An astrocytic cellular model of Lafora disease to study polyglucosan accumulation and inflammation.

Lafora disease (LD) is caused by mutations in either the laforin or malin gene. The hallmark of the disease is the accumulation of polyglucosan inclusions called Lafora Bodies (LBs). Malin knockout (KO) mice present polyglucosan accumulations in several brain areas, as do patients of LD. These structures are abundant in the cerebellum and hippocampus. Here, we report a large increase in glycogen synthase (GS) in these mice, in which the enzyme accumulates in LBs. Our study focused on the hippocampus where, under physiological conditions, astrocytes and parvalbumin-positive (PV+) interneurons expressed GS and malin. Although LBs have been described only in neurons, we found this polyglucosan accumulation in the astrocytes of the KO mice. They also had LBs in the soma and some processes of PV+ interneurons. This phenomenon was accompanied by the progressive loss of these neuronal cells and, importantly, neurophysiological alterations potentially related to impairment of hippocampal function. Our results emphasize the relevance of the laforin–malin complex in the control of glycogen metabolism and highlight altered glycogen accumulation as a key contributor to neurodegeneration in LD.

Lafora disease (LD) is a fatal form of progressive myoclonus epilepsy characterized by the accumulation of insoluble poorly branched glycogen-like inclusions named Lafora bodies (LBs) in the brain and peripheral tissues. In the brain, since its first discovery in 1911, it was assumed that these glycogen inclusions were only present in affected neurons. Mouse models of LD have been obtained recently, and we and others have been able to report the accumulation of glycogen inclusions in the brain of LD animals, what recapitulates the hallmark of the disease. In this work we present evidence indicating that, although in mouse models of LD glycogen inclusions co-localize with neurons, as originally established, most of them co-localize with astrocytic markers such as glial fibrillary acidic protein (GFAP) and glutamine synthase. In addition, we have observed that primary cultures of astrocytes from LD mouse models accumulate higher levels of glycogen than controls. These results suggest that astrocytes may play a crucial role in the pathophysiology of Lafora disease, as the accumulation of glycogen inclusions in these cells may affect their regular functionality leading them to a possible neuronal dysfunction.

Lafora disease (LD), the most devastating adolescence-onset epilepsy, is caused by mutations in the EPM2A or EPM2B genes, which encode the proteins laforin and malin, respectively. Loss of function of one of these proteins, which are involved in the regulation of glycogen synthesis, induces the accumulation of polyglucosan bodies (PGBs)-known as Lafora bodies (LBs) and associated with neurons-in the brain. Ageing and some neurodegenerative conditions lead to the appearance of another type of PGB called corpora amylacea, which are associated with astrocytes and contain neo-epitopes that can be recognized by natural antibodies. Here we studied the PGBs in the cerebral cortex and hippocampus of malin knockout mice, a mouse model of LD. These animals presented not only LBs associated with neurons but also a significant number of PGBs associated with astrocytes. These astrocytic PGBs were also increased in mice from senescence-accelerated mouse-prone 8 (SAMP8) strain and mice with overexpression of Protein Targeting to Glycogen (PTGOE ), indicating that they are not exclusive of LD. The astrocytic PGBs, but not neuronal LBs, contained neo-epitopes that are recognized by natural antibodies. The astrocytic PGBs appeared predominantly in the hippocampus but were also present in some cortical brain regions, while neuronal LBs were found mainly in the brain cortex and the pyramidal layer of hippocampal regions CA2 and CA3. Our results indicate that astrocytes, contrary to current belief, are involved in the etiopathogenesis of LD.

Many glycogen storage diseases, e.g., Lafora disease and Pompe disease, are characterized by aberrant glycogen‐like aggregates known as polyglucosan bodies (PGBs). Despite the clinical relevance of PGB accumulation, there are limited options to rapidly assess PGB levels in tissues and biofluids. Additionally, there are limitations to current pre‐clinical methods that rely on enzymatic degradation assays and mass spectrometry in quantitating PGBs. We recently developed novel sandwich ELISAs for the quantitation of both glycogen and PGBs. By utilizing the specificities of glycogen and PGB antibodies, we can specifically capture glycogen or PGBs and detect them utilizing a unique carbohydrate binding module (CBM). We establish the specificity of the assays and the sensitivity of the sandwich ELISAs compared to other techniques used for quantitation. We also demonstrate the applicability of the ELISA using multiple mouse models of Lafora disease and Pompe disease to quantify PGB loads in the brain. Moving forward, these ELISAs could be used in both research and clinical settings to accurately determine PGB levels.

