Neuroprotective properties of novel positive allosteric modulators of EAAT2

Romulo Martelli Falcucci

doi:10.17918/etd-7166

Excitatory amino acid transporters (EAATs) play a crucial role in the removal of synaptic glutamate to maintain extracellular concentrations below excitotoxic levels. Glutamate-mediated excitotoxicity has been associated with a number of diseases/conditions, including traumatic brain injury (TBI), stroke, Amyotrophic Lateral Sclerosis, HIV-associated neurocognitive disorders (HAND) among others. Compounds that increase the activity of EAAT2, the major glutamate transporter in the brain, could have therapeutic potential for neuroprotection. Previous work has identified molecular determinants implicated in EAAT2 transport stimulation. Additionally, a hybrid structure based approach has identified selected hit structures that interact with this region. Some of these novel compounds were shown to be selective positive allosteric modulators (PAMs) of the function of EAAT2, in glutamate transport assays in transfected COS-7 cells and in glial cultures. In this work, we have characterized the effects of novel PAMs of EAAT2 in a bilaminar culture model, in which rat cortical neurons are cultured in the presence of a glial feeder layer. To examine potential neuroprotective effects of these PAMs, cultures were subjected to excitotoxic and oxidative stress insults. Specifically, excitotoxic insults were performed with the application of 1, 10, 50 or 100 muM L-glutamate, and oxidative stress with the application of 10, 50 or 100 muM H₂O₂. Prolonged insults were carried out for 24 h, while acute insults were carried on for 20 min, in the presence or absence of a glial layer. Compounds were applied after the insults and neuronal survival was assessed by MAP-2 staining 24 h after the treatments. Our results indicate that the PAMs are neuroprotective in several conditions, including prolonged and acute excitotoxic insults, but not in oxidative stress conditions. Future directions include determining the in vitro therapeutic window of the PAMs, and the examination of the PAMs in other in vitro models, including oxygen-glucose deprivation and traumatic brain injury (stretch injury), among others. We also aim to profile these PAMs for ADME/ drug-like properties and pharmacokinetic properties, and to assess potential neuroprotection in in in vivo neuroprotection studies. In conclusion, these novel PAMs of EAAT2 have neuroprotection potential for translation in in vivo neurological diseases and conditions.

Neuroprotective properties of novel positive allosteric modulators of EAAT2

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Neuroprotective properties of novel positive allosteric modulators of EAAT2

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