FIGURE 2.

Potential implication of HMGB1 signaling in motor neuron-astrocyte communication in ALS. (A) The number of motor neurons showing HMGB1 into the nucleus or the cytoplasm was determined by immunohistochemistry on spinal cord sections from non-transgenic (non-Tg) or SOD1^WT and SOD1^G93A transgenic mice taken at 30 days (30 days, asymptomatic stage), ∼100 days (100 days, onset of motor deficits), and 130 days of age (130 days, symptomatic stage). Data (mean ± SEM) are expressed as percentage of motor neurons with nuclear or cytoplasmic HMGB1 on the total number of analyzed cells. ^∗∗p < 0.01 and ^∗p < 0.05 vs. 30 days SOD1^G93A; two-way ANOVA, followed by Bonferroni post hoc test, n = 3 mice per age, n = 190–313 motor neurons per genotype and age. Insets are representative immunofluorescent images of spinal cord sections double immunostained for SMI32 (red) and HMGB1 (green); nuclei were stained with Hoechst 33342 (blue). Scale bar, 10 μm. (B) Expression of BDNF and GDNF was determined in primary astrocytes from the spinal cord of non-Tg, SOD1^WT and SOD1^G93A mice. Cells were exposed to disulfide-HMGB1 (3 μg/ml) for 6 h. Total RNA was extracted and analyzed by RT-qPCR. Values (mean ± SEM) were normalized relative to HPRT and expressed as percentage of control (ctrl), i.e., the corresponding culture type challenged with saline. ^∗∗∗p < 0.001, ^∗∗p < 0.01 vs. ctrl; ^###p < 0.001 vs. HMGB1 in non-Tg and SOD1^WT astrocytes; two-way ANOVA, followed by Bonferroni post hoc test, n = 5–17 experiments, in triplicate.