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. 2016 Mar 21;291(20):10836–10846. doi: 10.1074/jbc.M115.698779

FIGURE 6.

FIGURE 6.

Enhancing NAD+ salvage pathway reverts the toxicity of astrocytes expressing ALS-linked mutant SOD1. A, confluent non-transgenic (NonTG) and hSOD1G93A (G93A) spinal cord astrocyte monolayers were treated with vehicle (Control), 5 mm NMN, or 5 mm NR. 24 h later purified motor neurons from non-transgenic E12.5 mice were plated on top of the astrocyte monolayer. B, the same experimental setup as in A was used, and purified motor neurons from hSOD1G93A E12.5 mice were plated on top of the astrocyte monolayer. For A and B, motor neuron survival was assessed 72 h later. *, significantly different from non-transgenic control (p < 0.05). C, confluent spinal cord astrocyte monolayers obtained from non-transgenic, hSOD1G93A, and hSOD1H46R/H48Q (H46R/H48Q) mice were transfected with adenovirus expressing GFP, NAMPT, or mNAMPT. 24 h later purified motor neurons from non-transgenic E12.5 mice were plated on top of the astrocyte monolayer. Motor neuron survival was assessed 72 h later. *, significantly different from non-transgenic GFP (p < 0.05). D and E, confluent non-transgenic (N) and hSOD1G93A (G) astrocytes were transfected with a negative control (NC), Sirt1, or Sirt3 siRNA, and 48 h later protein levels were determined by Western blotting. Actin levels were used as a loading control. A representative image is presented, and the bottom panels show the quantification of at least three experiments for each siRNA. *, significantly different from non-transgenic negative control (p < 0.05). F, confluent non-transgenic and hSOD1G93A spinal cord astrocytes monolayers were treated as in D and E, and 24 h later purified motor neurons from non-transgenic E12.5 mice were plated on top of the astrocyte monolayer. Motor neuron survival was assessed 72 h later. *, significantly different from non-transgenic negative control (p < 0.05). G and H, confluent hSOD1G93A astrocytes were transfected with plasmids coding for Sirt1, Sirt3, or an empty control plasmid, and 48 h later protein levels were determined by Western blotting. Actin levels were used as a loading control. The right panels show the quantification. To detect endogenous sirtuin expression (empty vector samples), the levels on the overexpressing samples had to be overexposed. Thus, the -fold change is likely underestimated. *, significantly different from empty control (p < 0.05). I, confluent non-transgenic and hSOD1G93A spinal cord astrocyte monolayers were transfected with plasmids coding for Sirt1, Sirt3, or an empty control plasmid, and 24 h later purified motor neurons from non-transgenic E12.5 mice were plated on top of the astrocyte monolayer. *, significantly different from non-transgenic empty (p < 0.05). For all panels, each data bar represents the mean ± S.D. (error bars) of at least three independent experiments.