MnSOD protein content upregulation in the white gastrocnemius (2.2-fold) and in Cu/Zn-SOD protein content upregulation in the red gastrocnemius (2.6-fold). The marked increase in MnSOD in the red gastrocnemius (4-fold), although more than 2-fold higher than its reported activity in rat liver mitochondria [49], is most likely a compensatory upregulation to the 2-fold increase in MDA observed in the tissue of this diseased animal model. However, catalase enzyme activity was unchanged in our study, which is in accordance with a CR study (60% of AL) in rats that found no difference in cardiac catalase enzyme activity [52]. Although catalase activity in skeletal muscle is somewhat minimal in comparison to other tissues [41], its lack of increase may have added to the elevated lipid peroxidation in the CR mice. The increase in oxidative stress, as measured by MDA, may be the reason for the faster clinical onset and shorter lifespan observed in the CR mice. Further support for the detriment imposed by the elevated oxidative stress in this model, is the compensatory upregulation in UCP3 protein content we observed in CR females. UCPs are mitochondrial anion-carrier proteins located in the inner mito- chondrial membrane and are activated by free radicals and free fatty acids [21]. In physiological situations of oxidative stress, superoxide can induce the activation of UCP3, thus regulating mitochondrial uncoupling, which decreases the electrochemical gradient inside the intermembrane space, subsequently reducing free radical production by the ETC [21]. In turn, UCP3 uncoupling is reduced in a feedback loop. UCP3 gene expression is up-regulated before the onset of disease (90 d) in skeletal muscle of G86R mice, as well as in SALS patients [54]. UCP3 upregulation seems to be a protective response to heightened oxidative stress [54]. It is interesting that this upregulation is specific to skeletal muscle, which exhibits heightened oxidative stress in the G93A mouse [32], and also presents with a reduction in ATP levels through a decrease in the mitochondrial respiratory control ratio [54]. We have shown that UCP3 protein content is elevated in CR female skeletal muscle. This finding is in line with studies showing an association of CR with increased levels of UCP3. UCP3 protein content in rat muscle mitochondria increases in response to long-term CR (60% of AL) [55], as well long-term CR (,60% of AL) increased UCP3 mRNA content in white adipose tissue by 2-fold [56]. These findings, in conjunction with our observed increase in UCP3 in CR females, are consistent with UCP3’s role as a compensatory protective mechanism against free radical species [56]. However, there was no change in UCP3 protein content in CR males, which further supports the premise that the G93A female sex is protective under CR. The failure to upregulate UCP3 may have exacerbated disease outcomes in CR males, making males more vulnerable to the damaging effects of oxidative stress. As previously mentioned, the protective ability of CR females to upregulate antioxidant enzymes and UCP3 was insufficient to delay the possible oxidative stress-induced decrease in life span in these animals. The role of oxidative stress in the pathogenesis of ALS is well-supported [57,58]. However, neuroinflammation has a strong role in ALS through the pathological hallmark of microglial proliferation [59,60], and neuroinflammation, which can be both a cause and consequence of enhanced free radical production [61]. TNF-a is an inflammatory cytokine that can activate an extrinsic, caspase-mediated apoptotic pathway upon binding to its cell- surface receptor [62]. This pathway involves the cleavage of caspase 8, which can cleave Bid (an antiapoptotic protein) and subsequently stimulate Bax to induce the release of cytochrome c, and cause cell death [62]. The detrimental role of TNF-a in ALS is supported by the use of thalidomide and lenalidomide to Caloric Restriction and ALS successfully inhibt TNF-a production [63,64] and significantly increase lifespan in G93A mice [65]. Under normal conditions, protein levels of TNF-a are increased in the spinal cords of 80 d and 120 d old G93A mice, suggesting that inflammatory pathways are activated prior to onset and during the symptomatic stage of disease [66]. When life-long CR (26 mo, 60% of AL) was applied to rats, the age-associated increase in plasma TNF-a was attenuated, signifying CR’s protective anti-inflammatory role [67]. However, we ascertained that the protein content of TNF-a was significantly higher (3.5- fold) in the quadriceps of CR vs. AL G93A mice, with no sex differences. The CR-induced elevation in this inflammatory cytokine predicts an increase in apoptotic pathways [62]. Thus, in the G93A mouse, energy restriction not only increases lipid peroxidation, but inflammation as well, potentially initiating a caspase-cascade leading to cell death. One of the novel aspects of this study is the CR-associated increase in MDA and TNF-a. The elevated oxidative stress and inflammation present in the skeletal muscle of G93A mice suggest that this diseased mouse model lacks an adequate defense system to respond to cellular insults, such as oxidative stress and inflammation. Hsps are molecular chaperones which assist in the support of other proteins, including maintenance of proper folding and protein conformation during conditions of stress [68,69]. Motor neurons are more vulnerable to stress than neurons in the hippocampus, cerebral cortex and substantia nigra [70]. This has implications for disease pathogenesis in ALS, as motor neurons have a reduced ability to upregulate Hsp70 in conditions of stress, making these neurons more susceptible to cellular insults [71]. Hence, when motor neurons were transfected with mSOD1, this resulted in the formation of toxic aggregates [72,73], which were delayed by gene transfer of Hsp70 [74]. When Hsp70 is over- expressed in cerebral ischemic and ischemia-like injury models, there is a decrease in inflammation and apoptosis, as well as an increase in antiapoptotic protein Bcl-2 [75]. Similarly, exogenous delivery of Hsp70 increased lifespan in G93A mice, indicating the importance of this protein in the pathogenesis of ALS [76]. In aged rat skeletal muscle, there is a diminution of Hsp70, indicating that older animals are less protected from cellular stresses. CR (60% of AL) in old rats increased the protein content of Hsp70 by 43% vs. old AL rats, thus the effect of age on Hsp70 was attenuated by CR [77]. CR is proposed to protect neurons against age-associated stresses by inducing the activation of Hsps [78], yet the vulnerability of motor neurons in ALS disease pathology may be incapable of this protective response [71]. Furthermore, the presence of Hsps in the cytosol and intermem- brane space of the mitochondria help prevent cell death. However, the binding of mSOD1 to Hsp70 may impede Hsp70’s normal antiapoptotic function, potentially promoting apoptosis and making the mitochondria more susceptible to stress [79]. In our study, both female and male CR mice had significantly decreased levels of Hsp70 in the quadriceps compared to AL mice. CR male mice had significantly less Hsp70 protein content than CR females, indicating that they are less protected from cell stress. It is possible that the failure of G93A mice to upregulate Hsp70 led to the faster clinical onset and disease progression observed under energy restriction and that once again, males were poorly defended against the heightened lipid peroxidation and inflam- mation compared to females. Our results coincide with the previous studies mentioned, which illustrate the reduced ability of motor neurons to upregulate Hsp70 under stress conditions [71], and the inhibition of Hsp70’s antiapoptotic role through its binding with mSOD1 [79]. PLoS ONE | www.plosone.org 14 February 2010 | Volume 5 | Issue 2 | e9386