Figure 1. ALS-associated SOD1 mutations activate cellular Nox activity.

(A) Rate of NADPH-dependent O₂^•– production by total endomembranes isolated from the brain, spinal cord, and liver of nontransgenic or transgenic mice overexpressing SOD1^WT or SOD1^G93A (mean ± SEM; n = 3 per group). (B) DHE fluorescent detection of O₂^•– in lumbar spinal cord sections from nontransgenic and transgenic mice overexpressing SOD1^WT or SOD1^G93A. DAPI staining shows cell nuclei in each section. (C) Rate of NADPH-dependent O₂^•– production in total endomembranes isolated from SH-SY (neuronal) or MO59J (glial) cells at 48 hours following infection with adenoviral vectors expressing LacZ, SOD1^WT, SOD1^L8Q, or SOD1^G93A. (D) Cell death was quantified in SH-SY and MO59J cells using trypan-blue exclusion at 72 hours after infection with the indicated adenoviral vectors. (E) Using conditions specified in C and D, the rate of NADPH-dependent O₂^•– production and cell death was assessed in the presence or absence of the Nox inhibitor apocynin (100 μM). Values are mean ± SEM (n = 6 per group). (F) Assessment of GTP-bound Rac1 (activated form) in spinal cord lysates from 2 nontransgenic or 3 SOD1^G93A transgenic mice (120 days old) and from MO59J cells overexpressing WT or mutant SOD1 proteins (at 36 hours after adenoviral infection). The 2 left lanes are controls for the Rac activation assay, in which nontransgenic spinal cord lysates were preincubated with the indicated non-hydrolysable guanine nucleotide analogs.