Abstracts

Rationale: High Mobility Group Box 1 (HMGB1) is a non histonic chromatin-binding protein that regulates gene transcription. Following cell injury or during seizures, HMGB1 rapidly translocates from the nucleus to the cytoplasm from where it is released extracellularly. HMGB1 function is determined by its redox state. We investigated whether HMGB1 traslocation, and its disulfide isoform that promotes seizures and cell loss, are generated by reactive oxygen species (ROS) during oxidative stress. Moreover, we studied whether prevention of disulfide HMGB1 generation reduces cell loss during epileptogenesis.Methods: We focused our study during epileptogenesis evoked by electrically induced self-sustained limbic status epilepticus (SE) in electrode implanted adult male Sprague-Dawley rats. We analyzed the following molecules in the hippocampus before the onset of spontaneous seizures: HMGB1 translocation and key markers of oxidative stress (Nrf2, iNOS, Xct) by immunohistochemistry (IHC, n=4 rats); disulfide HMGB1 and GSSG/GSH ratio by LC-MS/MS and HPLC, respectively (n=9-10 rats/each group). Electrode-implanted but not stimulated rats (sham, n= 4 for IHC; n=10/each group for HPLC and LC-MS/MS) were used as controls. In a different cohort of rats, we administered a combination of anti-oxidant drugs during epileptogenesis for blocking oxidative stress and measuring HMGB1 disulfide levels in brain and blood. We tested whether this anti-oxidant treatment afforded neuroprotection by quantifying Nissl-stained neurons during epileptogenesis vs sham rats (n=5 rats/each group), and in vitro using primary cultures of hippocampal neurons exposed to 1 mM H2O2 for 1 h vs medium alone (n=5-10 wells/each group from two independent experiments). In vitro cell loss was quantified by MTT assay.Results: Oxidative stress, HMGB1 translocation in astrocytes, and the generation of brain and blood disulfide HMGB1 were all precluded by the anti-oxidant treatment (p<0.01 vs sham). The treatment prevented the 25% loss of hilar interneurons occuring in the hippocampus during epileptogenesis, and the 85% neuronal cell loss induced in vitro by H202 (p<0.01). The treatment was safe since it did not affect body weight, motor activity or cognitive performance in the Morris water maze both in sham- and in SE-exposed rats.Conclusions: Our data show that oxidative stress is involved in disulfide HMGB1 generation after an epileptogenic insult, and suggest that HMGB1 translocation/release and its disulfide isoform are generated by ROS, thereby contributing to cell loss during epileptogenesis. These results foster further studies on the treatment’s effects on epilepsy development and the associated cognitive deficits.