Fig. 2.

CT4 removes misfolded SOD1 endogenously expressed in neurons. a Dose-dependent knockdown on SOD1 endogenously expressed in neurons. Neurons prepared from transgenic mice expressing G93A-hSOD1 were treated with CT4 at 0 μM, 0.2 μM, 1 μM, or 5 μM for 3 h. b Time-course of CT4-directed knockdown of misfolded SOD1 in primary neurons expressing G93A-hSOD1. n = 9 cultures prepared from 3 mice. Blots in a, b were probed with the A5C3 antibody. Data were analyzed with one-way ANOVA followed by Bonferroni’s multiple comparison post-test. *P < 0.05; **P < 0.01. n = 9 cultures prepared from 3 mice. c Levels of misfolded SOD1 in neurons expressing G93A-hSOD1 after treatment with CT4 (5 μM). The neurons were immunostained with B8H10 (red) and MAP2 (green) antibodies. Scale bar = 20 μm. d Immunofluorescence staining with antibodies to pNF-H (green) and misfolded SOD1 (B8H10, red) after treatment with CT4 in myelinating culture. Scale bar = 100 μm (for lower magnification) and 20 μm (for higher magnification). e, f Knockdown of misfolded SOD1 in the sciatic nerve and spinal cord of the G93A-hSOD1 mice treated by intravenous injection of CT4 or mCT4 at 10 mg/kg body weight daily for three days at the age of 100 days. e Sciatic nerves were harvested at 24 h afterwards. Sections of sciatic nerves were immunostained with antibodies to MBP (green), and misfolded SOD1 (red) was detected with the B8H10 antibody. Shown are staining intensities of misfolded SOD1 in axons. n = 4 mice. Scale bars equal 20 μm (for lower magnification) and 5 μm (for higher magnification). f Spinal cord sections were immunolabeled with an antibody to CHAT (Green), and misfolded SOD1 (red) was detected with the B8H10 antibody. Shown are staining intensities of misfolded SOD1 in CHAT-positive neurons. n = 4 mice. Scale bar: 100 μm (lower magnification) and 10 μm (higher magnification). Data in d–f represented mean ± SD and was analyzed with a two-tailed Student’s t-test. **p < 0.01