Epilepsy is the third most common neurological disorder of humans worldwide and its lifetime prevalence is ~2% (>65 million people). About 10% of Americans experience a seizure during their lifetime and ~3% of them develop epilepsy by the time they are 80 years of age. In the US, epilepsy management costs $15.5 billion/ year. About 1/3 of patients do not respond to any AEDs. The majority of the AEDs that do not cure epilepsy are ion-channel targeted drugs. This suggests the need for development of drugs that target alternative pathways to cure epilepsy. This is a major unmet clinical obligation due to poor understanding of the mechanisms of epileptogenesis. Neuroinflammation is emerging as a mechanistic target for drug development for temporal lobe epilepsy (TLE), the most common type of epilepsy associated with cognitive impairment. Recently, we demonstrated the upregulation of Src family kinase Fyn, PKC?, and gp91phox in the hippocampus during epileptogenesis in the mouse and rat kainate (KA) models of TLE. The PKC? activates NADPH oxidase (NOX2) by phosphorylating the cytosolic p47phox subunit, which forms a functional NOX2 complex with the membrane-associated gp91phox. This drives the production of ROS/RNS and activates NF-kB in microglia to initiate neuroinflammation, which is known to reduce seizure threshold. Therefore, our overarching hypothesis is that Fyn, the upstream of PKC?-NOX2-NFkB signaling pathway, is a potential therapeutic target for epileptogenesis/epilepsy. We propose to test the hypothesis in a rat model of TLE. KA-induced status epilepticus (SE) in rat causes epilepsy (spontaneous, recurrent seizures (SRS)) through a well-characterized process of neuroinflammation, nitro-oxidative stress, and neurodegeneration (?the disease promoters?). We will characterize the time-course activation of the Fyn-PKC?-NOX2 pathway components in microglia during epileptogenesis (Aim 1), and validate the role of Fyn kinase in epileptogenesis by utilizing microglia-specific rAAV-fyn-shRNA, and a novel Fyn/Src kinase inhibitor, saracatinib (SAR/AZD0530), in a rat model of TLE (Aim 2). SAR is blood-brain barrier permeable and has excellent bioavailability in the brain at a low dose. Its clinical tolerability and oral bioavailability has been demonstrated in phase IIa clinical trials for Alzheimer's disease, breast cancer and bone cancer patients. In our preliminary studies, SAR significantly reduced SE severity, SRS episodes, and prevented epileptogenesis (in >50% of the rats) in experimental models. We will determine antiepileptogenic effects of rAAV- fyn-shRNA and SAR (also anti-epileptic effects) in real-time by employing continuous video-EEG telemetry for 3 months, and the Morris water maze test to assess cognition in both male and female rat KA model of TLE. We will measure cytokines, ROS, and RNS from the serum and CSF, and the other disease promoters and the Fyn-PKC?-NOX2 components in different cell types (microglia, monocytes/macrophages, astrocytes, and neurons) in the hippocampus and cortex. The proposed project embodies a translational approach and it will yield a novel anti-epileptogenic and/or antiepileptic agent.
Epilepsy is a chronic debilitating and the third most common neurological disorder of humans and animals worldwide, and both male and female of all age groups are affected by epilepsy. The vast majority of the current antiepileptic drugs are ion-channel targeted, which do not effect in 1/3 of people with epilepsy and in the remaining 2/3 do not prevent the development of epilepsy or cure the disease. Therefore there is an unmet clinical need, which we will address by targeting the neuroinflammatory (Fyn/Src kinase) pathway in the rat kainate model of temporal lobe epilepsy, and we will measure the following outcomes: i) the time-course activation of the Fyn-PKC?-NOX2 pathway components during epileptogenesis; ii) validation of the role Fyn in epileptogenesis by utilizing microglia-specific fyn knockdown approach, and a novel pharmacological inhibitor of Fyn/Src kinase, saracatinib, in real-time by employing continuous (24/7) video-EEG monitoring for 3 months to quantify spontaneous seizures; iii) cognitive deficits versus recovery by intervention; and, iv) the reactive oxygen and nitrogen species, neuroinflammation (reactive gliosis, cytokines, and chemokines), and neurodegeneration.
