Accumulation of intracellular damage by reactive oxygen species accelerates biological aging, leading to the development of age-related lung diseases such as idiopathic pulmonary fibrosis (IPF). Mitochondrial dysfunction and mitochondria-related oxidative stress has been implicated in the pathogenesis of many age-related diseases. Selective autophagic degradation of mitochondria (mitophagy) is critical to maintain a proper pool of the organelle and preserve cellular energy homeostasis. Oxidative stress resulting from age-dependent defects in the quality of proteins and degradation of mitochondria promotes alveolar epithelial cell damage potentiating lung injury. Our research found diminished autophagy corresponding with elevated levels of oxidized proteins and lipofuscin in response to lung injury in old and middle-aged mice compared to younger animals. More importantly, older mice exposed to lung injury are characterized by deficient mitophagic responses. The pro-fibrotic cytokine transforming growth factor beta 1 (TGFβ1) plays a pivotal role in driving fibroblast-to-myofibroblast differentiation (FMD), an important feature of pulmonary fibrosis. TGFβ1-mediated FMD is characterized by reduced autophagy flux, altered mitophagy and defects in mitochondrial function. In accordance, PINK1 expression is reduced in the aging murine lung and biopsies from IPF patients compared to controls. "nOur research also revealed a decline in mitochondrial protein deacetylase sirtuin 3 (SIRT3) expression in the lungs of aging mice. Low levels of SIRT3 transcripts were observed in two different animal models of pulmonary fibrosis. SIRT3 expression was reduced in fibrotic regions of lung tissues from patients with fibrotic diseases. We demonstrated that down-regulation of SIRT3 by TGFβ1 promotes acetylation of major oxidative stress response regulators, such as superoxide dismutase 2 (SOD2) and isocitrate dehydrogenase 2 (IDH2), and that resveratrol induced SIRT3 expression and ameliorated acetylation changes induced by TGFβ1. Knockdown of SIRT3 expression by siRNA exacerbated TGFβ1-induced FMD. By contrast, promotion of SIRT3 expression attenuated the effect of TGFβ1 on myofibroblast differentiation. Finally, SIRT3-deficient mice were more susceptible to pulmonary fibrosis in response to bleomycin and had increased collagen deposition compared to control mice. Collectively, our research indicates that an age-related decline in autophagy, SIRT3 expression, and mitochondrial homeostasis may contribute to the promotion and/or perpetuation of pulmonary fibrosis. / Meredith L Sosulski

Manganese superoxide dismutase (MnSOD), is a mitochondria-localized antioxidant enzyme that scavenges superoxide anions generated in the respiratory chains, has been known to play an important role in the radioprotection, and function as a tumor suppressor gene in many types of cancer. Sirtuin 3 (Sirt3) is a mitochondrial NAD+-dependent deacetylase that regulates mitochondrial oxidative metabolism, MnSOD activity, and tumorigenesis. The current study was designed to examine the role of MnSOD and Sirt3 in regulating mitochondrial metabolism and steady-state levels of O2*- in thymocyte responses to ionizing radiation (IR) and lymphomagenesis. Loss of MnSOD in thymocytes resulted in the decreased levels of viability when mice were exposed to 0.1 or 1 Gy 137Cs radiation. In contrast, loss of Sirt3 did not affect thymocyte radiosensitivity or radiation-induced superoxide levels when mice were exposed to a single dose of 137Cs radiation (0.1 or 1 Gy). Interestingly, the Sirt3-/- thymocytes demonstrated a compromised ability to induce an adaptive response following whole body exposure to a 0.1 Gy dose of 137Cs radiation when challenged 4 h later with a 1.5 Gy dose of 137Cs radiation. Of note, both Sirt3-/- and MnSOD-/- thymocytes did not demonstrate increased T cell lymphoma in C57BL/6 mice at 15-18 months following low dose (0.1 Gy or 1 Gy) radiation exposure to low linear energy transfer (LET) 137Cs or high LET 300 MeV/n Silicon (67 kev/µM) irradiation. Interestingly, the loss of Sirt3 in Bax overexpressing transgenic thymocytes did cause significant alterations in mitochondrial oxidative metabolism including increased levels of superoxide, increased mitochondrial membrane potential, and increased oxygen consumption, as well as decreased levels of MnSOD activity and decreased steady-state levels of ATP. Sirt3 was also found to be upregulated in Lck-Bax transgenic pre-malignant thymocytes, and downregulation of Sirt3 was noted in lymphomas from Lck-Bax mice. Furthermore, a significant acceleration of thymic lymphoma development was observed in Lck-Bax transgenic animals lacking Sirt3. In conclusion, the data presented here support the hypothesis that increased levels of superoxide in thymocytes induced by loss of Sirt3 or MnSOD and/or exposure to low dose radiation was not sufficient to induce T cell lymphomas. However, loss of Sirt3 significantly accelerated thymic lymphoma formation induced in Lck-Bax overexpressing C57BL/6 mice, showing that Sirt3 could act as a tumor suppressor in this model system. These results also support the speculation that mitochondrial oxidative metabolism regulated by Sirt3 leads to increased steady-state levels of O2*- and may contribute to the promotion of carcinogenesis in the Lck-Bax model of T cell lymphoma development.

