Project Summary: Glycogen metabolism is impaired in >20 individual rare genetic diseases. Several of these diseases are caused by the formation of insoluble glycogen, which deposits in polyglucosan bodies (PBs). Without treatment currently available, PB accumulation causes pathology in liver, muscle, heart, and/or brain tissue. The mechanisms underlying the prevention of pathogenic insoluble glycogen are poorly understood. The PI’s work with established mouse models of polyglucosan body diseases links both glycogen phosphate and branching directly to glycogen solubility and imply a functional interdependence of phosphate and branching. The objective of this proposal is to identify how phosphate covalently linked to glycogen and glycogen branching impacts glycogen solubility in health and disease, and whether genetic modulation of each factor can decrease pathogenic PB accumulation in vivo. Utilizing novel in vitro and in vivo approaches, the proposed work will test the central hypothesis that glycogen phosphorylation and glycogen branching 1) are interrelated cellular processes that affect the solubility of glycogen, and 2) that when genetically manipulated can improve the physiological functionality of glycogen. Aim 1 characterizes the impact of glycogen phosphate, branching, and associated proteome on the precipitation risk of soluble glycogen in mouse models with insoluble glycogen accumulation. Analyses and experimental manipulation of these parameters will provide a mechanistic explanation for the structural changes in soluble glycogen that lead to glycogen insolubility. Aim 2 focuses on the impact and regulation of phosphorylation during glycogen synthesis, to interrogate glycogen phosphate as part of a GBE1-regulated protection mechanism of the cell to prevent glycogen insolubility. Aim3 determines the potential of enhanced branching in the prevention of insoluble glycogen. The impact of branching on glycogen precipitation risk will be characterized, and a new therapeutic approach for polyglucosan body diseases will be provided. This proposal uses established mouse models with PB-triggered pathology. In addition, two new mouse lines were generated, to separately modulate glycogen phosphate and branching in vivo. Combined with state-of-the-art glycogen biochemistry and proteomics, these new tools provide a unique opportunity to tease apart the interrelations of glycogen phosphate and branching and their effects on glycogen solubility. The proposed work can (1) shift the paradigm of glycogen phosphate being detrimental for glycogen solubility to phosphate as a protection mechanism from glycogen insolubility, (2) reveal regulatory connections between glycogen branching and phosphorylation, as well as (3) lead to the discovery of unknown glycogen kinases. It will (4) lay the ground work for new therapeutic approaches for polyglucosan body diseases and (5) provide a better grasp of vital cellular processes related to glycogen metabolism with im...