The discovery that Zn/Cu superoxide dismutase (SOD1) gene mutations are responsible for 15–20% of familial forms of amyotrophic lateral sclerosis (ALS)1 led to extensive studies of the susceptibility of the motor neuron to oxidative stress. The role of the normal SOD1 function—the conversion of toxic superoxide into less damaging hydrogen peroxide—in ALS pathogenesis remains unclear. It is, however, known that the restricted expression of mutant SOD1 in either motor neurons, microglia or astrocytes has repeatedly been demonstrated to be insufficient for an effective triggering of ALS symptoms.2 Microglia, in particular, have the capacity to recognize a stressed neuron and either attempt to restore the function (immune response) or release toxic factors to prune the compromised neurons. In the case of ALS, this is particularly damaging because the neurons already have difficulty coping with superoxide radicals, which wildtype SOD1 would typically reduce and remove. It was observed that one of the redox-related genes, which is specifically upregulated in activated microglia in spinal cords responding in ALS, is NOX2.3 The NADPH oxidase (NOX) enzymes operate by generating reactive oxygen species in a coordinated manner, often in response to inflammatory signals or microorganisms (Figure 1). Thus, by knocking out NOX2 or other redox-related genes, it could be predicted that motor neurons would have less damaging and fewer insults from activated microglia. In an article by Marden et al4, a hemizygous mouse that harbors a G93A SOD1 mutation was crossed with a mouse null for the NOX1 or the NOX2 genes. This result had a dramatic effect on the lifespan, in particular of NOX2-null mutant SOD1 mice, which survived on average 229 days compared with 132 days for the mice only with a G93A point mutation. This increase of almost 100 days is one of the largest effects observed for SOD1 mutant mice; most manipulations influence lifespan by at most 10–20 days. The exact nature of this benefit is not fully understood and should be the source of compelling future research.

Figure 1
figure 1

Effect of NOX genes on motor neuron physiology and motor neuron disease. (a) Prior to the onset of the disease, the motor neuron in mutant (G93A) SOD1 transgenic mice becomes stressed over time and sends signals to astrocytes and microglia. Microglia, in response to this stress (similar to their response to foreign attack by microorganisms), increase levels of NOX2, part of the NADPH oxidase complex. This complex generates toxic superoxide radicals that are converted to hydrogen peroxide by SOD1. (b) Increased superoxide and hydrogen peroxide concentration signals the recruitment and activation of additional microglia. Microglia activation and infiltration are often hallmarks of some of the detrimental effects of disease progression in the mouse. These reactive microglia can release toxic factors, including glutamate, a range of cytokines leading to high oxidative stress and excitotoxicity. This could lead to activation of receptors (GluR2, AMPA or IGF1 receptors) in the surface of motor neurons resulting in activation of signaling pathways (such as IGF1/Akt pathway) that target motor neurons for destruction. Knocking down NOX2 may prevent the generation of superoxide anions, further activation of microglia and inhibition of some of these signaling cascades, thus preventing death of motor neurons. (i) IGF1/Akt pathway, (ii) GluR2 AMPA receptors.

To examine the role that redox genes have in prolonging the lifespan of G93A SOD1 mice, these animals were compared with animals that are heterozygous or homozygous for mutant NOX1 or NOX2 genes on the mutant G93A SOD1 mouse background. Median lifespan was extended by 11 days in heterozygous NOX1 mice and by 33 days in NOX1-null homozygous mice. These levels were significantly different but much less than the levels observed for NOX2-deficient mice (54 days for heterozygous mice and 97 days for homozygous NOX2-null mice). Given the pronounced effects for NOX2-null mice, the morphological and behavioral effects on these mice were then carefully scrutinized. Superoxide production increases at the end-stage of disease for G93A SOD1 mice, but interestingly the levels were diminished to non-disease state following NOX2 deletion. In addition, motor neuron counts, coordination and stride length were all increased in these mice. Muscle weight and size was also restored to near levels observed for wildtype mice with no SOD1 mutation.

These results are particularly encouraging in light of recent experiments that were quite similar but less spectacular. Wu et al3 also crossed NOX2 null mice with G93A mice, but they observed a more modest increase in lifespan of 14 days. Why do the mice bred by Marden et al4 live dramatically longer? One key difference is the background strains used by the two groups (B6SJL4 versus congenic C57BL/6J3 strains). Modifier genes and other protective elements have been suggested to be present on the B6SJL background,5 which could act additively or synergistically with the NOX2 gene deletion for a more severe effect. This would correlate with human sporadic ALS cases where multiple genetic factors almost certainly exist and which interact to cause disease. Another explanation for the disparity in the results may have something to do with another unexpected feature of the mice studied by Marden et al4: 12.5% (1/8) of heterozygous mice and over 75% (19/25) of G93A mice homozygous for the NOX2 deletion were affected with a severe eye infection positive for Staphylococcus aureus that leads to rapid death if left untreated. Eye abnormalities are not something present in ALS (in humans or in mice). In mice with a noticeable eye infection, an antibiotic treatment using gentamicin and ceftazidime was administered, followed by 14 enrofloxacin subcutaneous injections if the symptoms were not alleviated. Some of the mice without eye infections also had an increased lifespan. However, the antibiotic treatment could certainly have influenced the progression of the disease in the treated mice, particularly in combination with NOX2 gene loss. This is something worth examining further.

Given the remarkable benefits observed from an absence of the NOX2 gene in the SOD1 G93A mice, the potential effects in humans must be considered. The presence of both NOX1 and NOX2 on the X chromosomes has important implications if considering how modifier genes may act. Male ALS patients have an increased predisposition to ALS (1.3–1.6:1 ratio) in sporadic forms of the disease.6 Thus, if the NOX genes are bona fide modifier genes, regulatory changes in these genes, which increase NOX gene production, would have a greater effect in males and help explain this gender bias. In addition, ALS patients who have SOD1 mutations (∼2% of all ALS cases) could have their age of onset of disease or absolute susceptibility influenced by different NOX genotypes.

Another critical question is: How can these results translate to therapeutics? As is often the case with mice models, a fair deal of caution must be exercised when correlating mouse effects to human, especially when considering the unexpected discoveries (eye disease) and variable effects (based on mouse strain differences) described above. Nonetheless, these findings provide exciting potential for experiments that decrease NOX genes in ALS patients to prevent activation of microglia and build-up of superoxide, particularly in patients positive for SOD1 mutations. The theme of oxidative stress as an influential aspect in ALS has received a great deal of attention. The interplay between toxic SOD1 and NADPH oxidases releasing superoxide in surrounding microglia suggests that even more consideration for oxidative stress in ALS is warrantedâ–ª