Clinical Onset Clinical score was significantly higher in CR vs. AL mice (P,0.0001). The age of mice at clinical onset was 8561 d for AL mice and 8061 d for CR mice (P = 0.005), 5 days younger for the CR mice, a 6% difference. Males showed a trend for reaching clinical onset 3 days younger than females (P = 0.096; females, 8361 d; males, 8062 d). The clinical onset curves were signifi- cantly different between the groups (Logrank test, P = 0.003), with the rate of attaining clinical onset (i.e., the hazard ratio) in the CR mice being 2-fold higher (95% CI: 1.4, 4.7) than the AL mice (Fig. 3). CR females had a 2-fold (95% CI: 1.2, 5.3) higher rate of attaining clinical onset as compared with AL females (P = 0.016). There was no sex difference in the rate of attaining clinical onset. Disease Progression There was no sex difference in disease progression for either endpoint (clinical score of 4 or 5). Using clinical score of 4 as endpoint, progression of the disease from clinical onset to endpoint was 8 d faster for the CR (3762 d) vs. AL (4563 d) mice (P = 0.011), an 18% difference. Using clinical score of 5 as endpoint, progression of the disease from clinical onset to endpoint was 6 d faster for the CR (4261 d) vs. AL (4863 d) mice (P = 0.014), a 13% difference. From clinical onset (clinical score of 2) to euthanasia, ability to move strongly and negatively correlated with clinical score (AL females, r = 20.997; AL males, r = 20.996; CR females, r = 20.997; CR males, r = 20.994) (Fig. 4). There was no difference between the curves. There was a strong relation between clinical score and PaGE (Fig. 4). For the females, the relationship was sigmoidal (AL females, r2 = 0.990; CR females, r2 = 0.995; curves were signifi- cantly different, P,0.0001). For the males, the relationship was linear (AL males, r = 20.984; CR males, r = 20.995; slopes were significantly different, P = 0.0011). Survival There was no sex difference in survival for either endpoint (clinical score of 4 or 5). The age of mice at clinical score of 4 was Figure 3. Probablility of clinical onset in CR vs. AL G93A mice. Probability of clinical onset in 23 ad libitum (AL: N, 16 females; &, 7 males) and 31 calorie restricted (CR, 60% of ad libitum: #, 21 females; %, 10 males) G93A mice. The rate of attaining clinical onset (i.e., the hazard ratio) in the CR mice was 2-fold higher (95% CI: 1.4, 4.7) than the AL mice. CR females had a 2-fold (95% CI: 1.2, 5.3) higher rate of attaining clinical onset as compared with AL females (P = 0.016). There was no sex difference in the rate of attaining clinical onset. doi:10.1371/journal.pone.0009386.g003 Caloric Restriction and ALS 12962 d for the AL mice and 11661 d for the CR mice (P,0.0001), 13 d younger for the CR mice. This is equivalent to a 10% decrease in life span. Survival curves were significantly different between the groups (P,0.0001; Fig. 5), with the rate of reaching endpoint in the CR mice (i.e., the hazard ratio) being 3.1- fold higher (95% CI: 2.9, 10.7) than the AL mice. The rate of reaching endpoint was 3.1-fold (95% CI: 2.6, 13.5) higher in the CR vs. AL females (P,0.0001) and 3.4-fold (95% CI: 1.9, 20.7) higher in the CR vs. AL males (P = 0.003). The age of mice at clinical score of 5 was 13262 d for the AL mice and 12061 d for the CR mice (P,0.0001), 12 days younger for the CR mice. This is equivalent to a 9% decrease in life span. Survival curves were significantly different between the groups (P,0.0001; Fig. 5), with the rate of reaching endpoint in the CR mice (i.e., the hazard ratio) being 3.1-fold higher (95% CI: 2.6, 9.8) than the AL mice. The rate of reaching endpoint was 2.9-fold (95% CI: 2.1, 10.2) higher in the CR vs. AL females (P = 0.0001) and 4-fold (95% CI: 2.8, 34.2) higher in the CR vs. AL males (P = 0.0004). There were no significant differences in the anthropometric data between the group which was followed to endpoint vs. the group that was sacrificed at age 99 d. Mitochondrial Oxidative Capacity In quadriceps, there were no differences in CS protein content and enzyme activity, cytochrome c oxidase subunit- I and IV protein content and COX activity between CR and AL or female and male mice (data not shown). However, COX to citrate synthase activity ratio was lower in CR vs. AL males (34%, P = 0.034; Fig. 6, panel A). Interestingly, cytochrome c oxidase subunit-IV to citrate synthase protein content ratio was higher in CR vs. AL males (2.3-fold, P = 0.059; Fig. 6, panel B). The protein content of UCP3 was significantly higher in CR vs. AL females (1.5-fold, P = 0.042; Fig. 6, panel C). However, unlike the CR females, UCP3 protein content was not significantly higher in CR vs. AL males. Lipid Peroxidation Spectrophotometric analysis of lipid peroxidation in red and white gastrocnemius revealed that MDA, a proxy measure of lipid damage, was higher in both female and male mice under CR vs. AL. MDA was higher in red gastrocnemius (83%, P,0.001; Fig. 7, panel A) as a result of CR, with females having higher levels than males (32%, P = 0.045). 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). In the white gastrocnemius, MDA was higher (14%, P = 0.095; Fig. 7, panel B) due to CR, with no sex differences. Protein Oxidation PC, an indication of oxidative damage to proteins, did not increase as a result of CR. In the red gastrocnemius, CR significantly lowered PC (62%, P,0.001; Fig. 8, panel A), with no sex differences. Within each sex, PC was lower in CR vs. AL females (50%, P = 0.012), and in CR vs. AL males (74%, P,0.001). In the white gastrocnemius, CR lowered PC (30%, P = 0.003; Fig. 8, panel B), with no sex differences. PC was lower in CR vs. AL females (43%, P,0.001) only. Antioxidant Enzymes The protein content of the antioxidant enzyme MnSOD displayed a significant upregulation in red gastrocnemius. MnSOD was significantly higher in CR vs. AL red gastrocnemius (3-fold, P = 0.031; Fig. 9, panel A), with a substantial upregulation in the PLoS ONE | www.plosone.org 6 February 2010 | Volume 5 | Issue 2 | e9386