Neuroprotective effects of chlorogenic acid against oxidative stress in rats subjected to lithium-pilocarpine-induced status epilepticus.

Epilepsy is a condition marked by sudden, self-sustained, and recurring brain events, showcasing unique electro-clinical and neuropathological phenomena that can alter the structure and functioning of the brain, resulting in diverse manifestations. Antiepileptic drugs (AEDs) can be very effective in 30% of patients in controlling seizures. Several factors contribute to this: drug resistance, individual variability, side effects, complexity of epilepsy, incomplete understanding, comorbidities, drug interactions, and no adherence to treatment. Therefore, research into new AEDs is important for several reasons such as improved efficacy, reduced side effects, expanded treatment options, treatment for drug-resistant epilepsy, improved safety profiles, targeted therapies, and innovation and progress. Animal models serve as crucial biological tools for comprehending neuronal damage and aiding in the discovery of more effective new AEDs. The utilization of antioxidant agents that act on the central nervous system may serve as a supplementary approach in the secondary prevention of epilepsy, both in laboratory animals and potentially in humans. Chlorogenic acid (CGA) is a significant compound, widely prevalent in numerous medicinal and food plants, exhibiting an extensive spectrum of biological activities such as neuroprotection, antioxidant, anti-inflammatory, and analgesic effects, among others. In this research, we assessed the neuroprotective effects of commercially available CGA in Wistar rats submitted to lithium-pilocarpine-induced status epilepticus (SE) model. After 72-h induction of SE, rats received thiopental and were treated for three consecutive days (1st, 2nd, and 3rd doses). Next, brains were collected and studied histologically for viable cells in the hippocampus with staining for cresyl-violet (Nissl staining) and for degenerating cells with Fluoro-Jade C (FJC) staining. Moreover, to evaluate oxidative stress, the presence of malondialdehyde (MDA) and superoxide dismutase (SOD) was quantified. Rats administered with CGA (30 mg/kg) demonstrated a significant decrease of 59% in the number of hippocampal cell loss in the CA3, and of 48% in the hilus layers after SE. A significant reduction of 75% in the cell loss in the CA3, shown by FJC+ staining, was also observed with the administration of CGA (30 mg/kg). Furthermore, significant decreases of 49% in MDA production and 72% in the activity of SOD were seen, when compared to animals subjected to SE that received vehicle. This study introduces a novel finding: the administration of CGA at a dosage of 30 mg/kg effectively reduced oxidative stress induced by lithium-pilocarpine, with its effects lasting until the peak of neural damage 72 h following the onset of SE. Overall, the research and development of new AEDs are essential for advancing epilepsy treatment, improving patient outcomes, and ultimately enhancing the quality of life for individuals living with epilepsy.

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.

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.

Neuroprotective properties of RT10, a fraction isolated from Parawixia bistriata spider venom, against excitotoxicity injury in neuron-glia cultures.

BACKGROUND: L-Glutamate (L-Glu), the major excitatory neurotransmitter in the mammalian Central Nervous System (CNS), is essential to cognitive functions. However, when L-Glu is accumulated in large concentrations at the synaptic cleft, it can induce excitotoxicity that results in secondary damage implicated in many neurological disorders. Current therapies for the treatment of neurological disorders are ineffective and have side effects associated with their use; therefore, there is a need to develop novel treatments. In this regard, previous studies have shown that neuroactive compounds obtained from the venom of the spider Parawixia bistriata have neuroprotective effects in vitro and in vivo. In this sense, this work aimed to evaluate potential neuroprotective effects of fraction RT10, obtained from this spider venom, on primary cultures of neuron and glial cells subjected to glutamate excitotoxicity insults. METHODS: Primary cultures of neurons and glia were obtained from the cerebral tissue of 1-day-old postnatal Wistar rats. After 7 days in vitro (DIV), the cultures were incubated with fraction RT10 (0.002; 0.02; 0.2 and 2 µg/µL) or riluzole (100 µM) for 3-hours before application of 5 mM L-Glu. After 12 hours, the resazurin sodium salt (RSS) test was applied to measure metabolic activity and proliferation of living cells, whereas immunocytochemistry for MAP2 was performed to measure neuronal survival. In addition, the cells were immunolabeled with NeuN and GFAP in baseline conditions. RESULTS: In the RSS tests, we observed that pre-incubation with RT10 before the excitotoxic insults from L-Glu resulted in neuroprotection, shown by a 10% reduction in the cell death level. RT10 was more effective than riluzole, which resulted in a cell-death reduction of 5%. Moreover, qualitative analysis of neuronal morphology (by MAP2 staining, expressed as fluorescence intensity (FI), an indirect measure of neuronal survival) indicate that RT10 reduced the toxic effects of L-Glu, as shown by a 38 % increase in MAP2 fluorescence when compared to L-Glu insult. On the other hand, the riluzole treatment resulted in 17% increase of MAP2 fluorescence; therefore, the neuroprotection from RT10 was more efficacious. CONCLUSION: RT10 fraction exhibits neuroprotective effects against L-Glu excitotoxicity in neuron-glia cultured in vitro.

