Figure 1.

Upregulation of α-adducin in SOD1^G93A astrocytes mediates non–cell autonomous degeneration of motor neurons. (a) Lysates of spinal cord from symptomatic SOD1^G93A transgenic mice and control non-transgenic mice were subjected to immunoblotting using an antibody that recognizes phosphorylation events in cells following exposure to oxidative stress. Lysates in lanes 5–8 were incubated with λ-phosphatase, which largely eliminated the 105-kDa immunoreactive band. (b) Lysates of spinal cords from symptomatic SOD1^G93A and control mice were subjected to immunoprecipitation using the α-adducin antibody followed by immunoblotting with our phospho-antibody. (c) Immunoblots from spinal cord lysates showed an increase in α-adducin and phosphorylated α-adducin relative to the internal control proteins ERK and 14-3-3β in symptomatic SOD1^G93A mice as compared with control wild-type littermates (120 d). (d) Immunoblots revealed that α-adducin and phosphorylated α-adducin were predominately expressed in primary glial cultures enriched with the astrocyte marker GFAP relative to primary motor neuron cultures enriched with the neuron marker β-tubulin. HSP60 was used as an internal control (lower panel). Blots shown in a–d are cropped. Full-length blots are presented in Supplementary Figure 11. (e) Immunohistochemistry of symptomatic SOD1^G93A lumbar spinal cord sections revealed that phosphorylated-Ser436 α-adducin (phospho-α-adducin) colocalized with the astrocyte protein GFAP. Dashed line indicates ventral horn. The boxed areas are shown at high magnification; scale bars represent 50 μm. (f) Co-cultured astrocytes and motor neurons were subjected to immunocytochemistry with antibodies recognizing the motor neuron nuclear protein Islet1 (red) and the dendrite protein Map2 (green); scale bar represents 50 μm. Wild-type astrocytes transfected with the control U6 or α-adducin RNAi plasmid had little or no effect on motor neuron morphology or survival (upper and lower left panels). Control U6 SOD1^G93A astrocytes induced non–cell autonomous motor neuron cell death and dendrite abnormalities (upper right panel). α-adducin knockdown in SOD1^G93A astrocytes protected motor neurons against the non–cell autonomous cell death and dendrite abnormality (lower right panel). (g,h) Quantification of motor neuron survival was derived from n ≥ 900 cells per condition and values presented are the average of three independent experiments; two-tailed unpaired t test, P = 0.0095. Quantification of dendrite length was derived from n ≥ 240 images per condition and values presented are the average of three independent experiments; two-tailed unpaired t test, P = 0.0001. Data are presented as mean ± s.e.m. **P < 0.01, ***P < 0.001.