The significant decrease in Hsp70, together with the heightened peroxidative damage and inflammation we observed in G93A mice, may be the catalysts for apoptotic cell death under CR. In symptomatic G93A mice, Bax levels are increased and Bcl-2 levels are decreased in the spinal cord as compared to age-matched nontransgenic controls [80]. Bcl-2, an antiapoptotic protein, and Bax, a proapoptotic protein, can moderate levels of apoptosis by preventing or promoting the release of cytochome c [62]. The elevation in TNF-a protein content observed in CR mice may elicit cell death through the activation of apoptotic proteins [62]. Furthermore, the decrease in Hsp70 in the CR mice is indicative of increased apoptosis, since the presence of Hsp prevents the recruitment of caspase 9 to the apoptosome [81]. In our study, there was a significant increase in Bax protein content in CR vs. AL mice. Normally, CR functions to decrease oxidative stress by inhibiting apoptosis [62] through a reduction in Bax (in the aging mouse heart) [82] and elevation in Bcl-2 (in the aging rat kidney) [83]. We observed that the ratio of Bax/Bcl-2 also increased, signifying enhanced cell death in the skeletal muscle of CR mice by increasing the activation of mitochondrial permeability transition pores [84]. Moreover, there was no change in Bcl-2 protein content between CR and AL mice. Our finding corresponds with another study which did not find an effect of CR (70% of AL) on Bcl-2 gene expression in male rat hepatocytes [85]. The unaltered protein content of Bcl-2 suggests a lack of protection from apoptosis, since the over-expression of Bcl-2 prolongs lifespan in G93A mice [86]. Additionally, 2B4 cells treated with an apoptotic signal, dexamethasone, resulted in lipid peroxidative damage, which was prevented by the over-expression of Bcl-2 [87]. The elevated lipid peroxidation in our mice may be the result of an imbalance between proapoptotic and antiapoptotic proteins. This observation is consistent with the increased MDA, in conjunction with significantly elevated levels of Bax and the ratio of Bax/Bcl-2 protein content in CR vs. AL females. It is likely that the heightened lipid peroxidation led to higher levels of Bax in females, although these levels were not significantly different between CR females and males. However, the protein content of Bax/Bcl-2 in CR females is indicative of higher apoptosis. Activation of this proapoptotic protein may potentially trigger apoptotic cell death and is a possible explanation of why CR hastened the clinical onset of disease and disease progression in G93A mice Conversely, there were no significant differences in the protein content of caspase 9, cleaved caspase 9 or the ratio of cleaved caspase 9/caspase 9. During apoptosis, translocation of Bax from the cytosol to the mitochondria in the spinal cords of G93A mice [88] triggers the release of cytochrome c from the mitochondria [89]. Once in the cytosol, cytochrome c interacts with Apaf-1 and dATP forming an apoptosome that recruits caspase 9 [90]. This initiator caspase can then activate downstream effector caspases 3 and 7 [91], which can cleave caspase 9 into its smaller 37 kDa fragment, signaling mitochondrial-dependent apoptotic cell death [88]. Guegan et al (2001) illustrated that the protein content of caspase 9 begins to decrease in the spinal cords from symptomatic G93A mice (3 mo of age) compared to nontransgenic control mice and that caspase 9 continues to decrease further in end stage G93A mice (5 mo of age). Alternatively, cleaved caspase 9 protein content begins to increase in symptomatic G93A mice, with highly expressed levels in end stage mice [88]. The increase in cleaved References Caloric Restriction and ALS caspase 9 protein content signifies an elevation in apoptosis in the later stages of the disease. In our study, G93A mice were sacrificed at 99 d (,3 mo), and while we detected both the cleaved and uncleaved forms of caspase 9, we did not observe any significant changes in these proteins. Although we have demonstrated increases in Bax protein content, the failure to obtain an increase in the ratio of cleaved caspase 9/caspase 9 between the CR and AL groups may be due to the timing of tissue collection. This is in accordance with a study which found changes in Bax protein content, but no change in caspase 3 activity with training in 3- month old rat skeletal muscle [92]. Additionally, life-long CR (60% of AL) in 26-month old rats (starting at 3.5 mo of age) did not alter caspase 9 activity in the brain compared to age-matched rats on an AL diet, despite an attenuation in DNA fragmentation with CR [93]. A similar study examining life-long CR (60% of AL) in rat showed decreased levels of cleaved caspase 3 with CR in 26- month old gastrocnemius, but no significiant differences in the protein content of caspase 9 or cleaved caspase 9 [94]. Furthermore, short-term CR (2 mo; 60% of AL) increased apoptosis in the liver of male rats in the absence of significant changes in caspase 9 or cleaved caspase 9 protein content [95]. These studies provide support that apoptosis could occur in the absence of any significant alterations in caspase 9 activity or cleaved caspase 9 protein content with CR. Thus, we show that CR increases Bax protein content, with no difference in caspase 9 or cleaved caspase 9 levels in our diseased animal model, illustrating that not every component of the mitochondrial- mediated apoptotic pathway is necessarily affected by CR. We conclude that CR, equivalent to 60% of AL, hastens clinical onset by 2-fold, disease progression by 13-18% and endpoint by 3.1-fold. We ascertained that CR increases mitochondrial biogenesis, lipid peroxidation and inflammation, while decreasing mitochondrial oxidative capacity, cellular stress response in the G93A mouse, leading to heightened apoptosis. We postulate that these series of events may explain the faster clinical onset and shorter lifespan observed in this animal model. The substantial upregulation of antioxidant enzymes and UCP3, a marker of mitochondrial energetics, may be a protective response to the heightened oxidative damage within the CR females. In contrast, dysregulation in cellular redox status and less protection from heat shock proteins exacerbates disease outcomes in CR males. Our study is unique from other intervention studies in that we have investigated the effect of CR in vivo by analyzing anthropometric, functional, biochemical and molecular outcome measures. Ulti- mately, this is the first study to illustrate that CR is detrimental in G93A mice and that if we were to extrapolate our findings to patients with the disease, CR would be contraindicated. Acknowledgments We thank Bart Hettinga and Celina Wu for technical assistance. We thank Chad Barber and Scott Christie for assisting with food preparation and Julie Hall and Jennifer Crozier for assisting with the PaGE testing. Author Contributions Conceived and designed the experiments: MAT MJH. Performed the experiments: BPP AS SR MJH. Analyzed the data: BPP AS SR. Contributed reagents/materials/analysis tools: MAT MJH. Wrote the paper: BPP AS MJH. 1. Haverkamp LJ, Appel V, Appel SH (1995) Natural history of amyotrophic lateral sclerosis in a database population. Validation of a scoring system and a model for survival prediction. Brain 118 (Pt 3): 707–719. 2. Simpson EP, Yen AA, Appel SH (2003) Oxidative Stress: a common denominator in the pathogenesis of amyotrophic lateral sclerosis. Curr Opin Rheumatol 15: 730–736. PLoS ONE | www.plosone.org 15 February 2010 | Volume 5 | Issue 2 | e9386