Mechanisms of disease-modifying effect of saracatinib (AZD0530), a Src/Fyn tyrosine kinase inhibitor, in the rat kainate model of temporal lobe epilepsy.

We have recently demonstrated the role of the Fyn-PKCδ signaling pathway in status epilepticus (SE)-induced neuroinflammation and epileptogenesis in experimental models of temporal lobe epilepsy (TLE). In this study, we show a significant disease-modifying effect and the mechanisms of a Fyn/Src tyrosine kinase inhibitor, saracatinib (SAR, also known as AZD0530), in the rat kainate (KA) model of TLE. SAR treatment for a week, starting the first dose (25 mg/kg, oral) 4 h after the onset of SE, significantly reduced spontaneously recurring seizures and epileptiform spikes during the four months of continuous video-EEG monitoring. Immunohistochemistry of brain sections and Western blot analyses of hippocampal lysates at 8-day (8d) and 4-month post-SE revealed a significant reduction of SE-induced astrogliosis, microgliosis, neurodegeneration, phosphorylated Fyn/Src-419 and PKCδ-tyr311, in SAR-treated group when compared with the vehicle control. We also found the suppression of nitroxidative stress markers such as iNOS, 3-NT, 4-HNE, and gp91phox in the hippocampus, and nitrite and ROS levels in the serum of the SAR-treated group at 8d post-SE. The qRT-PCR (hippocampus) and ELISA (serum) revealed a significant reduction of key proinflammatory cytokines TNFα and IL-1β mRNA in the hippocampus and their protein levels in serum, in addition to IL-6 and IL-12, in the SAR-treated group at 8d in contrast to the vehicle-treated group. These findings suggest that SAR targets some of the key biomarkers of epileptogenesis and modulates neuroinflammatory and nitroxidative pathways that mediate the development of epilepsy. Therefore, SAR can be developed as a potential disease-modifying agent to prevent the development and progression of TLE.

Sharma S ，Carlson S ，Gregory-Flores A ，Hinojo-Perez A ，Olson A ，Thippeswamy T ... -  《-》

Role of the Fyn-PKCδ signaling in SE-induced neuroinflammation and epileptogenesis in experimental models of temporal lobe epilepsy.

Status epilepticus (SE) induces neuroinflammation and epileptogenesis, but the mechanisms are not yet fully delineated. The Fyn, a non-receptor Src family tyrosine kinase (SFK), and its immediate downstream target, PKCδ are emerging as potential mediators of neuroinflammation. In order to first determine the role of Fyn kinase signaling in SE, we tested the efficacy of a SFK inhibitor, saracatinib (25mg/kg, oral) in C57BL/6J mouse kainate model of acute seizures. Saracatinib pretreatment dampened SE severity and completely prevented mortality. We further utilized fyn-/- and fyn+/+ mice (wildtype control for the fyn-/- mice on same genetic background), and the rat kainate model, treated with saracatinib post-SE, to validate the role of Fyn/SFK in SE and epileptogenesis. We observed significant reduction in SE severity, epileptiform spikes, and electrographic non-convulsive seizures in fyn-/- mice when compared to fyn+/+ mice. Interestingly, significant reductions in phosphorylated pSrc-416 and PKCδ (pPKCδ-507) and naive PKCδ were observed in fyn-/- mice as compared to fyn+/+ mice suggesting that PKCδ signaling is a downstream mediator of Fyn in SE and epileptogenesis. Notably, fyn-/- mice also showed a reduction in key proinflammatory mediators TNF-α, IL-1β, and iNOS mRNA expression; serum IL-6 and IL-12 levels; and nitro-oxidative stress markers such as 4-HNE, gp91phox, and 3-NT in the hippocampus. Immunohistochemistry revealed a significant increase in reactive microgliosis and neurodegeneration in the hippocampus and hilus of dentate gyrus in fyn+/+ mice in contrast to fyn-/- mice. Interestingly, we did not observe upregulation of Fyn in pyramidal neurons of the hippocampus during post-SE in fyn+/+ mice, but it was upregulated in hilar neurons of the dentate gyrus when compared to naïve control. In reactive microglia, both Fyn and PKCδ were persistently upregulated during post-SE suggesting that Fyn-PKCδ may drive neuroinflammation during epileptogenesis. Since disabling the Fyn kinase prior to SE, either by treating with saracatinib or fyn gene knockout, suppressed seizures and the subsequent epileptogenic events, we further tested whether Fyn/SFK inhibition during post-SE modifies epileptogenesis. Telemetry-implanted, SE-induced, rats were treated with saracatinib and continuously monitored for a month. At 2h post-diazepam, the saracatinib (25mg/kg) or the vehicle was administered orally and repeated twice daily for first three days followed by a single dose/day for the next four days. The saracatinib post-treatment prevented epileptogenesis in >50% of the rats and significantly reduced spontaneous seizures and epileptiform spikes in the rest (one animal did not respond) when compared to the vehicle treated group, which had >24 seizures in a month. Collectively, the findings suggest that Fyn/SFK is a potential mediator of epileptogenesis and a therapeutic target to prevent/treat seizures and epileptogenesis.

Sharma S ，Carlson S ，Puttachary S ，Sarkar S ，Showman L ，Putra M ，Kanthasamy AG ，Thippeswamy T ... -  《-》

1400W, a highly selective inducible nitric oxide synthase inhibitor is a potential disease modifier in the rat kainate model of temporal lobe epilepsy.

