tat-nr2b9c and Status-Epilepticus

Antiepileptogenic agents that prevent the development of epilepsy following a brain insult remain the holy grail of epilepsy therapeutics. We have employed a label-free proteomic approach that allows quantification of large numbers of brain-expressed proteins in a single analysis in the mouse (male C57BL/6J) kainate (KA) model of epileptogenesis. In addition, we have incorporated two putative antiepileptogenic drugs, postsynaptic density protein-95 blocking peptide (PSD95BP or Tat-NR2B9c) and a highly selective inducible nitric oxide synthase inhibitor, 1400W, to give an insight into how such agents might ameliorate epileptogenesis. The test drugs were administered after the induction of status epilepticus (SE) and the animals were euthanized at 7 days, their hippocampi removed, and subjected to LC-MS/MS analysis. A total of 2,579 proteins were identified; their normalized abundance was compared between treatment groups using ANOVA, with correction for multiple testing by false discovery rate. Significantly altered proteins were subjected to gene ontology and KEGG pathway enrichment analyses. KA-induced SE was most robustly associated with an alteration in the abundance of proteins involved in neuroinflammation, including heat shock protein beta-1 (HSP27), glial fibrillary acidic protein, and CD44 antigen. Treatment with PSD95BP or 1400W moderated the abundance of several of these proteins plus that of secretogranin and Src substrate cortactin. Pathway analysis identified the glutamatergic synapse as a key target for both drugs. Our observations require validation in a larger-scale investigation, with candidate proteins explored in more detail. Nevertheless, this study has identified several mechanisms by which epilepsy might develop and several targets for novel drug development. OPEN PRACTICES: This article has been awarded Open Data. All materials and data are publicly accessible as supporting information. Learn more about the Open Practices badges from the Center for Open Science: https://osf.io/tvyxz/wiki.

Topics: Amidines; Animals; Anticonvulsants; Benzylamines; Epilepsy; Hippocampus; Kainic Acid; Male; Mice, Inbred C57BL; Nitric Oxide Synthase Type II; Peptides; Proteomics; Status Epilepticus

N-methyl-D-aspartate receptors (NMDARs) mediate essential neuronal excitation, but overactivation of NMDARs results in excitotoxic cell death in a variety of pathologic conditions, including status epilepticus (SE). Although NMDAR antagonists attenuate SE-induced brain injury, undesirable side effects have limited their clinical efficacy. Tat-NR2B9c was designed to disrupt protein interactions involving postsynaptic density protein 95 in the NMDAR signaling complex while not interfering with function of the NMDAR ion channel. We examined the ability of Tat-NR2B9c to provide neuroprotection in the hippocampus of rats after 60 minutes of SE induced by the repeated injection of low doses of pilocarpine (10 mg/kg). Tat-NR2B9c was administered 3hours after the termination of SE, and neuronal densities were assessed 14 days later by stereologic analysis of NeuN-positive cells. After SE, pyramidal cell densities were reduced by 70% in CA1, 34% in CA3, 58% in CA4, and 88% in the piriform cortex. In Tat-NR2B9c-treated rats, neuronal densities in CA1, a subregion of CA3, and CA4 were decreased by only 38%, 4%, and 26%, respectively. Tat-NR2B9c did not reduce cell loss in the posterior piriform cortex. The results indicate that targeted disruption of the NMDAR signaling complex represents a potential therapeutic approach for limiting neuronal cell loss after SE.

Topics: Animals; Cell Count; Cell Death; Disks Large Homolog 4 Protein; Hippocampus; Immunohistochemistry; Intracellular Signaling Peptides and Proteins; Male; Membrane Proteins; Microscopy, Confocal; Nerve Degeneration; Nerve Tissue Proteins; Neuroprotective Agents; Peptides; Pilocarpine; Pyramidal Cells; Rats; Rats, Wistar; Receptors, N-Methyl-D-Aspartate; Recombinant Fusion Proteins; Status Epilepticus; Time Factors