Redox modifier genes in amyotrophic lateral sclerosis in mice

Abstract

Amyotrophic lateral sclerosis (ALS), one of the most common adult-onset neurodegenerative diseases, has no known cure. Enhanced redox stress and inflammation have been associated with the pathoprogression of ALS through a poorly defined mechanism. Here we determined that dysregulated redox stress in ALS mice caused by NADPH oxidases Nox1 and Nox2 significantly influenced the progression of motor neuron disease caused by mutant SOD1(G93A) expression. Deletion of either Nox gene significantly slowed disease progression and improved survival. However, 50% survival rates were enhanced significantly more by Nox2 deletion than by Nox1 deletion. Interestingly, female ALS mice containing only 1 active X-linked Nox1 or Nox2 gene also had significantly delayed disease onset, but showed normal disease progression rates. Nox activity in spinal cords from Nox2 heterozygous female ALS mice was approximately 50% that of WT female ALS mice, suggesting that random X-inactivation was not influenced by Nox2 gene deletion. Hence, chimerism with respect to Nox-expressing cells in the spinal cord significantly delayed onset of motor neuron disease in ALS. These studies define what we believe to be new modifier gene targets for treatment of ALS.

Figure 1. Deletion of NADPH oxidase genes (Nox1 or Nox2) enhances survival and survival index in ALS mice and significantly reduces superoxide production in spinal cords of end-stage SOD1^G93A mice.

(A) Survival curves for hemizygous SOD1^G93A transgenic mice with the indicated Nox1 and Nox2 genotypes. Median 50% survival rates were as follows: Nox1-WT (n = 8), 129 days; Nox1-HET (n = 6), 140 days, P < 0.03 versus Nox1-WT; Nox1-KO (n = 7), 162 days, P < 0.004 versus Nox1-WT; Nox2-WT (n = 16), 132 days; Nox2-HET (n = 8), 186 days, P < 0.0001 versus Nox2-WT; Nox2-KO (n = 24), 229 days, P < 0.0001 versus Nox2-WT. (B) Survival index for mice in A; onset of disease was defined as 10% weight loss from peak weight. Values are mean ± SEM. *P = 0.059, ^†P < 0.0001 versus WT. (C and D) NADPH-dependent superoxide production in spinal cords of hemizygous SOD1^G93A transgenic mice with WT, HET, and KO Nox2 genotypes. The relative mean rate ± SEM of superoxide production is plotted. (C) Ages at the time of spinal cord harvest were as follows: Nox2-WT, 125, 132, and 124 days; Nox2-HET, 186, 179, and 183 days; Nox2-KO, 224, 223, and 242 days (n = 3 per genotype). All mice were at the stage of clinical death at the time of harvest. ^†P < 0.008, *P < 0.045 versus WT; Student’s t test. (D) All mice were harvested at 120 days of age (n = 4 per genotype). ^†P < 0.0001 versus WT; Student’s t test.

Figure 2. Nox2 deficiency rescues motor neuron death in the spinal cords of mice hemizygous for the SOD1^G93A transgene.

Motor neurons were quantified in the lumbar region of the spinal cord using Cresyl violet staining at 120 days for the following genotypes: SOD1^G93A/Nox2-WT, SOD1^G93A/Nox2-KO, Nox2-WT, and Nox2-KO. In total 3 animals were included in each group and they were all derived from the same breeder pair (2 independent litters). (A) Representative photomicrographs of spinal cord section stained in Cresyl violet. Bottom panels show higher-magnification views of the boxed regions above. (B) Morphometric data on motor neuron counts for the given genotypes. Results depict the mean ± SEM for the indicated number of male and female animals quantified in each group. (C) Immunohistochemical staining for CD11b in spinal cords of the same mice shown in A. Scale bars: 200 μm (A and C, upper panels); 100 μm (A and C, lower panels).

Figure 3. Disease phenotyping of Nox2 genotypes on the SOD1^G93A ALS background.

(A) Survival data of male and female mice for the given genotypes. Boxes denote mice treated for eye infections with antibiotics; those marked with an X denote mice that were unsuccessfully treated and died from eye infections. Circles denote mice that never contracted eye infection. Numbers denote mean survival in days. (B) Rotarod data demonstrating the mean latency time maintained on the rotarod for each given genotype as a function of age. Results are mean ± SEM for the same mice as in A. (C) Stride length data demonstrating the mean stride distance for each given genotype as a function of age. Results are mean ± SEM for the same mice as in A. (D) Mean stride lengths at 120 days for the indicated genotypes. Representative raw stride length data is shown at right.