Lafora disease (LD; OMIM#254780) is a rare form of progressive myoclonus epilepsy characterized by the accumulation of insoluble deposits of aberrant glycogen (polyglucosans), named Lafora bodies (LBs), in the brain but also in peripheral tissues. It is assumed that the accumulation of LBs is related to the appearance of the characteristic pathological features of the disease. In mouse models of LD, we and others have reported an increase in the levels of reactive astrocytes and activated microglia, which triggers the expression of the different pro-inflammatory mediators. Recently, we have demonstrated that the TNF and IL-6 inflammatory signaling pathways are the main mediators of the neuroinflammatory phenotype associated with the disease. In this work, we present evidence that the activation of these pathways produces a dysregulation in the levels of different subunits of the excitatory ionotropic glutamatergic receptors (phopho-GluN2B, phospho-GluA2, GluK2) and also an increase in the levels of the GABA transporter GAT1 in the hippocampus of the Epm2b-/- mice. In addition, we present evidence of the presence of activated forms of the Src and Lyn protein kinases in this area. These effects may increase the excitatory glutamatergic signaling and decrease the inhibitory GABAergic tone, leading to hyper-excitability. More importantly, the enhanced production of these subunits occurs in non-neuronal cells such as activated microglia and reactive astrocytes, pointing out a key role of glia in the pathophysiology of LD.

Lafora disease (LD; OMIM#254780) is a rare form of progressive myoclonus epilepsy characterised by the accumulation of insoluble deposits of glycogen in the brain and peripheral tissues. In mouse models of LD, we have identified neuroinflammation as a secondary hallmark of the disease, characterised by increased levels of reactive astrocytes and activated microglia. Our previous work demonstrated that the TNF and IL-6 inflammatory signalling pathways are the primary drivers of this neuroinflammatory phenotype. In this work, we aimed to investigate whether TNF and IL-6 pathway activation contributes to alterations in the glutamatergic and GABAergic signalling pathways. We performed immunofluorescence and western blot analyses on the hippocampus of a mouse model of LD to evaluate potential changes in proteins associated with glutamatergic and GABAergic signalling pathways. Our findings reveal dysregulation in the expression of subunits of excitatory glutamatergic receptors (phospho-GluN2B and GluK2), as well as an increase in the levels of the GABA transporter GAT1. In addition, we detected activated forms of the Src and Lyn protein kinases in the hippocampus. More importantly, these alterations predominantly occur in nonneuronal cells, such as reactive astrocytes and microglia, underscoring the critical involvement of glial cells in the pathophysiology of LD. The observed upregulation of glutamatergic receptor subunits likely amplifies excitatory glutamatergic signalling, whereas the increased expression of GAT1 may reduce the inhibitory GABAergic tone. These changes contribute to the characteristic hyperexcitability of LD.

Adult polyglucosan body disease (APBD) and Lafora disease (LD) are autosomal recessive glycogen storage neurological disorders. APBD is caused by mutations in the glycogen branching enzyme (GBE1) gene and is characterized by progressive upper and lower motor neuron dysfunction and premature death. LD is a fatal progressive myoclonus epilepsy caused by loss of function mutations in the EPM2A or EPM2B gene. These clinically distinct neurogenetic diseases share a common pathology. This consists of time-dependent formation, precipitation, and accumulation of an abnormal form of glycogen (polyglucosan) into gradually enlarging inclusions, polyglucosan bodies (PBs) in ever-increasing numbers of neurons and astrocytes. The growth and spread of PBs are followed by astrogliosis, microgliosis, and neurodegeneration. The key defect in polyglucosans is that their glucan branches are longer than those of normal glycogen, which prevents them from remaining in solution. Since the lengths of glycogen branches are determined by the enzyme glycogen synthase, we hypothesized that downregulating this enzyme could prevent or hinder the generation of the pathogenic PBs. Here, we pursued an adeno-associated virus vector (AAV) mediated RNA-interference (RNAi) strategy. This approach resulted in approximately 15% reduction of glycogen synthase mRNA and an approximately 40% reduction of PBs across the brain in the APBD and both LD mouse models. This was accompanied by improvements in early neuroinflammatory markers of disease. This work represents proof of principle toward developing a single lifetime dose therapy for two fatal neurological diseases: APBD and LD. The approach is likely applicable to other severe and common diseases of glycogen storage.

Targeting Gys1 with AAV-SaCas9 Decreases Pathogenic Polyglucosan Bodies and Neuroinflammation in Adult Polyglucosan Body and Lafora Disease Mouse Models.

