Fig 2: Scheme of programmed necrotic neuronal death based on the present data and a previous report [7]. Increased ET-1 release and ETBreceptor expression induced by SE results in the up-regulation of ROCK1 expression, which subsequently increases LIMK2 expression independent of NOS activity. The up-regulated LIMK2 induces necrotic neuronal death by impairment of DRP1-mediated mitochondrial fission. Inhibition of LIMK2 expression and rescue of DRP1 function attenuate this programmed necrotic neuronal death

Fig 3: The effects of PDI knockdown on SNO-PDI, SNO-DRP1, and mitochondrial fusion in CA1 neurons under normal condition.PDI knockdown reduces PDI expression, the binding of PDI to DRP1, SNO-PDI, and SNO-DRP1 levels without changed DRP1 expression and nitrate/nitrite concentration. However, PDI knockdown elongates mitochondrial length in CA1 neurons, accompanied by elevated DRP1-S616 phosphorylation. a Quantitative values (mean ± S.E.M) of nitrate/nitrite levels in the hippocampus 7 days after PDI siRNA infusion. Open circles indicate each individual value. Horizontal bars indicate mean value. Error bars indicate SEM (n = 7, respectively). b Representative western blot for co-immunoprecipitation of PDI interaction with DRP1 following PDI knockdown. c Quantification of co-immunoprecipitation analyses of PDI interactions with DRP1 following PDI knockdown. Open circles indicate each individual value. Horizontal bars indicate mean value. Error bars indicate SEM (*p < 0.05 vs. control siRNA; n = 7, respectively). d Representative western blot for expression, S-nitrosylation, and phosphorylation levels of PDI and DRP1 following PDI knockdown. e–g Quantification of values (mean ± S.E.M) of expression, phosphorylation, and S-nitrosylation levels of PDI and DRP1 following PDI knockdown. Open circles indicate each individual value. Horizontal bars indicate mean value. Error bars indicate SEM (*p < 0.05 vs. control siRNA; n = 7, respectively). h Quantification of values (mean ± S.E.M) of mitochondrial length in CA1 neurons following PDI knockdown. Open circles indicate each individual value. Horizontal bars indicate mean value. Error bars indicate SEM (*p < 0.05 vs. control siRNA; n = 7, respectively). i Representative immunofluorescent photos for PDI and mitochondria (Mito) in CA1 neurons following PDI knockdown. The right panels are the high magnification photos of the rectangles in the middle panel

Fig 4: The effects of l-NAME on SNO-PDI, SNO-DRP1, Trx1 activity, and mitochondrial fusion in CA1 neurons 3 days after SE.SE significantly reduces expression levels of DRP1 and PDI, PDI-binding to DRP1, and phosphorylation levels of DRP1-S616 and S637 levels independent of Trx1 activity. Both SNO-PDI and SNO-DRP1 are more diminished by l-NAME treatment. a Representative western blot for the effect of l-NAME on expression, S-nitrosylation, and phosphorylation levels of PDI and DRP1 following SE. b–d Quantification of values (mean ± S.E.M) of expression, phosphorylation, and S-nitrosylation levels of PDI and DRP1. Open circles indicate each individual value. Horizontal bars indicate mean value. Error bars indicate SEM (*,#p < 0.05 vs. non-SE animals and vehicle, respectively; n = 7, respectively). e Quantification of values (mean ± S.E.M) of Trx1 activity. Open circles indicate each individual value. Horizontal bars indicate mean value. Error bars indicate SEM (n = 7, respectively). f Representative western blot for the effect of l-NAME on co-immunoprecipitation of PDI interaction with DRP1 following SE. g Quantification of co-immunoprecipitation analyses of PDI interactions with DRP1. Open circles indicate each individual value. Horizontal bars indicate mean value. Error bars indicate SEM (*p < 0.05 vs. non-SE animals; n = 7, respectively)

Fig 5: Effect of BQ788 and Y-27632 on dysfunction of mitochondrial fission at 3 days after SE. a Western blot images of DRP1, DRP1 S616 and DRP1 S637 in the hippocampus. As compared to vehicle, both BQ788 and Y-27632 (a ROCK inhibitor) attenuate the reductions in DRP1 and DRP1 S616 expression, but increase DRP1 S637 expression. b Quantitative values (mean ± S.E.M) of DRP1, DRP1 S616, DRP1 S637 level (n = 10 per each group). Significant differences from vehicle, *p < 0.05. c Quantitative values (mean ± S.E.M) of DRP1 S616/S637 ratio in the hippocampus (n = 10 per each group). Significant differences from vehicle, *p < 0.05. d Quantitative values (mean ± S.E.M) of mitochondrial length in the CA1 neurons (n = 10 per each group). Significant differences from non-SE animals, *p < 0.05 e-h Representative photographs of mitochondria and NeuN in the CA1 neurons. SE increases mitochondrial length and sphere formation. Both BQ788 and Y-27632 alleviate mitochondrial elongation and sphere formation induced by SE. Bar = 6.25 µm