Abstracts

Rationale: Tau protein hyperphosphorylation (p-tau) has been linked to neurodegenerative diseases following brain injury, including post-traumatic epilepsy (PTE). Identification of p-tau sites implicated in PTE may provide insight on kinase pathways that could be targeted for therapeutic interventions for PTE. In this EpiBioS4Rx Project 2 study, we investigated the site-specific and temporal patterns of p-tau forms after lateral fluid percussion injury (LFPI) induced brain trauma in adult male rats using antibodies to detect expression of p-tau in different sites, at Ser202/Thr205 (AT8) and Thr231 (AT180), total tau (tau5), as well as in the subunit PR55 of protein phosphatase 2 (PP2A). Methods: Male 11-week old Sprague-Dawley rats were used as naïve (n=11), sham (n=5-6/timepoint) and LFPI (n=5-6/timepoint) groups in each EpiBioS4Rx center. A left parietal 5mm craniotomy was performed in sham and LFPI rats. LFPI rats were subjected to ~3.1 atm pulse to induce brain injury. The brains were either perfused for immunohistochemistry (IHC) or flash frozen for Western blot (WB) analysis, at 2 days, 1, 2, 4 or 8 weeks post-surgery. Protein expression was assessed by IHC or WB using AT8, AT180, or anti-PP2A/PR55 antibodies. In the IHC studies, somatic and extrasomatic/extracellular signal densitometry of AT8, AT180, or PR55 immunoreactivity (-ir) was done at the ipsi- and contralateral primary motor (M1) and lateral [somatosensory (S2a, S2b); granular insular (GI)] cortices]. For WB, the ratio of AT8-ir/tau5-ir and PR55-ir to the loading control glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was calculated and the results were expressed as relative of the average sham value.  Results: In the IHC studies, we found increases in both somatic and extrasomatic/extracellular AT8-ir in the left M1 (p=0.01) and lateral cortices (p<0.05) of LFPI rats only at 2 days compared to all other groups. Somatic AT180-ir was increased after 8 weeks post-LFPI in the left S2a, S2b and GI (p<0.05) compared to naïve. In the WB studies, we found that AT8/tau5 ratio was increased in LFPI rats in the perilesional cortex at 2 days (p=0.03) and 8 weeks post-LFPI (p=0.03). In contrast, PR55-ir was not modified in either IHC or WB studies. Conclusions: We observed different patterns of p-tau sites post-LFPI: an early (2 days) focal increase in both somatic and extracellular AT8-ir, by both IHC and WB; and a late (8 weeks) increase in somatic AT180-ir, by IHC and AT8-ir, by WB in the ipsilateral to LFPI cortex. Our findings reveal site and time-specific p-tau changes post LFPI. The AT8 and AT180 p-tau sites may therefore be promising targets for the design of antiepileptogenic therapies following brain trauma.  Funding: NINDS U54 NS100064, NS091170, and US Department of Defense (W81XWH-13-1-0180)