Caloric Restriction Shortens Lifespan through an Increase in Lipid Peroxidation, Inflammation and Apoptosis in the G93A Mouse, an Animal Model of ALS Barkha P. Patel1,2, Adeel Safdar3,4,5, Sandeep Raha3, Mark A. Tarnopolsky3,4, Mazen J. Hamadeh1,2,3* 1 School of Kinesiology and Health Science, York University, Toronto, Ontario, Canada, 2 Muscle Health Research Centre, York University, Toronto, Ontario, Canada, 3 Department of Pediatrics, McMaster University, Hamilton, Ontario, Canada, 4 Department of Medicine, McMaster University, Hamilton, Ontario, Canada, 5 Department of Kinesiology, McMaster University, Hamilton, Ontario, Canada Abstract Caloric restriction (CR) extends lifespan through a reduction in oxidative stress, delays the onset of morbidity and prolongs lifespan. We previously reported that long-term CR hastened clinical onset, disease progression and shortened lifespan, while transiently improving motor performance in G93A mice, a model of amyotrophic lateral sclerosis (ALS) that shows increased free radical production. To investigate the long-term CR-induced pathology in G93A mice, we assessed the mitochondrial bioenergetic efficiency and oxidative capacity (CS – citrate synthase content and activity, cytochrome c oxidase - COX activity and protein content of COX subunit- I and IV and UCP3- uncoupling protein 3), oxidative damage (MDA – malondialdehyde and PC – protein carbonyls), antioxidant enzyme capacity (Mn-SOD, Cu/Zn-SOD and catalase), inflammation (TNF-a), stress response (Hsp70) and markers of apoptosis (Bax, Bcl-2, caspase 9, cleaved caspase 9) in their skeletal muscle. At age 40 days, G93A mice were divided into two groups: Ad libitum (AL; n = 14; 7 females) or CR (n = 13; 6 females), with a diet equal to 60% of AL. COX/CS enzyme activity was lower in CR vs. AL male quadriceps (35%), despite a 2.3-fold higher COX-IV/CS protein content. UCP3 was higher in CR vs. AL females only. MnSOD and Cu/Zn-SOD were higher in CR vs. AL mice and CR vs. AL females. MDA was higher (83%) in CR vs. AL red gastrocnemius. Conversely, PC was lower in CR vs. AL red (62%) and white (30%) gastrocnemius. TNF-a was higher (52%) in CR vs. AL mice and Hsp70 was lower (62%) in CR vs. AL quadriceps. Bax was higher in CR vs. AL mice (41%) and CR vs. AL females (52%). Catalase, Bcl-2 and caspases did not differ. We conclude that CR increases lipid peroxidation, inflammation and apoptosis, while decreasing mitochondrial bioenergetic efficiency, protein oxidation and stress response in G93A mice. Citation: Patel BP, Safdar A, Raha S, Tarnopolsky MA, Hamadeh MJ (2010) Caloric Restriction Shortens Lifespan through an Increase in Lipid Peroxidation, Inflammation and Apoptosis in the G93A Mouse, an Animal Model of ALS. PLoS ONE 5(2): e9386. doi:10.1371/journal.pone.0009386 Editor: Antoni L. Andreu, Hospital Vall d’Hebron, Spain Received September 16, 2009; Accepted November 9, 2009; Published February 24, 2010 Copyright: 2010 Patel et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This research was funded by Hamilton Health Sciences, Hamilton Health Sciences Foundation New Investigator Fund, the National Sciences and Engineering Research Council of Canada (http://www.nserc-crsng.gc.ca/) and York University-Faculty of Health. M. J. Hamadeh was recipient of the Hamilton Health Sciences Foundation William E. Noonan Research Career Award. A. Safdar was recipient of the Canadian Institute of Health Research - Institute of Aging Doctoral Research Award (http://www.cihr.ca). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: hamadeh@yorku.ca Introduction Amyotrophic lateral sclerosis (ALS) is a neuromuscular disease characterized by the degeneration of motor neurons in the brain and spinal cord and is associated with an increase in oxidative stress [1,2]. One mechanism which has been shown to decrease oxidative stress in animals and extend lifespan is caloric restriction (CR) [3–7]. Transgenic mice that overexpress the mutant human SOD1 gene (G93A mice) are an animal model of ALS and demonstrate elevations in free radical production [8]. Unexpect- edly, when applied to this model, long-term CR hastened clinical onset, disease progression and life span, while transiently improving motor performance [9,10]. Whereas short-term CR hastened clinical onset and shortened lifespan in male, but not female, G93A mice, and hastened disease progression with no effect on paw grip endurance [11]. The objective of this study was to explore how CR in the G93A mouse induces changes at the molecular level in genes involved in oxidative stress management, mitochondrial energetics, inflammation, stress response and apoptosis. Unraveling the mechanism(s) behind the accelerated clinical onset and disease progression which occurs as a result of CR is necessary to understanding why this intervention failed to extend lifespan. This study is significant in determining the impact of food intake on the progression of disease in this animal model of ALS. We hypothesized that CR would decrease mitochondrial oxidative capacity which will induce electron leakage from mitochondrial electron transport chain resulting in an increase in oxidative stress, and hence markers of oxidative stress in the skeletal muscle of G93A mice. Furthermore, we hypothesized that CR would: (a) increase antixodant enzymes (MnSOD and Cu/Zn-SOD) and UCP3 as a compensatory mechanism to counteract the increase in oxidative stress, (b) not affect catalase activity since this enzyme is resistant to changes in dietaty intake, (c) increase TNF-a content, (d) decrease Hsp70 content, (e) increase Bax and Bcl-2 content, and the ratio of Bax/Bcl-2, as well as caspase 9, cleaved caspase 9 PLoS ONE | www.plosone.org 1 February 2010 | Volume 5 | Issue 2 | e9386