SUMMARY 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.

The Hedgehog (Hh) pathway is a highly conserved signaling cascade crucial for cell fate determination during embryogenesis. Response to the Hh ligands is mediated by the receptor Patched-1 (Ptch1), a 12-pass transmembrane glycoprotein. Despite its essential role in Hh signaling and its activity as a tumor suppressor, Ptch1 remains largely uncharacterized. We demonstrate here that Ptch1 binds to itself to form oligomeric structures. Oligomerization is mediated by two distinct, structurally disordered, intracellular domains spanning amino acids 584 -734 ("middle loop") and 1162-1432 (C terminus). However, oligomerization is not required for Ptch1-dependent regulation of the canonical Hh pathway operating through Smo. Expression of a mutant protein that deletes both regions represses the Hh pathway and responds to the addition of Hh ligand independent of its inability to bind other factors such as Smurf2. Additionally, deletion of the cytoplasmic middle loop domain generates a Ptch1 mutant that, despite binding to Hh ligand, constitutively suppresses Hh signaling and increases the length of primary cilia. Constitutive activity because of deletion of this region is reversed by further deletion of specific sequences in the cytoplasmic C-terminal domain. These data reveal an interaction between the cytoplasmic domains of Ptch1 and that these domains modulate Ptch1 activity but are not essential for regulation of the Hh pathway.

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.

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.