The effects of long-term caloric restriction on molecular outcome measures in the copper/zinc-SOD mutant G93A mouse, an animal model of amyotrophic lateral sclerosis (ALS)

Caloric restriction (CR) extends lifespan through a reduction in oxidative stress. The G93A mouse, an animal model of amyotrophic lateral sclerosis (ALS), demonstrates elevations in free radical production, and hence is an ideal model to delineate the effects of CR. Unexpectedly, when applied to this model, long-term CR hastened clinical onset, disease progression and life span, while transiently improving motor performance. We investigated the effect of long-term CR in G93A mice on oxidative damage, antioxidant enzyme capacity, mitochondrial energetics, inflammation, stress response and apoptosis. G93A mice were randomly divided into two groups: AL (ad libitum, n = 14; 7 females) and CR (n = 13; 6 females), with a diet equal to 60% of AL. We measured oxidative stress markers malondialdehyde and protein carbonyls (MDA, PC), catalase enzyme activity and protein content of antioxidant enzymes (Mn-SOD, Cu/Zn-SOD), markers of mitochondrial energetics (UCP3), inflammation (TNF-alpha), stress response (Hsp70) and apoptosis (Bax, Bcl-2, caspase 9, cleaved caspase 9), in the skeletal muscle of 99-day-old G93A mice. MDA increased in CR vs. AL (83%, P < 0.001) and in female vs. male (32%, P = 0.045) red gastrocnemius, as well as in CR vs. AL white gastrocnemius (14%, P = 0.095). Conversely, PC decreased in CR vs. AL red (62%, P < 0.001) and white (30%, P = 0.003) gastrocnemius. MnSOD increased in CR vs. AL (3 fold, P = 0.031), and in female vs. male (2.5 fold, P = 0.026) red gastrocnemius, as well as in CR vs. AL white gastrocnemius (78%, P = 0.062). Likewise, Cu/Zn-SOD was higher in CR vs. AL red gastrocnemius (67%, P = 0.096) and increased in CR vs. AL females (2.6-fold, P = 0.020). In the quadriceps, UCP3 was higher in CR vs. AL females (1.5-fold, P = 0.042), TNF-alpha was higher in CR vs. AL mice (52%, P = 0.030), while Hsp70 was lower in CR vs. AL mice (62%, P = 0.002) and in males vs. females (37%, P = 0.030). Pro-apoptotic protein Bax was significantly higher in CR vs. AL mice (41%, P = 0.027) and in CR vs. AL females (52%, P = 0.048). Bax/Bcl-2 was significantly higher in CR vs. AL mice (68%, P = 0.040) and in CR vs. AL females (2.3-fold, P = 0.029). There were no differences in diet or sex for catalase activity and Bcl-2, caspase 9, cleaved caspase 9 or the ratio of cleaved caspase 9/caspase 9 protein content. We conclude that CR increases lipid peroxidation, inflammation and apoptosis, while decreasing protein oxidation and stress response in G93A mice. Females, but not males, show a compensatory upregulation in Mn-SOD, Cu/Zn-SOD and UCP3 protein content. Assuming we can extrapolate these results to humans, CR should be contraindicated for ALS patients.