Authors :
Presenting Author: Nikhil S. Rao, D.V.M., M.V.Sc. – Iowa State University
Marson Putra, MD, PhD. – Iowa State University; Christina Meyer, BS, MS – Iowa State University; Madison Van Otterloo, BS – Iowa State University; Thimmasettappa Thippeswamy, DVM, MVSc, PhD, FAES – Iowa State University
Rationale:
Organophosphates (OPs) are potent inhibitors of acetylcholinesterase that can induce status epilepticus (SE) by overstimulating cholinergic receptors, leading to epileptic phenotypes and cognitive impairment. A Src family tyrosine kinase (SFK), fyn, and tau have been implicated in mediating neuronal hyperexcitation and neurodegeneration in seizure-related disorders of epilepsy and Alzheimer’s disease (AD). However, the mechanism of excitotoxicity mediated by Fyn-tau interactions in OP model of epilepsy is unknown. In this study, we investigated the effects of diisopropylfluorophosphate (DFP)-induced seizures and neurotoxicity on Fyn-tau interactions and its downstream signaling pathways in the rat hippocampus and piriform cortex.
Methods:
Adult SD male rats were challenged with DFP (4 mg/kg, s.c.) and immediately treated with atropine sulfate (2mg/kg, i.m) and 2-PAM (25mg/kg) Animals were allowed to seize for one hour before administration of Midazolam (3 mg/kg, i.m.) to terminate behavioral seizures. Eight days post-DFP, the animals were sacrificed. We used proximity ligation assay (PLA) to identify the extent of Fyn-tau interactions, immunohistochemistry and Western blotting to measure hyperphosphorylated tau (pTau), phosphorylated SFK/Fyn (pSFK), phosphorylated NR2B (pNR2B), total NR2B, nNOS, total Fyn and total Tau levels in the PSD fraction of synaptosomes in the hippocampus and piriform cortex.
Results:
We found increased number of Fyn-tau association in PLA in the hippocampus and piriform cortex of DFP treated rats compared to controls. Immunohistochemically, we noted a significant increase in pTau (pY18) intensity in the hippocampus and piriform cortex of DFP treated rats compared to control. Moreover, we also observed a significant upregulation in pSFK (pY416) intensity in both these brain regions in DFP relative to control. In the hippocampal post-synaptic density (PSD) fraction, we noted significant increases in pSFK (pY416) and pTau (AT8) levels in DFP exposed versus control, indicating enhanced Fyn activation and tau hyperphosphorylation at the post-synaptic compartments. We also showed relatively elevated levels of post-synaptic nNOS and pNR2B in DFP-exposed rats, however, the differences were not statistically significant from controls.
Conclusions:
We have previously demonstrated enhanced Fyn-tau interactions during the early phase of epileptogenesis in a pentylenetetrazol mouse model. The findings from the DFP model of OPNA toxicity suggest a common pathomechanism by which seizures facilitate neurotoxicity. Therefore, Fyn-tau interaction can be a potential therapeutic target to counter OPNA-induced excitotoxicity.
Funding:
This project is supported by the W.E. Lloyd Endowment fund (SG2200008).