Neuroprotective properties of RT10, a fraction isolated from Parawixia bistriata spider venom, against excitotoxicity injury in neuron-glia cultures

L-Glutamate (L-Glu), the major excitatory neurotransmitter in the mammalian Central Nervous System (CNS), is essential to cognitive functions. However, when L-Glu is accumulated in large concentrations at the synaptic cleft, it can induce excitotoxicity that results in secondary damage implicated in many neurological disorders. Current therapies for the treatment of neurological disorders are ineffective and have side effects associated with their use; therefore, there is a need to develop novel treatments. In this regard, previous studies have shown that neuroactive compounds obtained from the venom of the spider Parawixia bistriata have neuroprotective effects in vitro and in vivo. In this sense, this work aimed to evaluate potential neuroprotective effects of fraction RT10, obtained from this spider venom, on primary cultures of neuron and glial cells subjected to glutamate excitotoxicity insults. Methods: Primary cultures of neurons and glia were obtained from the cerebral tissue of 1-day-old postnatal Wistar rats. After 7 days in vitro (DIV), the cultures were incubated with fraction RT10 (0.002; 0.02; 0.2 and 2 µg/µL) or riluzole (100 µM) for 3-hours before application of 5 mM L-Glu. After 12 hours, the resazurin sodium salt (RSS) test was applied to measure metabolic activity and proliferation of living cells, whereas immunocytochemistry for MAP2 was performed to measure neuronal survival. In addition, the cells were immunolabeled with NeuN and GFAP in baseline conditions. Results: In the RSS tests, we observed that pre-incubation with RT10 before the excitotoxic insults from L-Glu resulted in neuroprotection, shown by a 10% reduction in the cell death level. RT10 was more effective than riluzole, which resulted in a cell-death reduction of 5%. Moreover, qualitative analysis of neuronal morphology (by MAP2 staining, expressed as fluorescence intensity (FI), an indirect measure of neuronal survival) indicate that RT10 reduced the toxic effects of L-Glu, as shown by a 38 % increase in MAP2 fluorescence when compared to L-Glu insult. On the other hand, the riluzole treatment resulted in 17% increase of MAP2 fluorescence; therefore, the neuroprotection from RT10 was more efficacious. Conclusion: RT10 fraction exhibits neuroprotective effects against L-Glu excitotoxicity in neuron-glia cultured in vitro.(AU)

Identification of an allosteric modulator of the serotonin transporter with novel mechanism of action.

Serotonin transporters (SERTs) play an essential role in the termination and regulation of serotonin signaling in the brain. SERT is also the target of antidepressants and psychostimulants. Molecules with novel activities and modes of interaction with regard to SERT function are of great scientific and clinical interest. We explored structural regions outside the putative serotonin translocation pathway to identify potential binding sites for allosteric transporter modulators (ATMs). Mutational studies revealed a pocket of amino acids outside the orthosteric substrate binding sites located in the interface between extracellular loops 1 and 3 that when mutated affect transporter function. Using the structure of the bacterial transporter homolog leucine transporter as a template, we developed a structural model of SERT. We performed molecular dynamics simulations to further characterize the allosteric pocket that was identified by site-directed mutagenesis studies and employed this pocket in a virtual screen for small-molecule modulators of SERT function. In functional transport assays, we found that one of the identified molecules, ATM7, increased the reuptake of serotonin, possibly by facilitating the interaction of serotonin with transport-ready conformations of SERT when concentrations of serotonin were low and rate limiting. In addition, ATM7 potentiates 3,4-methylenedioxy-N-methylamphetamine (MDMA, "Ecstasy")-induced reversed transport by SERT. Taking advantage of a conformationally sensitive residue in transmembrane domain 6, we demonstrate that ATM7 mechanistically stabilizes an outward-facing conformation of SERT. Taken together these observations demonstrate that ATM7 acts through a novel mechanism that involves allosteric modulation of SERT function.

