Figure 1.

Exposure of primary spinal cord cultures to astrocyte conditioned media (ACM) derived from SOD1^G93A, SOD1^G86R, and TDP43^A315T expressing mice triggers death of motoneurons. (A) Flow diagram of experiment. Primary wild-type (WT) rat spinal cord cultures (4 DIV) were exposed for 3 days to ACM derived from transgenic mice overexpressing SOD1^G93A (ACM-SOD1^G93A), SOD1^G86R (ACM-SOD1^G86R), or TDP43^A315T (ACM-TDP43^A315T). Cells were fixed at 7 DIV, and cell survival was assayed with immunocytochemistry. (B) Fixed 7 DIV spinal cord cultures were double-labeled with anti-microtubule-associated protein 2 (MAP2) antibody (red) to visualize interneurons (arrowhead) and with the SMI-32 antibody (green) to identify motoneurons (arrow). Scale bar, 25 μm. (C) Representative images of MAP2⁺/SMI32⁺-labeled neurons in spinal cultures under control conditions (top image) or treated with the three different ACMs, as indicated in the figure. Scale bar, 200 μm. (D) Graph showing the percentage of motoneurons that survived after treatment with ACMs derived from SOD1^G93A, SOD1^G86R, and TDP43^A315T astrocytes, relative to motoneurons from sister cultures treated with control medium. (E) Graph showing the percentage of motoneurons that survived after treatment with media derived from mouse littermates that were negative for the mutated forms of SOD1 and TDP43 (ACM-NT-Control), or with media derived from transgenic mice carrying the non-pathological human wild-type SOD1 gene (ACM-SOD1^WT). Survival is shown relative to cultures treated with control media. Values represent mean ± s.e.m. from at least 3 independent experiments performed in duplicate, analyzed by One-Way ANOVA followed by a Tukey post-hoc test. ^***P < 0.001 relative to control medium at 7 DIV.