Role of glutamate excitotoxicity and glutamate transporter EAAT2 in epilepsy: Opportunities for novel therapeutics development.

Epilepsy is a complex neurological syndrome characterized by seizures resulting from neuronal hyperexcitability and sudden and synchronized bursts of electrical discharges. Impaired astrocyte function that results in glutamate excitotoxicity has been recognized to play a key role in the pathogenesis of epilepsy. While there are 26 drugs marketed as anti-epileptic drugs no current treatments are disease modifying as they only suppress seizures rather than the development and progression of epilepsy. Excitatory amino acid transporters (EAATs) are critical for maintaining low extracellular glutamate concentrations and preventing excitotoxicity. When extracellular glutamate concentrations rise to abnormal levels, glutamate receptor overactivation and the subsequent excessive influx of calcium into the post-synaptic neuron can trigger cell death pathways. In this review we discuss targeting EAAT2, the predominant glutamate transporter in the CNS, as a promising approach for developing therapies for epilepsy. EAAT2 upregulation via transcriptional and translational regulation has proven successful in vivo in reducing spontaneous recurrent seizures and offering neuroprotective effects. Another approach to regulate EAAT2 activity is through positive allosteric modulation (PAM). Novel PAMs of EAAT2 have recently been identified and are under development, representing a promising approach for the advance of novel therapeutics for epilepsy.

Elucidation of the Structure and Synthesis of Neuroprotective Low Molecular Mass Components of the Parawixia bistriata Spider Venom.

The South American social spider Parawixia bistriata produces a venom containing complex organic compounds with intriguing biological activities. The crude venom leads to paralysis in termites and stimulates l-glutamate uptake and inhibits GABA uptake in rat brain synaptosomes. Glutamate is the major neurotransmitter at the insect neuromuscular junction and at the mammalian central nervous system, suggesting a modulation of the glutamatergic system by the venom. Parawixin1, 2, and 10 (Pwx1, 2 and 10) are HPLC fractions that demonstrate this bioactivity. Pwx1 stimulates l-glutamate uptake through the main transporter in the brain, EAAT2, and is neuroprotective in in vivo glaucoma models. Pxw2 inhibits GABA and glycine uptake in synaptosomes and inhibits seizures and neurodegeneration, and Pwx10 increases l-glutamate uptake in synaptosomes and is neuroprotective and anticonvulsant, shown in in vivo epilepsy models. Herein, we investigated the low molecular mass compounds in this venom and have found over 20 small compounds and 36 unique acylpolyamines with and without amino acid linkers. The active substances in fractions Pwx1 and Pwx2 require further investigation. We elucidated and confirmed the structure of the active acylpolyamine in Pwx10. Both fraction Pwx10 and the synthesized component enhance the activity of transporters EAAT1 and EAAT2, and, importantly, offer in vitro neuroprotection against excitotoxicity in primary cultures. These data suggest that compounds with this mechanism could be developed into therapies for disorders in which l-glutamate excitotoxicity is involved.

Novel Positive Allosteric Modulators of Glutamate Transport Have Neuroprotective Properties in an in Vitro Excitotoxic Model.

Dysfunction of excitatory amino acid transporters (EAATs) has been implicated in the pathogenesis of various neurological disorders, such as stroke, brain trauma, epilepsy, and several neurodegenerative disorders. EAAT2 is the main transporter subtype responsible for glutamate clearance in the brain, and plays a key role in regulating neurotransmission and preventing excitotoxicity. Therefore, compounds that increase the activity of EAAT2 have therapeutic potential for neuroprotection. In previous studies, we used virtual screening approaches to identify novel positive allosteric modulators (PAMs) of EAAT2. These compounds were shown to selectively increase the activity of EAAT2 and increase Vmax of transport, without changing substrate affinity. In this work, our major effort was to investigate whether increasing the activity of EAAT2 by allosteric modulation would translate to neuroprotection in in vitro primary culture models of excitotoxicity. To investigate potential neuroprotective effects of one EAAT2 PAM, GT949, we subjected cultures to acute and prolonged excitotoxic insults by exogenous application of glutamate, or oxidative stress by application of hydrogen peroxide. GT949 administration did not result in neuroprotection in the oxidative stress model, likely due to damage of the glutamate transporters. However, GT949 displayed neuroprotective properties after acute and prolonged glutamate-mediated excitotoxicity. We propose that this compound prevents excess glutamate signaling by increasing the rate of glutamate clearance by EAAT2, thereby preventing excitotoxic damage and cell death. This novel class of compounds is therefore an innovative approach for neuroprotection with potential for translation in in vivo animal models of excitotoxicity.