Enhancing glutamate transport: mechanism of action of Parawixin1, a neuroprotective compound from Parawixia bistriata spider venom.

Previous studies have shown that a compound purified from the spider Parawixia bistriata venom stimulates the activity of glial glutamate transporters and can protect retinal tissue from ischemic damage. To understand the mechanism by which this compound enhances transport, we examined its effects on the functional properties of glutamate transporters after solubilization and reconstitution in liposomes and in transfected COS-7 cells. Here, we demonstrate in both systems that Parawixin1 promotes a direct and selective enhancement of glutamate influx by the EAAT2 transporter subtype through a mechanism that does not alter the apparent affinities for the cosubstrates glutamate or sodium. In liposomes, we observed maximal enhancement by Parawixin1 when extracellular sodium and intracellular potassium concentrations are within physiological ranges. Moreover, the compound does not enhance the reverse transport of glutamate under ionic conditions that favor efflux, when extracellular potassium is elevated and the sodium gradient is reduced, nor does it alter the exchange of glutamate in the absence of internal potassium. These observations suggest that Parawixin1 facilitates the reorientation of the potassium-bound transporter, the rate-limiting step in the transport cycle, a conclusion further supported by experiments showing that Parawixin1 does not stimulate uptake by an EAAT2 transport mutant (E405D) defective in the potassium-dependent reorientation step. Thus, Parawixin1 enhances transport through a novel mechanism targeting a step in the transport cycle distinct from substrate influx or efflux and provides a basis for the design of new drugs that act allosterically on transporters to increase glutamate clearance.

Neurochemical characterization of a neuroprotective compound from Parawixia bistriata spider venom that inhibits synaptosomal uptake of GABA and glycine.

The major contribution of this work is the isolation of a neuroprotective compound referred to as 2-amino-5-ureidopentanamide (FrPbAII) (M(r) = 174) from Parawixia bistriata spider venom and an investigation of its mode of action. FrPbAII inhibits synaptosomal GABA uptake in a dose-dependent manner and probably does not act on Na(+), K(+), and Ca(2+) channels, GABA(B) receptors, or gamma-aminobutyrate:alpha-ketoglutarate aminotransferase enzyme; therefore, it is not directly dependent on these structures for its action. Direct increase of GABA release and reverse transport are also ruled out as mechanisms of FrPbAII activities as well as unspecific actions on pore membrane formation. Moreover, FrPbAII is selective for GABA and glycine transporters, having slight or no effect on monoamines or glutamate transporters. According to our experimental glaucoma data in rat retina, FrPbAII is able to cross the blood-retina barrier and promote effective protection of retinal layers submitted to ischemic conditions. These studies are of relevance by providing a better understanding of neurochemical mechanisms involved in brain function and for possible development of new neuropharmacological and therapeutic tools.

Purification of a neuroprotective component of Parawixia bistriata spider venom that enhances glutamate uptake.

(1) In this study, we examined the effects of crude venom from the spider Parawixia bistriata on glutamate and GABA uptake into synaptosomes prepared from rat cerebral cortex. Addition of venom to cortical synaptosomes stimulated glutamate uptake and inhibited GABA uptake in a concentration-dependent manner. (2) The venom was fractionated using reverse-phase high-performance liquid chromatography on a preparative column. The fraction that retained glutamate uptake-stimulating activity was further purified on a reverse-phase analytical column followed by ion-exchange chromatography. (3) The active fraction, referred to as PbTx1.2.3, stimulated glutamate uptake in synaptosomes without changing the K(M) value, and did not affect GABA uptake. Additional experiments showed that the enhancement of glutamate uptake by PbTx1.2.3 occurs when ionotropic glutamate receptors or voltage-gated sodium and calcium channels are completely inhibited or when GABA receptors and potassium channels are activated, indicating that the compound may have a direct action on the transporters. (4) In an experimental model for glaucoma in which rat retinas are subjected to ischemia followed by reperfusion, PbTx1.2.3 protected neurons from excitotoxic death in both outer and inner nuclear layers, and ganglion cell layers. (5) This active spider venom component may serve as a basis for designing therapeutic drugs that increase glutamate clearance and limit neurodegeneration.