Myofiber-type-dependent 'boulder' or 'multitudinous pebble' formations across distinct amylopectinoses.

At least five enzymes including three E3 ubiquitin ligases are dedicated to glycogen's spherical structure. Absence of any reverts glycogen to a structure resembling amylopectin of the plant kingdom. This amylopectinosis (polyglucosan body formation) causes fatal neurological diseases including adult polyglucosan body disease (APBD) due to glycogen branching enzyme deficiency, Lafora disease (LD) due to deficiencies of the laforin glycogen phosphatase or the malin E3 ubiquitin ligase and type 1 polyglucosan body myopathy (PGBM1) due to RBCK1 E3 ubiquitin ligase deficiency. Little is known about these enzymes' functions in glycogen structuring. Toward understanding these functions, we undertake a comparative murine study of the amylopectinoses of APBD, LD and PGBM1. We discover that in skeletal muscle, polyglucosan bodies form as two main types, small and multitudinous ('pebbles') or giant and single ('boulders'), and that this is primarily determined by the myofiber types in which they form, 'pebbles' in glycolytic and 'boulders' in oxidative fibers. This pattern recapitulates what is known in the brain in LD, innumerable dust-like in astrocytes and single giant sized in neurons. We also show that oxidative myofibers are relatively protected against amylopectinosis, in part through highly increased glycogen branching enzyme expression. We present evidence of polyglucosan body size-dependent cell necrosis. We show that sex influences amylopectinosis in genotype, brain region and myofiber-type-specific fashion. RBCK1 is a component of the linear ubiquitin chain assembly complex (LUBAC), the only known cellular machinery for head-to-tail linear ubiquitination critical to numerous cellular pathways. We show that the amylopectinosis of RBCK1 deficiency is not due to loss of linear ubiquitination, and that another function of RBCK1 or LUBAC must exist and operate in the shaping of glycogen. This work opens multiple new avenues toward understanding the structural determinants of the mammalian carbohydrate reservoir critical to neurologic and neuromuscular function and disease.

Glycogen synthase downregulation rescues the amylopectinosis of murine RBCK1 deficiency.

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

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 mouse models of the disease. We synthesized an antisense oligonucleotide (Gys1-ASO) that targets the mRNA of the brain-expressed glycogen synthase 1 gene (Gys1). We administered Gys1-ASO by intracerebroventricular injection and analysed the pathological hallmarks of Lafora disease, namely glycogen accumulation, Lafora body formation, and neuroinflammation. Gys1-ASO prevented Lafora body formation in young mice that had not yet formed them. In older mice that already exhibited Lafora bodies, Gys1-ASO inhibited further accumulation, markedly preventing large Lafora bodies characteristic of advanced disease. Inhibition of Lafora body formation was associated with prevention of astrogliosis and strong trends towards correction of dysregulated expression of disease immune and neuroinflammatory markers. Lafora disease manifests gradually in previously healthy teenagers. Our work provides proof of principle that an antisense oligonucleotide targeting the GYS1 mRNA could prevent, and halt progression of, this catastrophic epilepsy.

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

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.

An inducible glycogen synthase-1 knockout halts but does not reverse Lafora disease progression in mice.

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

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.

Sensitive quantification of α-glucans in mouse tissues, cell cultures, and human cerebrospinal fluid.

The soluble α-polyglucan glycogen is a central metabolite enabling transient glucose storage to suit cellular energy needs. Glycogen storage diseases (GSDs) comprise over 15 entities caused by generalized or tissue-specific defects in enzymes of glycogen metabolism. In several, e.g. in Lafora disease caused by the absence of the glycogen phosphatase laforin or its interacting partner malin, degradation-resistant abnormally structured insoluble glycogen accumulates. Sensitive quantification methods for soluble and insoluble glycogen are critical to research, including therapeutic studies, in such diseases. This paper establishes methodological advancements relevant to glycogen metabolism investigations generally, and GSDs. Introducing a pre-extraction incubation method, we measure degradation-resistant glycogen in as little as 30 mg of skeletal muscle or a single hippocampus from Lafora disease mouse models. The digestion-resistant glycogen correlates with the disease-pathogenic insoluble glycogen and can readily be detected in very young mice where glycogen accumulation has just begun. Second, we establish a high-sensitivity glucose assay with detection of ATP depletion, enabling 1) quantification of α-glucans in cell culture using a medium-throughput assay suitable for assessment of candidate glycogen synthesis inhibitors, and 2) discovery of α-glucan material in healthy human cerebrospinal fluid, establishing a novel methodological platform for biomarker analyses in Lafora disease and other GSDs.

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.

MYORG is associated with recessive primary familial brain calcification.

