Identification of cellular signaling events dysregulated in Huntington’s disease.
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Huntington’s disease (HD) is a fatal neurodegenerative disorder resulting from a CAG repeat expansion in the first exon of the gene encoding the Huntingtin protein (Htt) with physical, emotional, and cognitive symptoms. Current standard-of-care regimens for HD are limited to symptom-mitigating therapies with little potential for increasing the overall quality of life. As such, there is an imminent need for the development of more effective treatment options, efforts for which are enabled by a greater understanding of the molecular basis of disease initiation, progression, and pathology. Alterations in numerous signal transduction pathways in HD result from aberrant kinase signaling. Protein phosphorylation is catalyzed by a class of enzymes called kinases, the cellular complement of which is referred to as its kinome. The kinases responsible for driving the fate of phosphoproteomes are central to elucidating various complex cellular events. The interactive capacity of the phosphate group makes the phosphorylated protein versatile in communicating. The study of kinome led to the development of a high throughput screening tool, peptide arrays. The arrays were exposed to lysates from cells / tissues where in the kinases from them phosphorylate the peptide spotted on the arrays. The degree of phosphorylation is measured for each spot on the array and compared to the controls thereby determining the upregulation or downregulation of signaling pathways in response to different biological treatments or conditions. The online tools used were a data analysis pipeline, Platform for Integrated, Intelligent Kinome Analysis-2 (PIIKA 2), and pathway analysis pipeline InnateDB. The kinases regulating the significantly (de)phosphorylating peptides were predicted through an online tool NetworKIN which utilized the output from PIIKA 2. Peptide arrays were utilized to identify the dysregulated kinase signaling in a) Neural stem cells (NSC) using a previously designed array with 298 peptides b) R6/2 HD mouse model across key developmental time points using customized arrays with 1268 peptides. In an effort to investigate disease-associated changes in signal transduction activity, global patterns of kinase activity (kinome analysis) were characterized within a NSC line derived from a patient with a confirmed diagnosis of HD. As indicated by kinome analysis and independently verified by phosphorylation-specific antibodies, cytoskeletal signaling, and in particular LIMK1/cofilin/slingshot signaling, was dysregulated in HD NSC’s. GSK3β was reported as a major upstream kinase potentially activated in the HD NSCs by NetworKIN analysis, an online tool. These changes in cytoskeletal associated signaling align with differences in dendrite formation between NSCs from HD and age-/sex-matched healthy controls (HC). Dendrites in the HD NSCs were 25% shorter relative to dendrites in control NSCs. The peptide array technique was then applied to R6/2 HD mouse model using the lysates from 8 key developmental time points (Embryonic 9 and 14; at birth; weeks 3, 4, 5, 7, 10) from both sexes. The subsequent confirmation of PIIKA 2 enhanced data transformation followed by pathway analysis revealed cytoskeletal dynamics as significantly dysregulated temporally. Changes in upstream regulators ROCK2 and PAK were prominent in the embryonic time points and LIMK1/cofilin/slingshot along with profilin showed alterations in the later time points especially the 3w and 4w, when the mutant huntingtin protein (mHtt) appears in the striatum the most affected cell type in HD brain. Collectively, these data highlight the potential role of cytoskeletal dynamics in HD pathology and shows that the targeted modulation of these signaling molecules may confer therapeutic benefit.
DegreeDoctor of Philosophy (Ph.D.)
SupervisorRoesler, William J
CommitteeLee, Jeremy S; Mousseau, Darrell D; Lukong, Kevin E; Wilson, Heather L
Copyright DateAugust 2019
Huntington disease, Kinome analysis