Figure 3.

Phosphorylated ASK-1 (P-ASK1) in the spinal cord of ALS patients. The control spinal cord shows a weak cytoplasmic staining for P-ASK1 in the anterior horn motor neurons (a). ALS patient spinal cords had increased immunoreactivity for P-ASK1 in the cell bodies and proximal neurites of anterior horn motor neurons (c). P-ASK1 immunostaining was more expressed in the glial cells (astrocytes and microglia) of ALS patients (d, arrows) than those of controls (b). Magnification: 40x. (e) Quantitative RT-PCR analysis of ASK1 mRNA levels in the lumbar spinal cord of ALS patients (n = 12) and controls (n = 6). Data were analyzed by t-test (^∗p < 0.05 ALS versus controls). (f) Immunocytochemistry for phosphorylated ASK-1 (P-ASK1, red) in astrocyte-spinal neuron cocultures expressing SOD1^G93A and nontransgenic control (CTR). Large motor neurons expressing SOD1^G93A identified by SMI32 labeling (green) and morphological criteria (maximum diameter and shape) showed greater immunoreactivity for P-ASK1 than those of controls. Three-day treatment with neutralizing antibodies anti-TNFR1 (AbTNFR1) or anti-TNFR2 (AbTNFR2) reduced immunoreactivity for P-ASK1 in the transgenic motor neurons. Magnification: 60x; scale bar: 10 μm. The graph (g) shows the quantification of P-ASK1 immunoreactivity. The bar graph represents mean ± SD (% of untreated control) of three independent experiments. Data were analyzed by two-way ANOVA followed by Tukey's multiple comparison test (^∗∗p < 0.01 compared to untreated CTR; ^§§p < 0.01 compared to SOD1^G93A). (h) Effect of the P-p38MAPK inhibitor SB239063 on motor neuron viability in astrocyte-spinal cord cocultures expressing SOD1^G93A. SB239063 totally protected transgenic motor neurons in SOD1^G93A cocultures after six days in vitro. Data were analyzed by two-way ANOVA followed by Tukey's multiple comparison test (^∗∗p < 0.01 compared to untreated CTR). All experiments with cell cultures were done as previously described [45].