Ian H. Kratter, Ph.D. - US grants

Huntington's disease (HD) is a progressive, adult-onset neurodegenerative disease caused by a polyglutamine (polyQ) expansion in the N-terminal region of the protein huntingtin (htt). Pathological hallmarks of HD include nuclear and cytosolic aggregates and significant and selective neurons loss. There is no known cure or disease-modifying treatment for HD, and the specific mechanisms behind the pathology and selective vulnerability remain unclear. Htt has a highly conserved phosphorylation site at serine 421. Prior work in cell lines and primary neurons discovered that significantly increasing phosphorylation of htt at this site eliminates mutant htt toxicity;conversely, decreasing phosphorylation enhances htt toxicity. Furthermore, phosphorylation levels are highest in the cerebellum, lower in the cortex, and lower still in the striatum-inversely proportional to the pattern of neurodegeneration in HD. Despite these intriguing findings and the lack of effective therapies, the relevance of S421 phosphorylation to HD pathogenesis in the brain is unknown. The goal of our study is to determine in vivo the role of the S421 phosphorylation site in mutant htt-induced neurodegeneration. We generated new lines of transgenic mice that express htt mutated at S421 either to mimic tonic phosphorylation (S421D) or to prevent phosphorylation (S421A). In Aim 1, we will characterize any behavioral defects and neuropathology developed by these mice and compares them to unmodified HD mice and wildtype controls. Our preliminary results indicate that tonic phosphorylation significantly ameliorates mutant htt-evoked behavioral dysfunction. Mechanistically, phosphorylation often plays a regulatory role in proteins, and S421 is located in close proximity to several reported htt cleavage sites. This is potentially significat because cleavage of htt generally increases its toxicity by creating N-terminal fragments that are commonly found in the nucleus and in aggregates. Accordingly, we hypothesize that S421 phosphorylation alters the generation or accumulation of particularly toxic N-terminal fragments. Indeed, my preliminary data show that the cleavage pattern of htt in young mice is altered by S421 modification. The in-vivo significance of various N-terminal fragments, however, is controversial. In Aim 2, we will make a detailed comparison of the fragments generated by each S421 mutant line. Specifically, we will compare fragment generation and accumulation in the nucleus and cytoplasm of tissues that are and are not susceptible to htt toxicity. Additionally, we will perform these experiments at select time points as mice age and acquire the HD phenotype. In this manner, we will determine which unique fragments correlate with the onset of behavioral deficits, aggregate formation, and/or neurodegeneration across different tissues. Upon completion of this project, we will have validated S421 as a therapeutic target for HD. We also will have determined whether phosphorylation is related to the selective neuropathology in HD and whether particular N-terminal fragments are most associated with pathology and/or mutant protein aggregation.