The Role of Glutamate Transporters in Neuroprotection after in vitro Stroke and Cocaine Seeking Behavior in vivo
Authors:   , Patel Bhumi , Mortensen Andreia , Mathiasen Joanne R. 
Published: July 2021

Glutamate, the main excitatory amino acid neurotransmitter in the CNS, has a large array of normal physiological functions. Consequently, glutamate dysfunction has profound effects both in disease and injury, and it has been implicated in the pathology of many acute and chronic CNS disorders, such as stroke, epilepsy, traumatic brain injury (TBI), HIV Associated Neurocognitive Disorder (HAND), and cocaine use disorder. Excitatory amino acid transporter 2 (EAAT2) is responsible for clearing the bulk of glutamate from the synaptic cleft, thereby playing a pivotal role in maintaining glutamate homeostasis and preventing excitotoxicity. Metabotropic glutamate receptors (mGluRs) are responsible for regulating synaptic transmission and neuronal excitability throughout the CNS, hence they also play a pivotal role in disorders associated with glutamate dysfunction. In this sense, compounds that increase the expression or functional activity of EAAT2 and modulators of mGluRs could be promising agents against disorders associated with glutamate dysfunction. My project investigated the effects of agonism of mGluRs and positive allosteric modulation (PAM) of EAAT2 in two different models that involve glutamatergic dysregulation: stroke and cocaine use disorder. Previous studies demonstrated that mGluR II agonists decrease glutamate release and offer neuroprotection against excitotoxicity, and that their activation has indirect effects on glutamate transport. In Aim 1 of my project, we first investigated the effect of mGluR modulators on glutamate uptake. DCG-IV, an mGluR II agonist, increased glutamate uptake in glial cultures with an EC50 of ~106 [mu]M. We then investigated its effect in an in vitro model of stroke. Additionally, our lab has identified novel PAMs of EAAT2, exemplified by NA-014, a selective EAAT2 PAM. Mixed neuron-glia cultures were subjected to 20 minutes of oxygen-glucose deprivation (OGD), a model that induces glutamate-mediated excitotoxicity. Neuronal survival was assessed following treatments with NA-014 and mGluR II agonist DCG-IV, using immunocytochemistry for MAP-2, a neuronal marker. Our results show that treatments with either DCG-IV or NA-014 resulted in a significant increase in neuronal survival, and combination treatment with both compounds further increased the levels of survival, suggesting an additive effect. Future in vitro studies will include the examination of other group II and III mGluR agonists for glutamate transport modulation and potential neuroprotective properties in combination with EAAT2 PAMs. It is well established that cocaine use leads to profound changes in glutamate transmission in the nucleus accumbens, and growing evidence suggests that glutamate transporters could be potential targets for the treatment of cocaine abuse. In Aim 2 of my project, we examined the effects of NA-014 on cocaine seeking behavior using the conditioned place preference (CPP) model. Male rats were administered three different concentrations of NA-014 (15, 30 and 60 mg/kg, IP), after 4 days of pre-conditioning with 10 mg/kg of cocaine, using a biased design. Our results show that 30 and 60 mg/kg NA-014 were effective doses in abolishing the development of CPP for cocaine, whereas the lower dose did not. This suggests that this compound has promise to be developed as therapy for cocaine seeking disorders. Future studies will include cocaine self-administration studies and examination of sex differences for the effect of NA-014. In conclusion, positive allosteric modulation of glutamate transporter EAAT2 seems to be a promising approach for further development of therapies for CNS disorders that involve dysfunctional glutamate signaling.