Abstracts

Rationale: Status epilepticus (SE) can induce neuronal death and epilepsy development. Among the possible mechanisms, SE kills neurons through neurotoxic activation of the NMDAR-PSD95-neuronal NO synthase (nNOS) complex, leading to peroxynitrite (ONOO-) production [measured by 3-nitrotyrosination (3-NT) level] that in turn may induce massive neurodegeneration through cytoskeletal degradation. Protein-protein interaction (PPI) inhibition of the NMDA-PSD95-nNOS complex is one of the most intriguing challenge to reduce neuronal death without affecting normal NMDAR function after excitotoxic insult in ischemia (Aarts 2002).Recent evidence suggests that cell injury may occur in patients with cortical dysplasia (CD), i.e., brain dysgenesis associated with severe drug-resistant epilepsy. We previously developed a rat model of CD based on prenatal methylazoxymethanol (MAM) induction of cortical dysgenesis and post-natal pilocarpine treatment leading to SE and spontaneous seizures (SRS; Colciaghi 2011). In MAM/pilocarpine (MP) rats the occurrence of SE and SRS induce steady NMDAR activation and neuronal/glial cell injury, suggesting that the NMDA-driven neurotoxic pathway may play a significant role in SE-related pathology (Colciaghi 2014; Nobili 2015). Thus, the present study verified whether the administration of the neuroprotective Tat-N-dimer (PSD95-nNOS PPI inhibitor, Bach 2012) to MP rats during SEcould preserve the brain from SE-induced damage. Methods: First experimental setting: epileptic MP rats (n=10) were sacrified 30’ after SE onset and compared to MP rats not experiencing SE (MDP-30’ n=10).Second experimental setting: MP rats received Tat-N-dimer (3 nmol/g, i.v.) 30’ after SE onset, sacrificed 18h later (MP-Tat-18h, n=12) and compared with epileptic untreated MP rats (MP-18h, n=12) and with non-epileptic MP control rats (MDP-18h, n=12).Experimental readouts: we performed 1) WB analysis of synaptic proteins to verify NMDA activation and nNOS membrane translocation; 2) c-fos IHC/WB analysis to verify excitotoxic neuronal activation; 3) WB of 3-NT and spectrin proteolysis as readout respectively of ONOO- formation and cytoskeleton degradation; 4) stereological quantification of FluoroJade (FJ+) neurons to assess neuronal vulnerability in different exp groups Results: Data from first experimental setting showed that SE was able to induce prompt and significant NMDA-NR2B subunit hyperphosphorylation, nNOS membrane translocation and increase of 3-NT in both neuronal and glial cells in the neocortex of MP-30’ vs MDP-30’ rats. This was paralleled by rapid c-fos overexpression and spectrin proteolysis. Data from Tat-N-dimer study revealed that interfering with the neurotoxic NMDA-nNOS activation was able to reduce significantly both the number of FJ+ degenerating neurons and c-fos expression and to affect SE-induced cytoskeletal degradation in MP-Tat-18h vs untreated MP-18h rats. Conclusions: Taken together, these data indicated that nNOS activation may be a main player in the early brain damage induced by SE in MP rats and suggest that NMDA-PSD95-nNOS complex inhibition may represent a new therapeutic strategy to protect the brain from seizure-induce damage while avoiding the undesirable effects of directly blocking NMDAR function.  Funding: Italian Minister of Health and AICE-FIRE 2016 grant  (Italian Association against Epilepsy and Italian Foundation for Epilepsy Research) to F. Colciaghi