Caloric Restriction and ALS Figure 6. Mitochondrial energetics in CR vs. AL G93A mice. (A) COX to citrate synthase activity ratio was lower in the quadriceps of CR vs. AL G93A male mice (34%, P = 0.034). (B) Cytochrome c oxidase subunit-IV to citrate synthase protein content ratio was higher in CR vs. AL G93A male mice (2.3-fold, P = 0.059). (C) UCP3 was higher in CR vs. AL female quadriceps (1.5-fold, P = 0.042). Data are presented as means 6 SEM. n = 27; AL, 7 males and 7 females; CR, 7 males and 6 females. Asterisks denote significant changes (P#0.05 vs. AL); dagger denotes strong trend (0.05,P#0.1 vs. AL male). doi:10.1371/journal.pone.0009386.g006 score of 4 as a proxy measure of endpoint would result in similar conclusions as using a clinical score of 5. Both the female sex and CR induced an increase in 24-h voluntary activity. We examined the effect of long-term CR on markers of mitochondrial bioenergetics, oxidative damage, antioxidant en- zyme capacity, inflammation, stress response and apoptosis in the skeletal muscle of G93A mice, an animal model of ALS. In this study, we ascertained that CR reduced the ratio of COX to citrate synthase activity in male G93A mice despite an up-regulation of Figure 7. Lipid peroxidation in CR vs. AL G93A mice. (A) MDA was higher in CR vs. AL (83%, P,0.001; main effect of diet), and in female vs. male (32%, P = 0.045; main effect of sex) red gastrocnemius. Within each sex, MDA was higher in CR vs. AL females (2-fold, P = 0.003), and in CR vs. AL males (56%, P = 0.005). (B) MDA was higher in CR vs. AL white gastrocnemius (14%, P = 0.095; main effect of diet), with no sex differences. Data are presented as means 6 SEM. Asterisks denote significant changes (P#0.05 vs. AL); dagger denotes strong trend (0.05,P#0.1 vs. AL). doi:10.1371/journal.pone.0009386.g007 the ratio of COX subunit-IV to citrate synthase protein content. Contrarily, CR females upregulated UCP3, a marker of mitochondrial energetics, which is activated in response to enhanced oxidative stress [21]. CR increased lipid peroxidation, but decreased protein oxidation. The increase in oxidative stress was accompanied by a substantial upregulation in the protein content of antioxidant enzymes MnSOD and Cu/Zn-SOD in CR vs. AL females. This upregulation in antioxidant enzymes was not observed in CR males. Furthermore, CR increased markers of inflammation and apoptosis, while decreasing Hsp70, a stress response protein. The life-extending properties of CR have been observed in insects, rodents and non-human primates, and are mainly attributed to a reduction in mitochondrial oxidant production and an increase in mitochondrial bioenergetic efficiency [3–7]. However, nutritional status in ALS patients is predictive of survival [22], with a low energy intake significantly correlating with death in this population [23,24]. Research involving G93A mice, the animal model of ALS used in this study, has provided strong PLoS ONE | www.plosone.org 9 February 2010 | Volume 5 | Issue 2 | e9386