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Name Dr. Hamad Yadikar
Organization or Institution University of Florida
Presentation Type Poster
Topic Biochemistry / Chem Bio.
Title

testing the effect of protein kinase inhibitors on traumatic brain injury-relevant cell-based model of tau hyperphosphorylation

Author(s)

Hamad Yadikar1,2, Isabel Torres1,2, Richard Yost4, Kevin K. Wang1,2,3

Author Institution(s)

Program for Neurotrauma, Neuroproteomics & Biomarkers Research1, The Departments of Psychiatry2 and Neuroscience3, University of Florida, Gainesville FL 32611
Department of Chemistry4, University of Florida, Gainesville FL 32611

Abstract

Traumatic brain injury (TBI) increases the subsequent risk of chronic neurodegenerative disorders, including Alzheimer disease and chronic traumatic encephalopathy (CTE). Tauopathies are neurodegenerative disorders that are related to the abnormal aggregation of hyperphosphorylated Tau proteins into oligomers. It was found in both human and animal model of TBI, Tau is also hyperphosphorylated post-injury. However, the biological mechanism through which Tau get hyperphosphorylated need further elucidation. In this study, okadiac acid (OA) (PP1/PP2A inhibitor) was used to induce Tau hyperphosphorylation and oligomerization as model for TBI. The objective was to setup a screening method for studying various protein kinase inhibitors on Tau hyperphosphorylation using rat primary cortical culture (CTX) and mouse neuroblastoma (N2A) cells. The inhibitors used were pan kinase inhibitors (k252a and STS), GSK3b inhibitors (LiCl, AR-A014418 and A-1070722), CDK5 inhibitor (Roscovitine), Calcineurin inhibitor (Cyclosporin), Calmodulin antagonist (Calmidazolium), Casein kinase II inhibitor (tTBB), fyn/src kinase inhibitor (Saracatinib) and calcium chelators (EGTA and EDTA). Treatment of OA for 24 hours achieved Tau hyperphosphorylation at multiple TBI-related phospho-epitopes such as pSer202/pThr205, pThr181, pSer202, pSer396/pSer404 and pThr231. The phosphorylation and oligomerization of Tau proteins was studied by Western blotting and identified by antibodies that detects total Tau at 102-140 kDa (DA9). In N2A cells, treatment with OA lead to the formation of Tau oligomers observed at ~ 170 kDa, which were inhibited when cells were pre-treated with tTBB. The hyperphosphorylation was partially blocked by EDTA, EGTA and k252a and completely inhibited by tTBB and cyclosporine. In CTX cells, LiCl, AR-A014418 and A-1070722 were the most potent inhibitors of Tau hyperphosphorylation followed by tTBB and roscovitine. Identification of protein kinases that inhibit Tau hyperphosphorylation in TBI can form the basis for screening of compounds and could provide potential therapeutic targets for the treatment of tauopathy-related neurodegenerative diseases.

 

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