Fig 2: Effects of Cu-Zn superoxide dismutase (SOD) on si-SCN2A vector transfection induced resting membrane electric potential. (A and B) Downregulated transcript and protein expression levels in SCN2A following transfection with the si-SCN2A vector, respectively. (C) No significant signals were observed in the si-SCN2A vector groups by immunofluorescence compared with the other groups. (D) Cu-Zn SOD concentration among the control, empty vector and si-SCN2A vector groups. Different letters (a or b) show significant differences among the groups (P<0.05). (E) Resting membrane electric potential detected by single-cell patch clamp showed a repeated discharge caused by si-SCN2A while the overexpression of Cu-Zn SOD prevents the repeated discharge.

Fig 3: NCX1 and NCX3 expression and function in rat primary motor neurons. A Immunolocalization of NCX1 and NCX3 isoforms in two different motor neurons within the same culture. B Superimposed representative traces of the effect of Na+-free on [Ca2+]i in motor neurons singly transfected with siControl, siNCX1 or siNCX3 (both at 10 nmol/L for 48 h). For details, please refer to Material and Methods. C Quantification of B expressed as ?% of increase. All the experiments were repeated at least three times; *p < 0.001 versus internal control (basal values of [Ca2+]i), **p < 0.05 versus All. D Quantification of basal values of [Ca2+]i of the treatments of B. *p < 0.05 versus All. E Immunolocalization of NCX1 and SOD1 in motor neuron-enriched culture at two different magnifications

Fig 4: Effect of SOD1 and ApoSOD1 on phospho-Akt expression and localization in rat primary motor neurons. A Immunolocalization of phospho-Akt (p-Akt) in MAP2-positive cells within a motor-neuron enriched culture under control conditions (a–c), exposed to SOD1 (400 ng/ml/10 min) (d–f) or ApoSOD1 (400 ng/ml/10 min) (g–i). Motor-neuron enriched culture were harvested before the treatment with SOD1 or ApoSOD1. White arrows indicate MAP2-positive neurons with a nuclear localization of p-Akt. Bar graph at the bottom represents the % of p-Akt-positive nuclei in each group. *p < 0.05 versus control. B Representative Western blotting and quantification of the effect of SOD1, and ApoSOD1 (both at 400 ng/ml/10 min) on p-Akt and Akt expression in the absence or presence of siNCX1 (10 nmol/L for 48 h). Data are expressed as mean ± SE of three different experimental sessions. *p < 0.05 versus siControl; **p < 0.05 versus siControl, SOD1 and ApoSOD1. C Bar graph depicting the effect of L-BMAA (300 µM/48 h) on cell survival in rat primary motor neurons pretreated with SOD1, or ApoSOD1 (both at 400 ng/ml/10 min) in the presence or absence of siNCX1 or after exposure to the NCX1 activator CN-PYB2 (10 nM). Data are expressed as mean ± S.E. of three different experimental sessions. *p < 0.05 versus control; **p < 0.05 versus L-BMAA alone; ***p < 0.05 versus L-BMAA + SOD1 or L-BMAA + ApoSOD1

Fig 5: Effect of SOD1 and ApoSOD1 on NCX1-mediated [Ca2+]i influx and [Na+]i in rat primary motor neurons. A Co-localization between plasmalemmal NCX1 and SOD1 in a representative rat primary motor neuron at two different magnifications (see arrows). B Quantification of SOD1 and ApoSOD1-induced [Na+]i increase in SBFI-loaded motor neurons (see representative images on the top). Data are quantified as ?% of increase in 35 cells for each group. All experiments were repeated at least three times; *p < 0.05 versus internal control (basal values of [Na+]i). C Superimposed representative traces of the effect on [Ca2+]i of SOD1 (400 ng/ml) in Na+-free solution and ApoSOD1 (400 ng/ml) in Na+-free solution both perfused on siControl neurons or siRNA-treated neurons against NCX1 (10 nmol/L for 48 h) loaded with Fura-2 (see representative images on the top). D Quantification of the effect of C reported as ?% of increase in 40 cells for each group. All the experiments were repeated at least three times; *p < 0.001 versus SOD1 or ApoSOD1 alone (in the presence of external Na+); **p < 0.05 versus “SOD1 + Na+-free” or “ApoSOD1 + Na+-free”