Objective: To investigate the genetic basis of the recessive form of primary familial brain calcification and study pathways linking a novel gene with known dominant genes that cause the disease. Methods: Whole exome sequencing and Sanger-based segregation analysis were used to identify possible disease causing mutations. Mutation pathogenicity was validated by structural protein modeling. Functional associations between the candidate gene, MYORG, and genes previously implicated in the disease were examined through phylogenetic profiling. Results: We studied nine affected individuals from two unrelated families of Middle Eastern origin. The median age of symptom onset was 29.5 years (range 21-57 years) and dysarthria was the most common presenting symptom. We identified in the MYORG gene, a homozygous c.1233delC mutation in one family and c.1060_1062delGAC mutation in another. The first mutation results in protein truncation and the second in deletion of a highly conserved aspartic acid that is likely to disrupt binding of the protein with its substrate. Phylogenetic profiling analysis of the MYORG protein sequence suggests co-evolution with a number of calcium channels as well as other proteins related to regulation of anion transmembrane transport (False Discovery Rate, FDR < 10-8) and with PDCD6IP, a protein interacting with PDGFR ß which is known to be involved in the disease. Interpretation: MYORG mutations are linked to a recessive form of primary familial brain calcification. This association was recently described in patients of Chinese ancestry. We suggest the possibility that MYORG mutations lead to calcification in a PDGFR ß-related pathway.

Lafora disease - from pathogenesis to treatment strategies.

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.

Novel Stress in Plants by Altering the Photoperiod.

Recent work has shown that changing the photoperiod induces stress in Arabidopsis thaliana. It has particularly dramatic consequences in cytokinin-deficient plants and clock mutants. Here, we argue that studying the impact of an altered photoperiod will provide novel insights into the circadian clock, factors regulating it, and pathways under its control.

Pathogenesis of Lafora Disease: Transition of Soluble Glycogen to Insoluble Polyglucosan.

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.

Circadian Stress Regimes Affect the Circadian Clock and Cause Jasmonic Acid-Dependent Cell Death in Cytokinin-Deficient Arabidopsis Plants.

The circadian clock helps plants measure daylength and adapt to changes in the day-night rhythm. We found that changes in the light-dark regime triggered stress responses, eventually leading to cell death, in leaves of Arabidopsis thaliana plants with reduced cytokinin levels or defective cytokinin signaling. Prolonged light treatment followed by a dark period induced stress and cell death marker genes while reducing photosynthetic efficiency. This response, called circadian stress, is also characterized by altered expression of clock and clock output genes. In particular, this treatment strongly reduced the expression of CIRCADIAN CLOCK ASSOCIATED1 (CCA1) and LATE ELONGATED HYPOCOTYL (LHY). Intriguingly, similar changes in gene expression and cell death were observed in clock mutants lacking proper CCA1 and LHY function. Circadian stress caused strong changes in reactive oxygen species- and jasmonic acid (JA)-related gene expression. The activation of the JA pathway, involving the accumulation of JA metabolites, was crucial for the induction of cell death, since the cell death phenotype was strongly reduced in the jasmonate resistant1 mutant background. We propose that adaptation to circadian stress regimes requires a normal cytokinin status which, acting primarily through the AHK3 receptor, supports circadian clock function to guard against the detrimental effects of circadian stress.

A novel protective function for cytokinin in the light stress response is mediated by the Arabidopsis histidine kinase2 and Arabidopsis histidine kinase3 receptors.

Cytokinins are plant hormones that regulate diverse processes in plant development and responses to biotic and abiotic stresses. In this study, we show that Arabidopsis (Arabidopsis thaliana) plants with a reduced cytokinin status (i.e. cytokinin receptor mutants and transgenic cytokinin-deficient plants) are more susceptible to light stress compared with wild-type plants. This was reflected by a stronger photoinhibition after 24 h of high light (approximately 1,000 µmol m(-2) s(-1)), as shown by the decline in maximum quantum efficiency of photosystem II photochemistry. Photosystem II, especially the D1 protein, is highly sensitive to the detrimental impact of light. Therefore, photoinhibition is always observed when the rate of photodamage exceeds the rate of D1 repair. We demonstrate that in plants with a reduced cytokinin status, the D1 protein level was strongly decreased upon light stress. Inhibition of the D1 repair cycle by lincomycin treatment indicated that these plants experience stronger photodamage. The efficiency of photoprotective mechanisms, such as nonenzymatic and enzymatic scavenging systems, was decreased in plants with a reduced cytokinin status, which could be a cause for the increased photodamage and subsequent D1 degradation. Additionally, slow and incomplete recovery in these plants after light stress indicated insufficient D1 repair. Mutant analysis revealed that the protective function of cytokinin during light stress depends on the Arabidopsis histidine KINASE2 (AHK2) and AHK3 receptors and the type B Arabidopsis response regulator1 (ARR1) and ARR12. We conclude that proper cytokinin signaling and regulation of specific target genes are necessary to protect leaves efficiently from light stress.