Pilocarpine-induced status epilepticus (SE), which results in temporal lobe epilepsy (TLE) in rodents, activates the JAK/STAT pathway. In the current study, we evaluate whether brief exposure to a selective inhibitor of the JAK/STAT pathway (WP1066) early after the onset of SE effects the severity of SE or reduces later spontaneous seizure frequency via inhibition of STAT3-regulated gene transcription. Rats that received systemic WP1066 or vehicle at the onset of SE were continuously video-EEG monitored during SE and for one month to assess seizure frequency over time. Protein and/or mRNA levels for pSTAT3, and STAT3-regulated genes including: ICER, Gabra1, c-myc, mcl-1, cyclin D1, and bcl-xl were evaluated in WP1066 and vehicle-treated rats during stages of epileptogenesis to determine the acute effects of WP1066 administration on SE and chronic epilepsy. WP1066 (two 50 mg/kg doses) administered within the first hour after onset of SE results in transient inhibition of pSTAT3 and long-term reduction in spontaneous seizure frequency WP1066 alters the severity of chronic epilepsy without affecting SE or cell death. Early WP1066 administration reduces known downstream targets of STAT3 transcription 24 hours after SE including cyclin D1 and mcl-1 levels, known for their roles in cell-cycle progression and cell survival, respectively. These findings uncover a potential effect of the JAK/STAT pathway after brain injury that is physiologically important and may provide a new therapeutic target that can be harnessed for the prevention of epilepsy development and/or progression.

Synaptic inhibition in the adult brain is primarily mediated by the γ-aminobutyric acid (GABA) type A receptor (GABAAR). The distribution, properties, and dynamics of these receptors are largely determined by their subunit composition. Alteration of subunit composition after a traumatic brain injury (TBI) may result in abnormal increased synaptic firing and possibly contribute to injury-related pathology. Several studies have shown that the Janus Kinase/Signal Transducer and Activator of Transcription (JAK/STAT) signaling pathway can alter GABAAR subunit expression. The present study investigated changes in JAK/STAT pathway activation after two different severities of experimental TBI in the mouse using the controlled cortical impact (CCI) model. It also investigated whether modulating the activation of the JAK/STAT pathway after severe controlled cortical impact (CCI-S) with a JAK/STAT inhibitor (WP1066) alters post-traumatic epilepsy development and/or neurological recovery after injury. Our results demonstrated differential changes in both the activation of STAT3 and the expression of the GABAAR α1 and γ2 subunit levels that were dependent on the severity of the injury. The change in the GABAAR α1 subunit levels appeared to be at least partly transcriptionally mediated. We were able to selectively reverse the decrease in GABAAR α1 protein levels with WP1066 treatment after CCI injury. WP1066 treatment also improved the degree of recovery of vestibular motor function after injury. These findings suggest that the magnitude of JAK/STAT pathway activation and GABAAR α1 subunit level decrease is dependent on injury severity in this mouse model of TBI. In addition, reducing JAK/STAT pathway activation after severe experimental TBI reverses the decrease in the GABAAR α1 protein levels and improves vestibular motor recovery.