Polyglucosans are glycogen molecules with overlong chains, which are hyperphosphorylated in the neurodegenerative Lafora disease (LD). Brain polyglucosan bodies (PBs) cause fatal neurodegenerative diseases including Lafora disease and adult polyglucosan body disease (ABPD), for which treatments, biomarkers, and good understanding of their pathogenesis are currently missing. Mutations in the genes for the phosphatase laforin or the E3 ubiquitin ligase malin can cause LD. By depleting PTG, an activator of the glycogen chain-elongating enzyme glycogen synthase (GYS1), in laforin- and malin-deficient LD mice, we show that abnormal glycogen chain lengths and not hyperphosphorylation underlie polyglucosan formation, and that polyglucosan bodies induce neuroinflammation. We provide evidence indicating that a small pool of overactive GYS1 contributes to glycogen insolubility in LD and APBD. In contrast to previous findings, metabolomics experiments using in situ-fixed brains reveal only modest metabolic changes in laforin-deficient mice. These changes are not replicated in malin-deficient or APBD mice, and are not normalized in rescued LD mice. Finally, we identify a pool of metabolically volatile malto-oligoglucans as a polyglucosan body- and neuroinflammation-associated brain energy source, and promising candidate biomarkers for LD and APBD, including malto-oligoglucans and the neurodegeneration marker CHI3L1/YKL40.

Mutations in the EPM2A and EPM2B genes, encoding laforin and malin proteins respectively, are responsible for Lafora disease, a fatal form of progressive myoclonus epilepsy with autosomal recessive inheritance. Neuroimaging studies of patients with Lafora disease have shown different degrees of brain atrophy, decreased glucose brain uptake and alterations on different brain metabolites mainly in the frontal cortex, basal ganglia and cerebellum. Mice deficient for laforin and malin present many features similar to those observed in patients, including cognitive, motor, histological and epileptic hallmarks. We describe the neuroimaging features found in two mouse models of Lafora disease. We found altered volumetric values in the cerebral cortex, hippocampus, basal ganglia and cerebellum using magnetic resonance imaging (MRI). Positron emission tomography (PET) of the cerebral cortex, hippocampus and cerebellum of Epm2a−/− mice revealed abnormal glucose uptake, although no alterations in Epm2b−/− mice were observed. Magnetic resonance spectroscopy (MRS) revealed significant changes in the concentration of several brain metabolites, including N-acetylaspartate (NAA), in agreement with previously described findings in patients. These data may provide new insights into disease mechanisms that may be of value for developing new biomarkers for diagnosis, prevention and treatment of Lafora disease using animal models.

Early Treatment with Metformin Improves Neurological Outcomes in Lafora Disease.

Lafora disease (LD) is a neurological disorder characterized by progressive myoclonus epilepsy. The hallmark of the disease is the presence of insoluble forms of glycogen (polyglucosan bodies, or PGBs) in the brain. The accumulation of PGBs is causative of the pathophysiological features of LD. However, despite the efforts made by different groups, the question of why PGBs accumulate in the brain is still unanswered. We have recently demonstrated that, in vivo, astrocytes accumulate most of the PGBs present in the brain, and this could lead to astrocyte dysfunction. To develop a deeper understanding of the defects present in LD astrocytes that lead to LD pathophysiology, we obtained pure primary cultures of astrocytes from LD mice from the postnatal stage under conditions that accumulate PGBs, the hallmark of LD. These cells serve as novel in vitro models for studying PGBs accumulation and related LD dysfunctions. In this sense, the metabolomics of LD astrocytes indicate that they accumulate metabolic intermediates of the upper part of the glycolytic pathway, probably as a consequence of enhanced glucose uptake. In addition, we also demonstrate the feasibility of using the model in the identification of different compounds that may reduce the accumulation of polyglucosan inclusions.

Gene therapy for Lafora disease in the Epm2a−/− mouse model

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

The significance of the development of polyglucosan bodies (PBs) in the CNS is incompletely understood. We present the clinicopathological features of three autopsy cases with numerous PBs other than the common corpora amylacea or Lafora bodies. The first patient had pleomorphic PBs in the neuronal processes of pallidum and substantia nigra which, thus, are consistent with Bielschowsky bodies. Bielschowsky bodies involved also the hypothalamus and tegmentum of midbrain and medulla. The present case was the first not associated with any clinical symptoms. The second patient also had incidental Bielschowsky bodies in the external pallidum, substantia nigra, and pallidothalamic, pallidonigral and nigrostriatal tracts. Additionally, unique clusters of small PBs appeared in the cerebral cortex, putamen, pallidum, and caudate nucleus. Immunostaining suggested that these small clustered PBs were located in the cytoplasm and processes of astrocytes. Ultrastructurally, these clustered PBs were in agreement with previous descriptions of PBs. The third patient had adult polyglucosan body disease. Most PBs in the white matter were corpora amylacea situated in astrocytic processes or axons. In the gray matter, many pleomorphic PBs resembling Bielschowsky bodies occurred in neuronal processes. In the peripheral nervous system, a few PBs were seen in myelinated axons.(ABSTRACT TRUNCATED AT 250 WORDS)