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.

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 (PBs) 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, APBD), and mutations in EPM2A or EPM2B a fatal progressive myoclonus epilepsy (Lafora disease, LD). RBCK1 deficiency (RD) causes an amylopectinosis with fatal skeletal and cardiac myopathy (Polyglucosan Body Myopathy 1, PGBM1, OMIM# 615895). RBCK1 is a component of the linear ubiquitin chain assembly complex (LUBAC), 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 (1) that the amylopectinosis of RD like in APBD and LD affects the brain, (2) that RD glycogen like in APBD and LD has overlong branches, (3) that unlike APBD but like LD, RD glycogen is hyperphosphorylated, and finally (4) that unlike laforin-deficient LD but like malin-deficient LD RD’s glycogen hyperphosphorylation is limited to precipitated polyglucosans. In summary, the fundamental glycogen pathology of RD recapitulates that of malin-deficient LD. Additionally, we uncover sex and genetic background effects in RD on organ and brain region specific amylopectinoses, and in the brain on consequent neuroinflammation and behavioral deficits. Finally, we exploit the portion of the basic glycogen pathology that is common to APBD, both forms of LD, and RD, 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.

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 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. Secondly, 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.