Modulation of glutamate transporters as a potential therapeutic intervention for neurological disorders

Abstract

Excitatory amino acid transporters (EAATs) are key proteins in the CNS that regulate glutamate levels and, therefore, control excitatory neurotransmission and limit excitotoxicity. EAATs are trimers, with each protomer consisting of a scaffold domain, and a transport domain that slides along the scaffold domain in an "elevator-like motion" to move synaptic glutamate into astrocytes and neurons. Our lab has discovered novel positive allosteric modulators (PAMs) of EAAT2, the main EAAT subtype responsible for glutamate clearance in the CNS. We hypothesize that these compounds act by altering the interactions between the scaffold and the transport domain. To test this, we used computational modeling, followed by site-directed mutagenesis and radioligand uptake assays. This allowed us to identify mutations in specific amino acid residues on astrocytic transporters EAAT1 and EAAT2 that result in increased activity without changes in expression. This suggests that interactions between the domains are responsible for enhanced efficiency of these transporters. These gain-of-function mutated EAAT1 and EAAT2 are currently being studied in single molecule Förster Resonance Energy Transfer (smFRET) approaches to further understand the kinetics and dynamics of EAATs. In our work, we also evaluated the neuroprotective properties of our EAAT2 PAMs in models of excitotoxicity. Previously, our lab has demonstrated that EAAT2 PAMs are protective in an in vitro stroke model. We expanded our studies to investigate potential neuroprotective properties an in vitro epilepsy model, using live calcium signaling measured in primary neuron/glia cultures. Lastly, we explored the ability of these compounds to attenuate cocaine seeking behavior in conditioned place preference behavioral experiments. Collectively, these studies expand our understanding of the mechanisms of these PAMs and broaden their potential therapeutic indications.