The Regulation of Glial Glutamate Transporters after in‐vitro Ischemic Stroke

Abstract

Stroke is the 3rd leading cause of death in the United States but adequate treatment is still lacking. Moreover, the pathological mechanisms involved are still not fully understood, highlighting the need for further studies. In this work, we employed an in vitro model of ischemic stroke to study the regulation of excitatory amino acid transporters (EAATs), which are key proteins that control the extracellular concentration of glutamate in the brain and thereby help maintain brain homeostasis. Glutamate dysregulation is a fundamental feature of many neurological disorders, leading to neuronal death through a mechanism known as excitotoxicity. This makes EAATs key players in mitigating glutamate imbalance in the brain and thereby have a major role in excitotoxic outcomes following ischemia. Our goal was to understand how the activity and expression of EAATs are modulated following different severities of ischemic insults. Glia cultures were subjected to oxygen‐glucose deprivation (OGD) for 30‐minutes, 1‐hour and 2‐hours. Our results demonstrated that increasing lengths of insult result in decreased levels of glutamate transport, suggesting an inability of these transporters to effectively clear excess glutamate. The expression of glial transporters EAAT1 and EAAT2 are differentially regulated: EAAT1 is downregulated after ischemia, with a more profound effect after 1‐hour OGD, whereas EAAT2 expression is up regulated after 30‐minutes and 1‐hour OGD before decreasing below control levels after 2‐hour OGD. These results suggest that these transporters may work together, likely as a compensatory mechanism. We also investigated the effects of Clavulanic Acid (CA), a compound that enhances EAAT2, which is the main subtype of EAAT in the CNS. CA pretreatment was only able to rescue EAAT activity and significantly increase EAAT2 levels in control conditions and after 30‐minutes of OGD, suggesting that this strategy only offers benefit in mild ischemic insults. Furthermore, we investigated reactive gliosis, which can be defined by the activation of glia cells in response to a pathology. Previous work has shown that reactive gliosis is a phenotype associated with ischemia, however we wanted to further define and evaluate this type of astrocytic response. In specific, we wanted to determine how reactive gliosis is affected following different ischemic insults. Results show that reactive gliosis, which was determined by an increased expression and fluorescence of GFAP, increases with OGD insult, with a significant difference seen after 2‐hours of OGD. This suggests the reactive phenotype is more prominent as the severity of ischemia increases. These findings advanced our knowledge on EAAT regulation following different severities of ischemic stroke in‐vitro. Future studies will investigate potential mechanisms associated with their differential regulation, such as transport reversal, internalization, and degradation pathways.