Mitochondrial superoxide production during the early and late radiation response is increasingly recognized as a critical driver of oxidative damage and injury processes in mammalian cells. The role of Sirtuin 3, a key mitochondrial regulatory deacetylase, in preventing mitochondrial superoxide generation in conditions of nutrient and oxidative stress may be critical during the radiation response in mammalian liver. Because several tumor types express lower than normal levels of Sirtuin 3, the involvement of Sirtuin 3 in the radiation response may also provide clues to improving cancer radiation therapy and understanding the process of carcinogenesis. Studies of how the SIRT3 loss impacts the hepatic radiation response may also provide insight into the role of superoxide in normal liver physiology as well as in conditions of pathology. Increased superoxide production has largely been associated with disease, but oftentimes without clear demonstration of mechanism or even clear descriptions of pathogenesis. Here we identify a target of Sirtuin 3, the mitochondrial antioxidant enzyme manganese superoxide dismutase, and delineate the role that Sirtuin 3-mediated increases in manganese superoxide dismutase may be playing in the prevention of injury following biologically relevant doses of low linear energy transfer and high linear energy transfer radiation types including Cs-137 and Fe and Si particle radiation. Loss of Sirtuin 3 appears to correlate with decreases in hepatocellular carcinoma 16 months after 0.1 and 1 Gy doses of particle radiation known to increase hepatocellular carcinoma rates. These results indicate that Sirtuin 3 is a critical regulator of superoxide metabolism in the liver following whole body irradiation.

Sirt3, une des sept sirtuines chez les mammifères, est une déacétylase mitochondriale NAD+-dépendante qui joue un rôle dans le contrôle des facteurs clés de plusieurs voies métaboliques. Sirt3 déacétyle et active un grand nombre d'enzymes mitochondriales impliquées dans l'activité de la chaîne respiratoire, la production d'ATP, le cycle de Krebs, ainsi que le cycle de l'urée. Parallèlement à son rôle dans le métabolisme énergétique, l'activité mitochondriale intervient également dans l'induction de l'apoptose ainsi que dans la régulation de la prolifération et la différenciation cellulaires. En particulier les travaux du laboratoire ont montré qu'il existe une véritable régulation de la différenciation myogénique par l'activité mitochondriale. Comme Sirt3 régule l'activité mitochondriale, nous nous sommes intéressés à étudier l'implication de cette sirtuine dans la différenciation des myoblastes. Dans une première partie, nous avons évalué l'expression endogène de Sirt3 au cours de la différenciation des myoblastes murins C2C12, puis étudié l'effet de son inhibition sur le processus de différenciation et sur l'activité mitochondriale. Nous avons montré que l'expression de Sirt3 endogène augmente après induction de la différenciation des C2C12. Une inhibition stable de l'expression de Sirt3 par interférence (Short hairpin Sirt3, shSirt3) entraîne : 1) un blocage de la différenciation terminale des C2C12 reflété par une chute significative de l'index de fusion ainsi que de l'expression des marqueurs myogéniques MyoD, Myogénine et troponine T ; 2) une diminution de l'activité mitochondriale reflétée par une altération de l'expression de PGC-1alpha, VDAC et citrate synthase, et une diminution des activités enzymatiques des complexes de la chaîne respiratoire et de la respiration maximale des myoblastes ; 3) une augmentation de la production de DROs. Ces résultats suggèrent un rôle important de Sirt3 dans la différenciation des myoblastes, en relation avec son influence sur l'activité mitochondriale.Dans une seconde partie, nous avons évalué l'importance de Sirt3 in vivo sur le développement et le métabolisme du tissu musculaire en étudiant le phénotype de souris surexprimant l'isoforme courte (MCK-SIRT3M3) ou l'isoforme longue (MCK-SIRT3M1) de Sirt3 spécifiquement dans le muscle squelettique. Nos premiers résultats obtenus à l'âge de 3 mois montrent que la capacité oxydative des souris MCK-SIRT3M1 est plus faible