Marco Giorgio

The p66shc adaptor protein controls oxidative stress response and life span in mammals.

Gene mutations in invertebrates have been identified that extend life span and enhance resistance to environmental stresses such as ultraviolet light or reactive oxygen species. In mammals, the mechanisms that regulate stress response are poorly understood and no genes are known to increase individual life span. Here we report that targeted mutation of the mouse p66shc gene induces stress resistance and prolongs life span. p66shc is a splice variant of p52shc/p46shc (ref. 2), a cytoplasmic signal transducer involved in the transmission of mitogenic signals from activated receptors to Ras. We show that: (1) p66shc is serine phosphorylated upon treatment with hydrogen peroxide (H2O2) or irradiation with ultraviolet light; (2) ablation of p66shc enhances cellular resistance to apoptosis induced by H2O2 or ultraviolet light; (3) a serine-phosphorylation defective mutant of p66shc cannot restore the normal stress response in p66shc-/- cells; (4) the p53 and p21 stress response is impaired in p66shc-/- cells; (5) p66shc-/- mice have increased resistance to paraquat and a 30% increase in life span. We propose that p66shc is part of a signal transduction pathway that regulates stress apoptotic responses and life span in mammals.

Electron transfer between cytochrome C and p66Shc generates reactive oxygen species that trigger mitochondrial apoptosis

Reactive oxygen species (ROS) are potent inducers of oxidative damage and have been implicated in the regulation of specific cellular functions, including apoptosis. Mitochondrial ROS increase markedly after proapoptotic signals, though the biological significance and the underlying molecular mechanisms remain undetermined. P66Shc is a genetic determinant of life span in mammals, which regulates ROS metabolism and apoptosis. We report here that p66Shc is a redox enzyme that generates mitochondrial ROS (hydrogen peroxide) as signaling molecules for apoptosis. For this function, p66Shc utilizes reducing equivalents of the mitochondrial electron transfer chain through the oxidation of cytochrome c. Redox-defective mutants of p66Shc are unable to induce mitochondrial ROS generation and swelling in vitro or to mediate mitochondrial apoptosis in vivo. These data demonstrate the existence of alternative redox reactions of the mitochondrial electron transfer chain, which evolved to generate proapoptotic ROS in response to specific stress signals.

Hydrogen peroxide: a metabolic by-product or a common mediator of ageing signals?

The reactive oxygen species that are generated by mitochondrial respiration, including hydrogen peroxide (H2O2), are potent inducers of oxidative damage and mediators of ageing. It is not clear, however, whether oxidative stress is the result of a genetic programme or the by-product of physiological processes. Recent findings demonstrate that a fraction of mitochondrial H2O2, produced by a specialized enzyme as a signalling molecule in the pathway of apoptosis, induces intracellular oxidative stress and accelerates ageing. We propose that genes that control H2O2 production are selected determinants of lifespan.

A p53-p66Shc signalling pathway controls intracellular redox status, levels of oxidation-damaged DNA and oxidative stress-induced apoptosis.

Correlative evidence links stress, accumulation of oxidative cellular damage and ageing in lower organisms and in mammals. We investigated their mechanistic connections in p66Shc knockout mice, which are characterized by increased resistance to oxidative stress and extended life span. We report that p66Shc acts as a downstream target of the tumour suppressor p53 and is indispensable for the ability of stress-activated p53 to induce elevation of intracellular oxidants, cytochrome c release and apoptosis. Other functions of p53 are not influenced by p66Shc expression. In basal conditions, p66Shc−/− and p53−/− cells have reduced amounts of intracellular oxidants and oxidation-damaged DNA. We propose that steady-state levels of intracellular oxidants and oxidative damage are genetically determined and regulated by a stress-induced signal transduction pathway involving p53 and p66Shc.

