Novel Model Systems Link Floridean Starch Metabolism to Laflora Disease: A glucan phosphate assay as a diagnotic tool for lafora disease

Lafora disease (LD) is an autosomal recessive, neurodegenerative disease that begins with epilepsy in the patient's second decade of life, is followed by rapid neurological deteriation, and ends with death within ten years of the first episode. A hallmark of LD is the accumulation of insoluble glucans/carbohydrates, called Lafora bodies (LBs), in the cytoplasm of all cells. While LBs form in all cells, only neurons undergo apoptosis. It is thought that LBs trigger neuronal apoptosis, neurodegneration, epilepsy, and the death of the patient. LD is caused by mutations in the gene encoding the glucan phosphatase laforin or the E3 ubiquitin ligase malin. Two mechanisms explain the molecular etiology of LD: 1) laforin dephosphorylates nascent glucans to allow proper glycogen synthesis and in its absence LBs form, and 2) laforin recruits the E3 ubiquitin ligase malin to sites of glycogen synthesis, and malin regulates the protein levels of many glycogen metabolism enzymes. While significant progress has been made in determining the molecular mechanisms causing LD, there is no cure or long-term treatment. However, multiple treatments are being tested in mouse models and at least one clinical trial is set to begin. All of the proposed therapies require a biomarker to determine if the therapy is producing functionallaforin and/or malin, but no such biomarker exists. The focus of this grant is to develop a laforin biomarker for pre-clinical and clinical trials. Successful completion of this proposal will provide an in vitro assay to evaluate the two aspects oflaforin functionality, i.e. the ability to bind and dephosphorylate glycogen. To obtain this goal, we will: 1) Establish which a-Iaforin antibody most efficiently immunoprecipitates mouse & human laforin. 2) Determine if laforin can bind & dephosphorylate glucans while bound by an antibody-resin. 3) Optimize the immunoprecipitation procedure. 4) Define the minimal amount of mouse and human cells needed to perform the assay. The assay will consist of immunoprecipitating laforin from tissue and using immunoprecipitated laforin to test its ability to bind and dephosphorylate glucans.