Status epilepticus (SE) initiates epileptogenesis to transform normal brain to epileptic state which is characterized by spontaneous recurrent seizures (SRS). Prior to SRS, progressive changes occur in the brain soon after SE, for example, loss of blood-brain barrier (BBB) integrity, neuronal hyper-excitability (epileptiform spiking), neuroinflammation [reactive gliosis, high levels of reactive oxygen/nitrogen species (ROS/RNS)], neurodegeneration and synaptic re-organization. Our hypothesis was that modification of early epileptogenic events will alter the course of disease development and its progression. We tested the hypothesis in the rat kainate model of chronic epilepsy using a novel disease modifying drug, 1400W, a highly selective inhibitor of inducible nitric oxide synthase (iNOS/NOS-II). In an in vitro mouse brain slice model, using a multi-electrode array system, co-application of 1400W with kainate significantly suppressed kainate-induced epileptiform spiking. In the rats, in vivo, 4h after the induction of SE with kainate, 1400W (20mg/kg, i.p.) was administered twice daily for three days to target early events of epileptogenesis. The rats were subjected to continuous (24/7) video-EEG monitoring, remotely, for six months from epidurally implanted cortical electrodes. The 1400W treatment significantly reduced the epileptiform spike rate during the first 12-74h post-SE, which resulted in >90% reduction in SRS in long-term during the six month period when compared to the vehicle-treated control group (257±113 versus 19±10 episodes). Immunohistochemistry (IHC) of brain sections at seven days and six months revealed a significant reduction in; reactive astrogliosis and microgliosis (M1 type), extravascular serum albumin (a marker for BBB leakage) and neurodegeneration in the hippocampus, amygdala and entorhinal cortex in the 1400W-treated rats when compared to the vehicle control. In the seven day group, hippocampal Western blots revealed downregulation of inwardly-rectifying potassium (Kir 4.1) channels and glutamate transporter-1 (GLT-1) levels in the vehicle group, and 1400W treatment partially reversed Kir 4.1 levels, however, GLT-1 levels were unaffected. In the six month group, a significant reduction in mossy fiber staining intensity in the inner molecular layer of the dentate gyrus was observed in the 1400W-treated group. Overall these findings demonstrate that 1400W, by reducing the epileptiform spike rate during the first three days of post-insult, potentially modifies epileptogenesis and the severity of chronic epilepsy in the rat kainate model of TLE.

Puttachary S ，Sharma S ，Verma S ，Yang Y ，Putra M ，Thippeswamy A ，Luo D ，Thippeswamy T ... -  《-》

The effects of Src tyrosine kinase inhibitor, saracatinib, on the markers of epileptogenesis in a mixed-sex cohort of adult rats in the kainic acid model of epilepsy.

Neurodegeneration and neuroinflammation are key processes of epileptogenesis in temporal lobe epilepsy (TLE). A considerable number (∼30%) of patients with epilepsy are resistant to currently available antiseizure drugs and thus there is a need to develop adjunct therapies to modify disease progression. A vast majority of interventional strategies to treat TLE have utilized males which limits the translational nature of the studies. In this study, we investigated the effects of repeated low-dose kainic acid (KA) injection on the initial status epilepticus (SE) and the effects of Src kinase inhibitor, saracatinib (SAR/AZD0530; 20 mg/kg, oral, daily for 7 days), in a mixed-sex cohort of adult Sprague Dawley rats during early epileptogenesis. There were no sex differences in response to KA-induced SE, and neither did the stage of estrus influence SE severity. KA-induced SE caused significant astrogliosis and microgliosis across the hippocampus, piriform cortex, and amygdala. SAR treatment resulted in a significant reduction of microgliosis across brain regions. Microglial morphometrics such as branch length and the endpoints strongly correlated with CD68 expression in the vehicle-treated group but not in the SAR-treated group, indicating mitigation by SAR. KA-induced SE caused significant neuronal loss, including parvalbumin-positive inhibitory neurons, in both vehicle (VEH) and SAR-treated groups. SAR treatment significantly mitigated FJB-positive neuronal counts as compared to the VEH group. There was an increase in C3-positive reactive astrocytes in the VEH-treated group, and SAR treatment significantly reduced the increase in the piriform cortex. C3-positive astrogliosis significantly correlated with CD68 expression in the amygdala (AMY) of VEH-treated rats, and SAR treatment mitigated this relationship. There was a significant increase of pSrc(Y419)-positive microglia in both KA-treated groups with a statistically insignificant reduction by SAR. KA-induced SE caused the development of classical glial scars in the piriform cortex (PIR) in both KA-treated groups, while SAR treatment led to a 42.17% reduction in the size of glial scars. We did not observe sex differences in any of the parameters in this study. SAR, at the dose tested in the rat kainate model for a week in this study mitigated some of the markers of epileptogenesis in both sexes.

Rao NS ，Putra M ，Meyer C ，Almanza A ，Thippeswamy T ... -  《Frontiers in Molecular Neuroscience》

Post-status epilepticus treatment with the Fyn inhibitor, saracatinib, improves cognitive function in mice.

Luo XM ，Zhao J ，Wu WY ，Fu J ，Li ZY ，Zhang M ，Lu J ... -  《BMC NEUROSCIENCE》