Caloric Restriction and ALS Figure 4. Correlation between clinical score and ability to move and between clinical score and paw grip endurance in CR vs. AL G93A mice. (A) Relation between clinical score (from clinical onset to euthanasia) and ability to move and (B) between clinical score and paw grip endurance (s) 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. For ability to move, slopes and intercepts were not different with the following pooled equation: ability to move = (21.4) x clinical score + (7.1). For paw grip endurance, data for the females followed a sigmoidal relationship (AL females, r2 = 0.990; CR females, r2 = 0.995; curves were significantly different, P,0.0001) whereas data for the males followed a linear relationship (slopes were significantly different, P = 0.0011). For AL males (r = 20.984, P,0.0001), paw grip endurance = (227.861.1) x clinical score + (133.563.8); for CR males (r = 20.995, P,0.0001), paw grip endurance = (231.860.7) x clinical score + (159.462.9), with the data presented as means 6 SD. Data are means of each group on the same day. doi:10.1371/journal.pone.0009386.g004 protein content in females vs. males (2.5-fold, P = 0.026). An interaction between diet and sex revealed that CR females were significantly different from CR males (P = 0.038), AL females (P = 0.031) and AL males (P = 0.020). Within each sex, MnSOD was significantly elevated in CR vs. AL females (4-fold, P = 0.015), but not in CR vs. AL males. In the white gastrocnemius, MnSOD was higher in CR vs. AL mice (78%, P = 0.062; Fig. 9, panel B), with no sex differences. MnSOD was higher in CR vs. AL females (2.2-fold, P = 0.038) only. Despite the upregulation in the protein content of MnSOD, Cu/Zn-SOD, another potent antioxidant enzyme, was only up-regulated in red gastrocnemius. Cu/Zn- SOD was higher in CR vs. AL red gastrocnemius (67%, P = 0.096; Fig. 9, panel C), with no sex differences. Cu/Zn-SOD was significantly higher in CR vs. AL females (2.6-fold, P = 0.020). In the white gastrocnemius, there was no change in Cu/Zn-SOD protein content (Fig. 9, panel D). Catalase enzyme activity was not measured in red gastrocne- mius due to insufficient amounts of tissue. In white gastrocnemius, catalase activity, measured from the kinetic decomposition of H2O2, was not different between female and male mice under CR or AL (Fig. 10). Protein Content of TNF-a – Marker of Inflammation TNF-a, a marker of inflammation, was significantly higher in the quadriceps of CR vs. AL mice (52%, P = 0.030; Fig. 11). There were no sex differences in the protein content of TNF-a. Protein Content of Genes Related to Cell Stress Response and Apoptosis The protein content of a molecular chaperone, Hsp70, generally up-regulated in response to cell stress, was reduced as a result of CR. In the quadriceps, Hsp70 was significantly lower in CR vs. AL (62%, P = 0.002; Fig. 12) and in male vs. female (37%, P = 0.030) mice. Within each sex, Hsp70 was significantly lower in CR vs. AL females (64%, P,0.001) and in CR vs. AL males (59%, P = 0.039). In relation to the heightened inflammation and decreased ability to respond to stress under CR, Bax, a pro-apoptotic protein, PLoS ONE | www.plosone.org 7 February 2010 | Volume 5 | Issue 2 | e9386