In vitro neuroprotective properties of positive allosteric modulators and expression enhancers of EAAT2

Abstract

Glutamate excitotoxicity has been shown to be associated with several acute and chronic disorders of the CNS, including stroke, epilepsy, traumatic brain injury (TBI), and Alzheimer's disease. Excitatory amino acid transporter 2 (EAAT2) is responsible for rapid removal of most extracellular glutamate, thus maintaining glutamate homeostasis and preventing excitotoxicity. Therefore, compounds that enhance its expression or function could serve as valuable neuroprotective agents. Previous studies identified an allosteric site on EAAT2, allowing for virtual screening approaches to identify novel compounds that were characterized as positive allosteric modulators (PAMs) of this transporter. Medicinal chemistry efforts generated several analogs with better drug-like properties. Previous studies in the lab further characterized the potency and selectivity of these compounds in glutamate uptake assays in transfected COS-7 cells and glial cultures. In this study, we investigated the potential neuroprotective properties of one of these compounds, NA-014, a selective EAAT2 PAM. We also investigated the potential neuroprotective properties of a natural compound, Parawixin10, that has previously been characterized as a PAM of EAAT1 and EAAT2, and clavulanic acid, a -lactam that has been identified as an enhancer of the expression of EAAT2. For these studies, mixed neuron-glia cultures were subjected to four different types of excitotoxic insults: exogenous glutamate application, exogenous application of H2O2 (an in vitro model of oxidative stress), oxygen-glucose deprivation (OGD, an in vitro stroke model), and low magnesium buffer (an in vitro model of epilepsy). Neuronal survival and dendritic arborization were assessed 24 hours following the insults using immunocytochemistry against MAP-2 and GFAP (neuronal and glial markers, respectively). Our results indicate that NA-014 is neuroprotective in the glutamate, OGD, and low magnesium in vitro models and has a therapeutic time window of approximately 8 hours. However, the compound lacks effectiveness in the oxidative stress model, and we suggest that this insult damages the glial transporters. Our results also show that Parawixin10 and clavulanic acid are both neuroprotective in the glutamate and OGD in vitro models. Ongoing and future studies aim to determine the extracellular glutamate concentration following insults and treatments with NA-014. Preliminary in vivo studies in models of TBI and neuropathic pain demonstrate that this compound has neuroprotective and nociceptive properties. Future studies will address whether this compound can be developed for stroke therapy and will further investigate the potential additivity of combining treatments of allosteric modulator and expression enhancer. Finally, future studies will optimize the drug-like properties of NA-014 and Parawixin10 through medicinal chemistry. In summary, these studies indicate that EAAT2 PAMs and expression enhancers display neuroprotective properties in several in vitro models of excitotoxicity, and they may be developed for clinical use in treating disorders involving excitotoxicity, like stroke, epilepsy, and traumatic brain injury (TBI).M.S., Pharmacology and Physiology -- Drexel University, 201