Brain-derived neurotrophic factor (BDNF) levels are elevated after status epilepticus (SE), leading to activation of multiple signaling pathways, including the janus kinase/signal transducer and activator of transcription pathway that mediates a decrease in GABA A receptor ␣1 subunits in the hippocampus (Lund et al., 2008). While BDNF can signal via its pro or mature form, the relative contribution of these forms to signaling after SE is not fully known. In the current study, we investigate changes in proBDNF levels acutely after SE in C57BL/6J mice. In contrast to previous reports (Unsain et al., 2008; Volosin et al., 2008; VonDran et al., 2014), our studies found that levels of proBDNF in the hippocampus are markedly elevated as early as 3 h after SE onset and remain elevated for 7 d. Immunohistochemistry studies indicate that seizure-induced BDNF localizes to all hippocampal subfields, predominantly in principal neurons and also in astrocytes. Analysis of the proteolytic machinery that cleaves proBDNF to produce mature BDNF demonstrates that acutely after SE there is a decrease in tissue plasminogen activator and an increase in plasminogen activator inhibitor-1 (PAI-1), an inhibitor of extracellular and intracellular cleavage, which normalizes over the first week after SE. In vitro treatment of hippocampal slices from animals 24 h after SE with a PAI-1 inhibitor reduces proBDNF levels. These findings suggest that rapid proBDNF increases following SE are due in part to reduced cleavage, and that proBDNF may be part of the initial neurotrophin response driving intracellular signaling during the acute phase of epileptogenesis.Key words: BDNF; cleavage; epilepsy; PAI-1; ProBDNF; tPA Significance StatementThe studies reported here are the first to demonstrate acute changes in the expression of proBDNF within 3 h of the onset of status epilpticus (SE) that occur within principle cells and glia in all hippocampal subfields. We further found evidence that reduced expression of tissue plasminogen activator, part of the extracellular proteolytic cascade, and increased expression of plasminogen activator inhibitor-1, an inhibitor of extracellular and intracellular cleavage, may contribute to reduced proBDNF cleavage and elevations in proBDNF levels. These findings suggest that proBDNF may be part of the initial neurotrophin response driving intracellular signaling acutely after SE and during the earliest phase of epileptogenesis.

In this study, we analyzed the impact that spontaneous seizures might have on the plasma membrane expression, composition and function of GABAA receptors (GABAARs). For this, tissue of chronically epileptic rats was collected within 3 hours of seizure occurrence (≤3 hours group) or at least 24 hours after seizure occurrence (≥24 hours group). A retrospective analysis of seizure frequency revealed that selecting animals on the bases of seizure proximity also grouped animals in terms of overall seizure burden with a higher seizure burden observed in the ≤3 hours group. A biochemical analysis showed that although animals with more frequent/recent seizures (≤3 hours group) had similar levels of GABAAR at the plasma membrane they showed deficits in inhibitory neurotransmission. In contrast, tissue obtained from animals experiencing infrequent seizures (≥24 hours group) had increased plasma membrane levels of GABAAR and showed no deficit in inhibitory function. Together, our findings offer an initial insight into the molecular changes that might help to explain how alterations in GABAAR function can be associated with differential seizure burden. Our findings also suggest that increased plasma membrane levels of GABAAR might act as a compensatory mechanism to more effectively maintain inhibitory function, repress hyperexcitability and reduce seizure burden. This study is an initial step towards a fuller characterization of the molecular events that trigger alterations in GABAergic neurotransmission during chronic epilepsy.

Summary Objective Temporal lobe epilepsy (TLE) is frequently medically intractable and often progressive. Compromised inhibitory neurotransmission due to altered GABAA receptor α4 (GABARα4) subunit expression has been emphasized as a potential contributor to the initial development of epilepsy following a brain insult (primary epileptogenesis), but the regulation of GABARα4 during chronic epilepsy, specifically, how expression is altered following spontaneous seizures, is less well understood. Methods Continuous video-EEG recordings from rats with pilocarpine-induced TLE were used to capture epileptic animals within 3 hours of a spontaneous seizure (SS) or greater than 24 hours after the last SS to determine whether recent occurrence of a seizure was associated with altered levels of GABARα4 subunit expression. We further evaluated whether this GABARα4 subunit plasticity is regulated by signaling mechanisms active in primary epileptogenesis, specifically, increases in brain-derived neurotrophic factor (BDNF) and early growth response factor 3 (Egr3). Results Elevated levels of GABARα4 subunit mRNA and protein were observed following spontaneous seizures, and were associated with higher levels of BDNF and Egr3 mRNA Significance These data suggest that spontaneous, recurrent seizures that define chronic epilepsy may influence changes in GABARα4 subunit expression, and that signaling pathways known to regulate GABARα4 expression after SE may also be activated after spontaneous seizures in chronically epileptic animals.