The Regulation of Glial Glutamate Transporters after in-vitro Ischemic Stroke
Authors:   , Gill Simran , Mortensen Andreia , McGonigle Paul 
Published: August 2021

Stroke is the 3rd leading cause of death in the United States however it still lacks adequate treatment. Ischemic stroke is one of the major types of stroke; ischemia involves the cessation of blood flow to the brain, which is usually the result of a blood clot. Neuronal death that is observed following ischemia is primarily due to glutamatergic imbalance-an excess of glutamate release leads to excitotoxicity and subsequent cell death. The excitatory amino acid transporters (EAATs) are responsible for the regulation of extracellular glutamate, and therefore play a major role in ischemic outcomes. My project was designed to better understand the modulation of EAATs following ischemic insults, as previous studies have shown conflicting results. More specifically, we explored how EAAT activity and expression is regulated following varying severities of ischemia in glia cultures: 30-minutes, 1-hour and 2-hours of oxygen-glucose deprivation (OGD), which could be considered mild, moderate and severe ischemic insults, respectively. Furthermore, we also sought to determine the effects of Clavulanic Acid (CA), an EAAT2 expression enhancer, on EAAT expression and activity after varying ischemic insults. Previous work by our lab has found that CA provides neuroprotection after 20 minutes of OGD in neuron-glia cultures. Our study showed that with increasing length of ischemic insult, there is a decrease in glutamate transport, suggesting that these transporters are unable to effectively clear excess glutamate. This could happen via transport reversal, internalization, degradation, and/or other mechanisms. This decrease in activity is only rescued by CA pretreatment following mild ischemia. The expression of EAAT1 is downregulated after ischemia, with a more profound effect at the 1-hour time point. On the other hand, EAAT2 expression is up regulated after 30-minutes and 1-hour OGD, and decreases below control levels after 2-hours of OGD. These results suggest that these glial transporters may work together, likely as a compensatory mechanism to mitigate the increased glutamate release and decreased uptake resulting from OGD. We also found that CA significantly increased EAAT2 levels in control conditions as well as after 30-minutes of OGD, suggesting that this strategy only offers benefit in mild ischemic insults. Future studies will include cell surface biotinylation, functionality assays of Na⁺/K⁺ATPase (that provide energy for transport), and electrophysiological approaches. Additionally, studies to evaluate ubiquitination, transcription factors (such as calcineurin, which has been associated with excitotoxic cell death mechanisms), as well as the activation of mTOR-Akt-NFkB signaling cascades, and miRNAs associated with EAAT modulation, can help us further understand the mechanisms associated with EAAT regulation during ischemia. We also we investigated reactive gliosis, which can be defined by the activation of glial cells in response to a pathology. Previous work has shown that reactive gliosis is a phenotype associated with ischemia, however we wanted to define the point at which this phenomenon occurs. Our 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. Future studies will include the use of more specific markers of reactive gliosis, such as BrdU as well as A1 and A2 subtype markers to take our analysis to the next step. Overall, the findings from this study helped further our knowledge regarding glutamate regulation following different severities of ischemic stroke in-vitro and could aid in the development of novel therapeutics.