PRE-CLINICAL ADVANCEMENTS IN BIOMARKERS, TOOLS, AND THERAPEUTICS FOR A METABOLIC NEURODEGENERATIVE DISEASE

Glycogen is the storage form of glucose and a highly important substrate for cellular metabolism. Characterization of the enzymes and mechanisms of glycogen metabolism began over 70 years ago and over the last 20 years, a previously unknown protein called laforin has emerged as an important contributor to glycogen metabolism homeostasis. Multiple labs demonstrated that laforin is a glycogen phosphatase and mutations in the gene encoding laforin cause the formation of aberrant glycogen-like aggregates called Lafora bodies (LBs). LBs are cytoplasmic, water-insoluble aggregates that drive neurodegeneration and early death in Lafora disease (LD) patients. The direct relationship between mutated laforin, LB formation, and LB neurotoxicity highlights the importance of glycogen metabolism in the brain. To translate this knowledge into effective LD therapeutics, a comprehensive analysis of pre-clinical biomarkers, a new set of tools, and advancements in therapeutic strategies are needed. Consistent with the clinical phenotype, the laforin KO mouse model possesses LBs that drive neurodegeneration. A few studies have pointed to inflammatory responses in both astrocytes and microglia. However, these data need to be considered thoroughly to understand their relationship to LD. Two primary measures of pre-clinical LD therapeutic efficacy are to ablate LBs and reverse astrocyte activation. In the present work, we provide a longitudinal analysis of LBs and soluble glycogen and provide physiological evidence of the propensity of LBs to aggregate. In this same study, we provide evidence to suggest that microglia, not astrocytes, are drivers of an inflammatory response in LD. Another potential therapeutic target is glycogen metabolism, however, the exact relationship between central carbon metabolism and laforin loss-of-function has yet to be defined and is essential for exploring therapeutic options to target it. Herein, we showcase a longitudinal discovery of changes to citric acid cycle intermediates specific to the brain of the aging laforin KO and WT mouse model. We find that metabolite load in adult mice is lower in laforin KOs compared to WT, however, the metabolic signatures of aged laforin KOs and WT mice overlap. Using a predictive biomarker analysis tool, we find that a recently proposed driver of metabolic dysregulation in laforin KO mice, glucosamine sequestration by PGBs, may also be driving aspects of metabolic disarray in aging. These results have generated hypotheses regarding damaged metabolic pathways in LD and aging that may be targeted for treatment. These data are yet another indicator that laforin is critical for brain glycogen metabolism. While laforin is known to remove phosphate from glycogen, how phosphate impacts normal glycogen homeostasis or how hyperphosphorylation is detrimental to glycogen homeostasis and LB formation remains under investigation. Progress in this discovery could be accelerated with additional tools. The study presented here showcases the generation and characterization of six laforin nanobodies and their epitopes and demonstrates that one inhibits laforin’s phosphatase activity. These laforin nanobodies could be useful tools for discovering the importance of glycogen phosphate and its modulation by laforin, WT laforin crystallization, laforin physical interactions, and laforin’s dynamic localization. Overall, these nanobodies represent an important set of tools that will open new avenues to illuminate the mechanism of laforin’s role in normal glycogen metabolism and LD. Other applications of the laforin nanobodies are in vitro detection assays for pre-clinical/clinical assessment of laforin recovery treatments. Two theoretical therapeutic options for laforin recovery include premature termination codon (PTC) readthrough therapy and adeno-associated viral vector (AAV) therapy. Both these therapeutic strategies aim to deliver corrected genetic information for proper translation of the protein of interest. In the study we present here, we generate one sandwich ELISA with two putative functions for either PTC readthrough or AAV pre-clinical/clinical development using the laforin nanobodies. This work has advanced pre-clinical biomarkers, tools, and therapeutics for LD, a metabolic neurodegenerative disease. The results of which will be the basic framework of effective therapeutic development for several possible treatment routes and mechanistic insights into glycogen metabolism