The focus of this grant is to determine how laforin, the only phosphatase in Kingdom Animalia with a carbohydrate binding domain, regulates changes in glycogen metabolism in vertebrates and invertebrates. Humans develop insoluble aberrant glycogen particles, called Lafora bodies, as a result of an autosomal recessive neurodegenerative disorder known as Lafora disease (LD). LD is the result of loss of function mutations to either of the genes that encode the dual specific phosphatase laforin or the E3 ubiquitin ligase malin. LD is one of five major progressive myoclonus epilepsies (PMEs) and presents as a single seizure in the second decade of the patient's life; this single event is followed by progressive central nervous system degeneration, intellectual decline, severe motor deterioration, and finally death within ten years. Laforin is reported as only being conserved among vertebrates; however, I recently identified laforin orthologs in five unicellular eukaryotes, including Cyanidioschyzon merolae and Toxoplasma gondii. Additionally, the biochemical composition of LBs closely resembles that of floridean starch; an insoluble carbohydrate molecule synthesized by the same unicellular eukaryotes that have laforin. Thus, there is a direct correlation between the presence of laforin and synthesis of floridean starch amongst invertebrates. I propose that laforin is involved in degrading insoluble carbohydrates, either to utilize it as an energy source for invertebrates or to protect cellular integrity in mammals, and that laforin's role in these two processes is conserved from invertebrates to humans. To determine laforin's function in glycogen metabolism Iwill: 1) Perform rigorous phylogenomic and functional analyses of laforin's invertebrate orthologs. I will identify every invertebrate laforin ortholog via bioinformatics and will determine the mRNA expression, protein expression and subcellular localization of laforin in C. merolae and T. gondii. 2) Define laforin's target in floridean starch metabolism in Toxoplasma gondii. I will generate multiple transgenic T. gondii organisms, identify any phenotypes that these organisms manifest and employ these organisms in biochemical experiments to identify laforin's substrate or a signal transduction pathway that it modulates. Additionally, I will utilize a T. gondii laforin knockout line to test if other laforin orthologs complement any observed phenotype. 3) Characterize the role of floridean starch in the human myoclonic epilepsy Lafora disease. I will extend myfindings from the invertebrate systems to the vertebrate system and LD. I will test any laforin substrate identified in the previous sections to see if it is also a substrate of laforin in the mammalian system. Additionally, I will fully characterize the interaction I previously identified between malin and laforin to determine both the cause and effect of this interaction. The findings from all three of my specific aims can be applied to identify new therapeutic targets to treat LD and possibly other polyglucosan diseases.
| DePaoli-Roach, Anna A; Segvich, Dyann M; Meyer, Catalina M et al. (2012) Laforin and malin knockout mice have normal glucose disposal and insulin sensitivity. Hum Mol Genet 21:1604-10 |
| Gentry, Matthew S; Dixon, Jack E; Worby, Carolyn A (2009) Lafora disease: insights into neurodegeneration from plant metabolism. Trends Biochem Sci 34:628-39 |
| Worby, Carolyn A; Gentry, Matthew S; Dixon, Jack E (2008) Malin decreases glycogen accumulation by promoting the degradation of protein targeting to glycogen (PTG). J Biol Chem 283:4069-76 |
| Gentry, Matthew S; Dowen 3rd, Robert H; Worby, Carolyn A et al. (2007) The phosphatase laforin crosses evolutionary boundaries and links carbohydrate metabolism to neuronal disease. J Cell Biol 178:477-88 |
| Kim, Youngjun; Gentry, Matthew S; Harris, Thurl E et al. (2007) A conserved phosphatase cascade that regulates nuclear membrane biogenesis. Proc Natl Acad Sci U S A 104:6596-601 |
| Cheng, Alan; Zhang, Mei; Gentry, Matthew S et al. (2007) A role for AGL ubiquitination in the glycogen storage disorders of Lafora and Cori's disease. Genes Dev 21:2399-409 |
