Caloric Restriction and ALS Figure 5. Probability of survival in CR vs. AL G93A mice. Probability of survival using as endpoint a clinical score of (A) 4 and (B) 5 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 reaching endpoint in the CR mice (i.e., the hazard ratio) was 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). doi:10.1371/journal.pone.0009386.g005 was elevated in CR mice. Bax was significantly higher in CR vs. AL mice (41%, P = 0.027; Fig. 13, panel A) and in CR vs. AL females (52%, P = 0.048). While there was no increase in the protein content of Bcl-2, an anti-apoptotic protein in the quadriceps (Fig. 13, panel B), the ratio of Bax/Bcl-2 protein content indicated heightened apoptosis under CR. Bax/Bcl-2 was significantly higher in CR vs. AL mice (68%, P = 0.040; Fig. 13, panel C), and in CR vs. AL females (2.3-fold, P = 0.029) only. During apoptosis, caspase 9 is activated to form cleaved caspase 9. However, we did not observe any changes in the protein content of caspase 9 (Fig. 13, panel D), cleaved caspase 9 (Fig. 13, panel E) or the ratio of cleaved caspase 9/caspase 9 for diet or sex (Fig. 13, panel F) Discussion This is the first study to illustrate that CR is detrimental. In the current study, CR equivalent to 60% of AL hastened clinical onset of disease, disease progression and endpoint in the G93A mouse, an animal model of ALS. We also ascertained that using a clinical PLoS ONE | www.plosone.org 8 February 2010 | Volume 5 | Issue 2 | e9386