ABSTRACT
It is well known that GluK2-containing kainate receptors play essential roles in seizure and cerebral ischemia-induced neuronal death, while GluK1-containing kainate receptors could increase tonic inhibition of post-synaptic pyramidal neurons. This research investigated whether GluK1 could inhibit activation of c-Jun N-terminal kinase 3 (JNK3) signaling pathway mediated by the GluK2 in cerebral ischemia-reperfusion. The results show that GluK1 activation by (RS)-2-amino-3-(3-hydroxy-5-tert-butylisoxazol-4-yl) propanoic acid (ATPA) at 1nmol per rat could inhibit the assembly of GluK2·Postsynaptic density 95·mixed lineage kinase 3 signaling module, activation of JNK3 and its downstream signal molecules. However, the inhibition of ATPA could be prevented by GluK1 antagonist NS3763, GluK1 antisense, and GABA(A) receptor antagonist bicuculline. In addition, ATPA played a neuroprotective role against cerebral ischemia. In sum, the findings indicate that activation of GluK1 by ATPA at specific dosages may promote GABA release, which then suppresses post-synaptic GluK2-JNK3 signaling-mediated cerebral ischemic injury via GABA(A)R.
Subject(s)
Isoxazoles/pharmacology , Neuroprotective Agents/pharmacology , Propionates/pharmacology , Receptors, Kainic Acid/agonists , Reperfusion Injury/metabolism , Signal Transduction/drug effects , Animals , Enzyme Activation/physiology , Male , Mitogen-Activated Protein Kinase 10/drug effects , Mitogen-Activated Protein Kinase 10/metabolism , Neurons/metabolism , Rats , Rats, Sprague-Dawley , Receptors, GABA-A/metabolism , Receptors, Kainic Acid/drug effects , Receptors, Kainic Acid/metabolism , Signal Transduction/physiology , gamma-Aminobutyric Acid/metabolism , GluK2 Kainate ReceptorABSTRACT
PURPOSE: Past work has demonstrated that kainic acid (KA)-induced seizures could cause the enhancement of excitation and lead to neuronal death in rat hippocampus. To counteract such an imbalance between excitation and inhibition, we designed experiments by activating the inhibitory gamma-aminobutyric acid (GABA) receptor to investigate whether such activation suppresses the excitatory glutamate signaling induced by KA and to elucidate the underlying molecular mechanisms. METHODS: Muscimol coapplied with baclofen was intraperitoneally administrated to the rats 40 min before KA injection by intracerebroventricular infusion. Subsequently we used a series of methods including immunoprecipitation, immunoblotting, histologic analysis, and immunohistochemistry to analyze the interaction, expression, and phosphorylation of relevant proteins as well as the survival of the CA1/CA3 pyramidal neurons. RESULTS: Coadministration of muscimol and baclofen exerted neuroprotection against neuron death induced by KA; inhibited the increased assembly of the GluR6-PSD-95-MLK3 module induced by KA; and suppressed the activation of MLK3, MKK7, and JNK3. DISCUSSION: Taken together, we demonstrate that coactivation of the inhibitory GABA receptors can attenuate the excitatory JNK3 apoptotic signaling pathway via inhibiting the increased assembly of the GluR6-PSD-95-MLK3 signaling module induced by KA. This provides a new insight into the therapeutic approach to epileptic seizure.
Subject(s)
Apoptosis/drug effects , Baclofen/pharmacology , GABA Agonists/pharmacology , Mitogen-Activated Protein Kinase 10/drug effects , Muscimol/pharmacology , Receptors, GABA/drug effects , Seizures/metabolism , Animals , Apoptosis/physiology , Disease Models, Animal , Disks Large Homolog 4 Protein , Humans , Immunohistochemistry , Intracellular Signaling Peptides and Proteins/drug effects , Kainic Acid/pharmacology , MAP Kinase Kinase Kinases/metabolism , Male , Membrane Proteins/metabolism , Mitogen-Activated Protein Kinase 10/antagonists & inhibitors , Mitogen-Activated Protein Kinase 10/metabolism , Rats , Rats, Sprague-Dawley , Receptors, GABA/metabolism , Receptors, GABA-A/drug effects , Receptors, Kainic Acid/drug effects , Seizures/chemically induced , GluK2 Kainate ReceptorABSTRACT
Identification of a viable lead is a critical step in drug discovery. The qualities of the lead set the stage for subsequent efforts to ameliorate therapeutic efficacy through potency, selectivity, pharmacokinetics, toxicity and side effects. In a retrospective view of drug research the lead identification has been realised mainly by in vivo methodologies. However, limitations of in vivo models were found to be critical factors when analysing attrition rates that prompted research groups to introduce in vitro tests and rational approaches at the frontline of discovery programs. Virtual screening (VS) methods merge in vitro high-throughput (HTS) and rational approaches. The VS methods can be classified as ligand and structure based techniques. Structure based approaches depart from the structural information of the target to identify potential interactions between the ligands and the protein. The advantages and disadvantages and the applicability of the structure based virtual screening approaches constituted the main aim of my studies. The glycogen synthase kinase 3beta (GSK-3beta), the beta-secretase and the c-jun N-terminal kinase 3 (JNK-3) were selected as primary targets for virtual screening. The performance of virtual screens can only be validated in parallel with HTS, therefore a head to head comparative analysis was my next goal.
