Original Contribution Gender and age-dependent differences in the mitochondrial apoptogenic pathway in Alzheimer's disease Ana Lloret a , Mari-Carmen Badía b , Nancy J. Mora a , Angel Ortega a , Federico V. Pallardó a , Maria-Dolores Alonso b , Hani Atamna c , Jose Viña a, ⁎ a Departamento de Fisiología, Facultad de Medicina, Avda. Blasco Ibáñez 15, 46010 Valencia, Spain b Departamento de Neurología, Hospital Clínico Universitario, Avda. Blasco Ibáñez,13, 46010 Valencia, Spain c Nutrition and Metabolism Center, Children's Hospital Oakland Research Institute, Oakland, CA, USA article info abstract Article history: Received 16 December 2007 Revised 18 February 2008 Accepted 29 February 2008 Available online 20 March 2008 Age-related mitochondrial oxidative stress is highly gender dependent. The aim of this study was to determine the role of gender in the mitochondrial contribution to neuronal apoptosis in Alzheimer's disease (AD). We used mitochondria isolated from brains of Wistar rats to study the toxicity of ß-amyloid peptide (Aß), and found that it increases mitochondrial peroxide production, nitration and oxidation of proteins, and release of cytochrome c. The toxic effects occurred in young males and in old females but not in young females, indicating their resistance to Aß. This resistance was abolished with age. These toxic effects of Aß were prevented by heme. Our findings provide a molecular mechanism for the contribution of Aβ to the mitochondrial dysfunction and oxidative stress seen in AD, as well as for the mitochondria-dependent pathway of apoptosis in AD. Gender and age-related differences seen in the development of AD can also be partially explained. © 2008 Elsevier Inc. All rights reserved. Keywords: Neurodegenerative disease Aging Free radicals Glutathione Heme Introduction Alzheimer's is an age-related disease of multifactorial origin. Extracellular deposits of ß-amyloid (Aß) peptide (known as senile plaques) are morphological hallmarks consistently found in postmortem examination of brain autopsies of these patients. Genetic, biochemical, and immunological findings support the mechanistic role of excess Aß in causing neurodegeneration in the brain of Alzheimer's disease [AD] patients [1]. Increased oxidative stress has been proposed to play an important role in Aß-induced neuronal death in AD [2–4]. There is growing evidence of intracellular toxicity of Aß in the pathophysiology of AD [5]. The intracellular accumulation of Aß is facilitated by the α7- acetylcholine receptor [6]. Recent data suggest that Aß might induce apoptotic cell death by altering mitochondrial physiology. Thus Aß needs functional mitochondria to induce oxidative stress and apoptosis [7,8]. There are several lines of evidence for the involvement of mitochondria in the pathophysiology of AD [7,8], particularly regarding oxidative stress, but the mechanism of their involvement in Aß-induced cell death is still unclear [9,10]. We previously established the importance of the interaction between estrogens and mitochondria to explain the higher longevity of females vs males [11]. The role of heme in the mechanism of Aß-induced AD and the mitochondrial decline has been recently proposed [12,13]. Adequate production of the various heme isoforms depends on estrogen. Therefore, in this study, we examined the role of heme in the mitochondrial toxicity inflicted by Aß. Experimental procedures Animals Young males and females (5- to 6-month-old) and old females (24- to 26-month-old) Wistar rats were used. Animals were housed at constant temperature and humidity and with a 12 h light/12 h dark cycle. They were fed on a standard laboratory diet (containing 590 g carbohydrates, 30 g lipids, and 160 g protein per kilogram of diet) and tap water ad libitum. In all cases, animals were treated in accordance with the recommendations for the good care of laboratory animals. Isolation of mitochondria After the animals were sacrificed by cervical dislocation, their brains were quickly removed. Mitochondria were obtained by differential centrifugation, as described by Rickwood et al. [14]. We measured viability of mitochondria using mitochondrial membrane potential by flow cytometry with rhodamine 123 (Sigma-Aldrich, St. Louis, MO) as marker. Free Radical Biology & Medicine 44 (2008) 2019–2025 Abbreviations: AD, Alzheimer's disease; Aß, beta-amyloid; DMEM, Dulbecco's modified Eagle's medium; GSH, reduced glutathione. ⁎ Corresponding author. Fax: +34963864642. E-mail address: Jose.vina@uv.es (J. Viña). 0891-5849/$ – see front matter © 2008 Elsevier Inc. All rights reserved. doi:10.1016/j.freeradbiomed.2008.02.017 Contents lists available at ScienceDirect Free Radical Biology & Medicine journal homepage: www.elsevier.com/locate/freeradbiomed