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. Author manuscript; available in PMC: 2016 Jan 6.
Published in final edited form as: Nat Neurosci. 2014 Oct 26;17(12):1710–1719. doi: 10.1038/nn.3853

Figure 5.

Figure 5

Na/K ATPase stimulates mitochondrial respiration and expression of inflammatory genes in SOD1G93A astrocytes. (a) Control SOD1G93A astrocytes (left) induced non–cell autonomous motor neuron cell death and dendrite abnormalities. Pharmacological inhibition of Na/K ATPase with ouabain (middle) and digoxin (right) was neuroprotective from the non–cell autonomous cell death and dendrite abnormalities induced by SOD1G93A astrocytes (left); scale bar represents 50 μm. Pharmacological inhibition of Na/K ATPase in control co-cultures non-transgenic astrocytes (Non-tg) and motor neurons with ouabain and digoxin did not alter motor neuron survival or morphology. (b,c) 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: ouabain, P = 0.0024; digoxin, P = 0.0001). Quantification of dendrite lengths was derived from n ≥ 240 images per condition and values presented are the average of three independent experiments (two-tailed unpaired t test: ouabain, P = 0.0002; digoxin, P = 0.0001). Data are presented as mean ± s.e.m. **P < 0.01, ***P < 0.001. (d) A representative plot of oxygen consumption measured in astrocytes from control non-transgenic, SOD1G93A and heterozygous-null SOD1G93A mice using Seahorse Bioscience XF Analyzer. Arrow indicates time when the mitochondrial uncoupler FCCP was added. (e) Basal and maximum oxygen consumption were quantified in control non-transgenic (n = 3), SOD1G93A (n = 3) and heterozygous-null SOD1G93A mice (n = 3) astrocytes using Seahorse Bioscience XF Analyzer (two-tailed unpaired t test: basal consumption, P = 0.0001; maximum consumption, P = 0.0001). Data are presented as mean ± s.e.m. ***P < 0.001. (f) Total RNA of astrocytes from control non-transgenic (n = 3), SOD1G93A (n = 3) and heterozygous-null SOD1G93A mice (n = 3) were subjected to qRT-PCR analyses using primers to a panel of pro-inflammatory genes. Gene expression was normalized to GAPDH expression. The expression of 18 inflammatory genes was upregulated in SOD1G93A astrocytes. Downregulation of α2-Na/K ATPase in SOD1G93A astrocytes significantly decreased expression of half of the upregulated inflammatory genes (two-tailed unpaired t test: SPP1, P = 0.0253; LCN2, P = 0.0013; CLM1, P = 0.0052; WNT1, P = 0.0198; CCL11, P = 0.0387; CXCL1, P = 0.0246; CCR4, P = 0.0314; Il1b2, P = 0.0421; Itgb2, P = 0.0215; II1r1, P = 0.0092). Data are presented as mean ± s.e.m. *P < 0.05, **P < 0.01, ***P < 0.001.

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