Deletion of the p66Shc longevity gene reduces systemic and tissue oxidative stress, vascular cell apoptosis, and early atherogenesis in mice fed a high-fat diet

Several experimental and clinical studies have shown that oxidized low-density lipoprotein and oxidation-sensitive mechanisms are central in the pathogenesis of vascular dysfunction and atherogenesis. Here, we have used p66Shc−/− and WT mice to investigate the effects of high-fat diet on both systemic and tissue oxidative stress and the development of early vascular lesions. To date, the p66Shc−/− mouse is the unique genetic model of increased resistance to oxidative stress and prolonged life span in mammals. Computer-assisted image analysis revealed that chronic 21% high-fat treatment increased the aortic cumulative early lesion area by ≈21% in WT mice and only by 3% in p66Shc−/− mice. Early lesions from p66Shc−/− mice had less content of macrophage-derived foam cells and apoptotic vascular cells, in comparison to the WT. Furthermore, in p66Shc−/− mice, but not WT mice, we found a significant reduction of systemic and tissue oxidative stress (assessed by isoprostanes, plasma low-density lipoprotein oxidizability, and the formation of arterial oxidation-specific epitopes). These results support the concept that p66Shc−/− may play a pivotal role in controlling systemic oxidative stress and vascular diseases. Therefore, p66Shc might represent a molecular target for therapies against vascular diseases.

Diabetes Promotes Cardiac Stem Cell Aging and Heart Failure, Which Are Prevented by Deletion of the p66shc Gene

Diabetes leads to a decompensated myopathy, but the etiology of the cardiac disease is poorly understood. Oxidative stress is enhanced with diabetes and oxygen toxicity may alter cardiac progenitor cell (CPC) function resulting in defects in CPC growth and myocyte formation, which may favor premature myocardial aging and heart failure. We report that in a model of insulin-dependent diabetes mellitus, the generation of reactive oxygen species (ROS) leads to telomeric shortening, expression of the senescent associated proteins p53 and p16INK4a, and apoptosis of CPCs, impairing the growth reserve of the heart. However, ablation of the p66shc gene prevents these negative adaptations of the CPC compartment, interfering with the acquisition of the heart senescent phenotype and the development of heart failure with diabetes. ROS elicit 3 cellular reactions: low levels activate cell growth, intermediate quantities trigger cell apoptosis, and high amounts initiate cell necrosis. CPC replication predominates in diabetic p66shc−/−, whereas CPC apoptosis and myocyte apoptosis and necrosis prevail in diabetic wild type. Expansion of CPCs and developing myocytes preserves cardiac function in diabetic p66shc−/−, suggesting that intact CPCs can effectively counteract the impact of uncontrolled diabetes on the heart. The recognition that p66shc conditions the destiny of CPCs raises the possibility that diabetic cardiomyopathy is a stem cell disease in which abnormalities in CPCs define the life and death of the heart. Together, these data point to a genetic link between diabetes and ROS, on the one hand, and CPC survival and growth, on the other.

Deletion of p66Shc Longevity Gene Protects Against Experimental Diabetic Glomerulopathy by Preventing Diabetes-Induced Oxidative Stress

p66Shc regulates both steady-state and environmental stress-dependent reactive oxygen species (ROS) generation. Its deletion was shown to confer resistance to oxidative stress and protect mice from aging-associated vascular disease. This study was aimed at verifying the hypothesis that p66Shc deletion also protects from diabetic glomerulopathy by reducing oxidative stress. Streptozotocin-induced diabetic p66Shc knockout (KO) mice showed less marked changes in renal function and structure, as indicated by the significantly lower levels of proteinuria, albuminuria, glomerular sclerosis index, and glomerular and mesangial areas. Glomerular content of fibronectin and collagen IV was also lower in diabetic KO versus wild-type mice, whereas apoptosis was detected only in diabetic wild-type mice. Serum and renal tissue advanced glycation end products and plasma isoprostane 8-epi-prostaglandin F2α levels and activation of nuclear factor κB (NF-κB) were also lower in diabetic KO than in wild-type mice. Mesangial cells from KO mice grown under high-glucose conditions showed lower cell death rate, matrix production, ROS levels, and activation of NF-κB than those from wild-type mice. These data support a role for oxidative stress in the pathogenesis of diabetic glomerulopathy and indicate that p66Shc is involved in the molecular mechanism(s) underlying diabetes-induced oxidative stress and oxidant-dependent renal injury.

