Aquaporin-4 (AQP4) is the glial water channel that is primarily expressed in astrocytes. Various roles of AQP4 have been previously discussed such as deletion of AQP4 induces enlarged extracellular space. However, the role of AQP4 in brain water diffusivity is not well described. In this study, we investigated the developmental changes of brain water diffusivity in relation to development and presence of AQP4 channels in AQP4+/+ and AQP4-/- animals using diffusion tensor imaging (DTI) and brain water content analyses. DTI results revealed a developmental decrease in water diffusivity in both AQP4+/+ and AQP4-/- genotypes. AQP4-/- mice exhibited an increased diffusivities as juveniles but later deceased in adulthood. Brain water content analysis revealed greater water content in AQP4-/- mice, which suggests the effect of enlarged extracellular space resulting in greater basal water content. The results highlight the role of AQP4 channels in the regulation and mediation of brain water mobility.

Posttraumatic epilepsy (PTE) is a long-term negative consequence of traumatic brain injury (TBI) in which recurrent spontaneous seizures occur after the initial head injury. PTE develops over an undefined period where circuitry reorganization in the brain causes permanent hyperexcitability. Unfortunately, current existing antiepileptogenic drugs (AEDs) have all failed at treating PTE, and thus, there is a critical need to identify biomarkers of PTE to ultimately develop new therapeutic strategies. The pathophysiology by which trauma leads to spontaneous seizures is unknown and clinically relevant models of PTE are key to understanding the molecular and cellular mechanisms underlying the development of PTE. Current animal studies of PTE are limited and comprehensive in vivo electrophysiological approaches remain absent. In the present study, I aimed to identify optical and electrographic biomarkers of PTE with correlation to hippocampal histopathology at 14, 30, 60, and 90 days post injury (dpi). Here, adult male CD1 wildtype (WT) and aquaporin-4 knockout (AQP4 KO) mice were subjected to a moderate-severe TBI in the right frontal cortex using the well-established controlled cortical impact (CCI) injury model. Additionally, mice underwent optical coherence tomography (OCT) imaging, in vivo video-electroencephalographic (vEEG) recordings, and immunohistochemistry and Western blot analysis for the key epileptogenic astrocytic channels AQP4 and Kir4.1. The main findings from these studies are: 1) successful implementation of CCI-based PTE in mice with chronic vEEG generated, for the first time,17% and 27% of WT and AQP4 KO mice with PTE, respectively (the highest yield of PTE reported); 2) AQP4 KO mice had a greater incidence of spontaneous seizures and PTE compared with WT mice; 3) AQP4 KO mice had longer spontaneous seizure duration compared with WT mice; 4) EEG power patterns are different between mice with and without PTE; and 5) AQP4, but not Kir4.1, is significantly upregulated in the frontal cortex and hippocampus of mice with PTE. Collectively, these findings identified specific PTE EEG phenotypes that may be modulated by AQP4 and carry significant implications for epileptogenesis after TBI which may serve as the first steps to developing surrogate biomarkers for PTE.

Epilepsy is one of the most common neurological disorders and is characterized by the occurrence of unprovoked seizures. Temporal lobe epilepsy (TLE) is the most common form of epilepsy with focal seizures. Unfortunately, some patients will develop refractory epilepsy that is pharmaco-resistant to current antiepileptic drugs (AEDs). Current AEDs work primarily by targeting neurons directly by inhibition of glutamatergic excitatory neurotransmission or enhancement of GABAergic inhibitory neurotransmission. Non-neuronal targets are an attractive alternative approach to treat epilepsy with potentially fewer deleterious effects. Neuronal hyperexcitability is a major contributor to epilepsy but increased evidence suggests that changes in astrocytic glutamate transporters can contribute to the development of epilepsy. This proposal aims to examine the cellular and molecular mechanisms associated with glutamate transporter dysregulation and their potential as a therapeutic target in epilepsy. I hypothesize that astrocytic glutamate transporter dysregulation contributes to the development of epilepsy and therefore can be targeted for the attenuation of epilepsy. Changes in astrocytic glutamate transporters were evaluated post-kainate induced status epilepticus. Additionally, post-translational modifications (PTMs) of these transporters that have previously been determined to cause both mislocalization and dysfunction of glutamate transporters in other models of neurological disease including SUMOylation, ubiquitination and palmitoylation were examined. For the first time, whether glutamate transporter modulation reduces seizures and attenuates pathological changes observed in the IHKA model of TLE using an AAV-Gfa2-GLT1-cHA viral vector and neuregulin (NRG-1) treatment was investigated. Finally, real-time glutamate spike activity to identify whether marked glutamate spike patterns can be used to predict epileptiform activity in epileptogenesis was examined. The main findings from these studies are: 1.) Synaptosomal GLT-1 protein is downregulated at a critical time point in epileptogenesis; 2.) Overexpression of GLT-1 in astrocytes delays neuronal death and granular cell dispersion in epileptogenesis; 3.) Overexpression of GLT-1 suppresses electrographic seizures and large behavioral seizures in epileptogenesis; 4.) Exogenous NRG-1 treatment induces upregulation of glutamate transporter EAAC1 and bi-hemispheric neuroprotection in epileptogenesis; and 5.) Glutamate peak events are increased in the epileptic brain and could be used as a biomarker in TLE.

Epilepsy is a group of chronic neurological disorders characterized by abnormally synchronized activity among neurons that presents as seizures. It is a major health problem that is estimated to affect 1 in 26 people during their lifetime. Many antiepileptic drugs (AEDs) currently exist, but approximately 30% of patients taking AEDs cannot control their seizures with drugs alone. In addition, adverse effects such as cognitive impairment are common. This may be because current AEDs act as central nervous system depressants and target neuronal channels to control tissue excitability. Therefore, new drugs based on non-neuronal targets may serve as novel therapeutic strategies with fewer deleterious effects. Astrocytes maintain glutamate and water homeostasis primarily through glutamate transporters and aquaporin-4 (AQP4), respectively. The two astrocyte-specific glutamate transporters are glutamate transporter-1 (GLT1) and glutamate aspartate transporter (GLAST), of which GLT1 is responsible for the majority of glutamate uptake in the forebrain. Alterations in both glutamate and water homeostasis have powerful effects on excitability, but the regulation of GLT1, GLAST, and AQP4 in epilepsy is not well understood. Furthermore, the β-lactam antibiotic ceftriaxone has previously been shown to upregulate GLT1 expression, but its efficacy in a chronic epilepsy model has not been well studied. Here I describe AQP4, GLT1, and GLAST hippocampal expression changes in a model of epilepsy. I begin by characterizing the intrahippocampal kainic acid (IHKA) model of epilepsy, focusing on astrocyte reactivity during the development of epilepsy (epileptogenesis). I then describe the highly polarized AQP4 expression in the healthy brain and show reduced hippocampal levels during epileptogenesis. After that, I discuss alterations of GLT1 levels after IHKA-induced epileptogenesis. Next, I discuss the lack of effect of β-lactam drugs on regulating AQP4 and GLT1 expression, thereby eliminating it as a potential antiepileptic therapeutic. I then describe the co-localization patterns of AQP4 and GLT1; these two proteins do not co-immunoprecipitate, suggesting the lack of a strong interaction between them. In the penultimate chapter, I briefly describe the minimal changes in GLAST expression during epileptogenesis. I conclude with a summary of my findings and suggested future directions for this line of research.