogen synthesis enzymes suggest uninvestigated regulatory mechanisms.

Oligomerization and carbohydrate binding of glucan phosphatases.

Glucan phosphatases are a unique subset of the phosphatase family that bind to and dephosphorylate carbohydrate substrates. Family members are found in diverse organisms ranging from single-cell red algae to humans. The nature of their functional oligomerization has been a source of considerable debate. We demonstrate that the human laforin protein behaves aberrantly when subjected to Size Exclusion Chromotography (SEC) analysis due to interaction with the carbohydrate-based matrix. This interaction complicates the analysis of laforin human disease mutations. Herein, we show that SEC with Multi-Angle static Light Scattering (SEC-MALS) provides a method to robustly define the oligomerization state of laforin and laforin variants. We further analyzed glucan phosphatases from photosynthetic organisms to define if this interaction was characteristic of all glucan phosphatases. Starch EXcess-four (SEX4) from green plants was found to lack significant interaction with the matrix and instead exists as a monomer. Conversely, Cm-laforin, from red algae, exists as a monomer in solution while still exhibiting significant interaction with the matrix. These data demonstrate a range of oligomerization behaviors of members of the glucan phosphatase family, and establish SEC-MALS as a robust methodology to quantify and compare oligomerization states between different proteins and protein variants in this family.

A novel EPM2A mutation yields a slow progression form of Lafora disease.

Lafora disease (LD, OMIM 254780) is a rare disorder characterized by epilepsy and neurodegeneration leading patients to a vegetative state and death, usually within the first decade from the onset of the first symptoms. In the vast majority of cases LD is related to mutations in either the EPM2A gene (encoding the glucan phosphatase laforin) or the EPM2B gene (encoding the E3-ubiquitin ligase malin). In this work, we characterize the mutations present in the EPM2A gene in a patient displaying a slow progression form of the disease. The patient is compound heterozygous with Y112X and N163D mutations in the corresponding alleles. In primary fibroblasts obtained from the patient, we analyzed the expression of the mutated alleles by quantitative real time PCR and found slightly lower levels of expression of the EPM2A gene respect to control cells. However, by Western blotting we were unable to detect endogenous levels of the protein in crude extracts from patient fibroblasts. The Y112X mutation would render a truncated protein lacking the phosphatase domain and likely degraded. Since minute amounts of laforin-N163D might still play a role in cell physiology, we analyzed the biochemical characteristics of the N163D mutation. We found that recombinant laforin N163D protein was as stable as wild type and exhibited near wild type phosphatase activity towards biologically relevant substrates. On the contrary, it showed a severe impairment in the interaction profile with previously identified laforin binding partners. These results lead us to conclude that the slow progression of the disease present in this patient could be either due to the specific biochemical properties of laforin N163D or to the presence of alternative genetic modifying factors separate from pathogenicity.

The unique evolution of the carbohydrate-binding module CBM20 in laforin.

Laforin catalyses glycogen dephosphorylation. Mutations in its gene result in Lafora disease, a fatal progressive myoclonus epilepsy, the hallmark being water-insoluble, hyperphosphorylated carbohydrate inclusions called Lafora bodies. Human laforin consists of an N-terminal carbohydrate-binding module (CBM) from family CBM20 and a C-terminal dual-specificity phosphatase domain. Laforin is conserved in all vertebrates, some basal metazoans and a small group of protozoans. The present in silico study defines the evolutionary relationships among the CBM20s of laforin with an emphasis on newly identified laforin orthologues. The study reveals putative laforin orthologues in Trichinella, a parasitic nematode, and identifies two sequence inserts in the CBM20 of laforin from parasitic coccidia. Finally, we identify that the putative laforin orthologues from some protozoa and algae possess more than one CBM20.