Mitochondrial pathways for ROS formation and myocardial injury: the relevance of p66(Shc) and monoamine oxidase

Although mitochondria are considered the most relevant site for the formation of reactive oxygen species (ROS) in cardiac myocytes, a major and unsolved issue is where ROS are generated in mitochondria. Respiratory chain is generally indicated as a main site for ROS formation. However, other mitochondrial components are likely to contribute to ROS generation. Recent reports highlight the relevance of monoamine oxidases (MAO) and p66Shc. The importance of these systems in the irreversibility of ischemic heart injury will be discussed along with the cardioprotective effects elicited by both MAO inhibition and p66Shc knockout. Finally, recent evidence will be reviewed that highlight the relevance of mitochondrial ROS formation also in myocardial failure and atherosclerosis.

p66Shc-generated oxidative signal promotes fat accumulation

Reactive oxygen species (ROS) and insulin signaling in the adipose tissue are critical determinants of aging and age-associated diseases. It is not clear, however, if they represent independent factors or they are mechanistically linked. We investigated the effects of ROS on insulin signaling using as model system the p66Shc-null mice. p66Shc is a redox enzyme that generates mitochondrial ROS and promotes aging in mammals. We report that insulin activates the redox enzyme activity of p66Shc specifically in adipocytes and that p66Shc-generated ROS regulate insulin signaling through multiple mechanisms, including AKT phosphorylation, Foxo localization, and regulation of selected insulin target genes. Deletion of p66Shc resulted in increased mitochondrial uncoupling and reduced triglyceride accumulation in adipocytes and in vivo increased metabolic rate and decreased fat mass and resistance to diet-induced obesity. In addition, p66Shc-/- mice showed impaired thermo-insulation. These findings demonstrate that p66Shc-generated ROS regulate the effect of insulin on the energetic metabolism in mice and suggest that intracellular oxidative stress might accelerate aging by favoring fat deposition and fat-related disorders.

The cardioprotective effects elicited by p66Shc ablation demonstrate the crucial role of mitochondrial ROS formation in ischemia/reperfusion injury

Although a major contribution to myocardial ischemia-reperfusion (I/R) injury is suggested to be provided by formation of reactive oxygen species (ROS) within mitochondria, sites and mechanisms are far from being elucidated. Besides a dysfunctional respiratory chain, other mitochondrial components, such as monoamine oxidase and p66(Shc), might be involved in oxidative stress. In particular, p66(Shc) has been shown to catalyze the formation of H(2)O(2). The relationship among p66(Shc), ROS production and cardiac damage was investigated by comparing hearts from p66(Shc) knockout mice (p66(Shc-/-)) and wild-type (WT) littermates. Perfused hearts were subjected to 40 min of global ischemia followed by 15 min of reperfusion. Hearts devoid of p66(Shc) were significantly protected from I/R insult as shown by (i) reduced release of lactate dehydrogenase in the coronary effluent (25.7+/-7.49% in p66(Shc-/-) vs. 39.58+/-5.17% in WT); (ii) decreased oxidative stress as shown by a 63% decrease in malondialdehyde formation and 40+/-8% decrease in tropomyosin oxidation. The degree of protection was independent of aging. The cardioprotective efficacy associated with p66(Shc) ablation was comparable with that afforded by other antioxidant interventions and could not be increased by antioxidant co-administration suggesting that p66(Shc) is downstream of other pathways involved in ROS formation. In addition, the absence of p66(Shc) did not affect the protection afforded by ischemic preconditioning. In conclusion, the absence of p66(Shc) reduces the susceptibility to reperfusion injury by preventing oxidative stress. The present findings provide solid and direct evidence that mitochondrial ROS formation catalyzed by p66(Shc) is causally related to reperfusion damage.