et celle des souris MCK-SIRT3M3 plus élevée par rapport aux souris sauvages. Les souris MCK-SIRT3M3 présentent une atrophie musculaire dès l'âge de trois mois alors que la capacité musculaire et l'activité mitochondriale dans les muscles de ces souris ne sont pas modifiées. Avec l'âge, le phénotype des souris surexprimant l'isoforme M3 dans le muscle est plus marqué : l'atrophie s'accentue, le nombre de mitochondries augmente, et l'expression de la myosine de type 1 augmente alors que l'expression des myosines de type II diminue. Ces données indiquent que l'isoforme courte de Sirt 3 aurait une influence dans le développement et le métabolisme du muscle squelettique de souris. / Sirt3, one of the seven mammalian sirtuins, is a mitochondrial nicotinamide adenine dinucleotide (NAD+)-dependent deacetylase, and has been shown to control multiple key metabolic pathways. Sirt3 deacetylates and activates a large number of mitochondrial enzymes implicated in the activity of respiratory chain and ATP production, TCA and Urea cycles. We have previously shown that mitochondrial activity is importantly involved in the regulation of myoblast differentiation. Since Sirt3 modulates mitochondrial activity, we have investigated its influence on myoblast differentiation. First, we have evaluated endogen Sirt3 expression during C2C12 myoblast differentiation and then we examined the effect of its inhibition on the differentiation processes and on mitochondrial activity. We have shown that Sirt3 protein expression increased after the induction of myoblast differentiation. A stable inhibition of Sirt3 expression, using short hairpin Sirt3 (shSirt3) in C2C12 myoblasts resulted in: 1) abrogation of terminal differentiation reflected by a sharp decrease of the fusion index and a significant decrease of Myogenin, MyoD and Troponin T protein expression; 2) a decrease in mitochondrial activity reflected by alterations in PGC1-alpha, VDAC and citrate synthase expression, and a decrease in respiratory chain complexes activity and myoblast maximal respiration, 3) an increase in ROS production. These data suggest that Sirt3 plays an important role in the regulation of myoblast differentiation through its influence on mitochondrial activity.In a second part, to investigate the role of Sirt3, in vivo, in myogenesis and in mitochondrial activity, we have studied the effect of Sirt3 isoforms (short and long, MCK-SIRT3M3 and MCK-SIRT3M1 respectively) overexpression exclusively in skeletal muscle tissue of transgenic mice. We show that basal metabolism is lower MCK-SIRT3M1 mice and higher in MCK-SIRT3M3 compared to WT mice at 3 months of age. In 3 month-old MCK-SIRT3M3 mice, skeletal muscle is atrophied while muscle capacity and mitochondrial activity are not altered. Skeletal muscle phenotype evolves with age, in MCK-SIRT3M3 mice : increase in muscle atrophy, mitochondrial content. These data suggest that Sirt3 short isoform plays an important role in skeletal muscle development and metabolism in mice.

Dans les pays industrialisés, les rétinopathies ischémiques proliférantes telles que la rétinopathie diabétique et la rétinopathie du prématuré sont les principales causes de cécité chez les individus en âge de travailler et la population pédiatrique. Ces pathologies sont caractérisées par une dégénérescence microvasculaire initiale suivie d’une hyper-vascularisaton compensatoire disproportionnée et pathologique. Les sirtuines constituent une importante famille de protéines impliquées dans le métabolisme et la réponse au stress. Plus particulièrement, sirtuine 3 (SIRT3) est une déacétylase mitochondriale primordiale qui agit au cœur du métabolisme énergétique et de l’activation de nombreuses voies métaboliques oxydatives. Nos résultats démontrent pour la première fois qu’une déficience en SIRT3 diminue la sévérité des lésions vasculaires dans le modèle murin de rétinopathie induite par l’oxygène (OIR). En plus de stimuler l’angiogénèse, l’absence de SIRT3 est aussi associée à une augmentation de la glycolyse, possiblement en activant la famille de gènes 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase (PFKFB). Nous suggérons que le manque de SIRT3 est impliqué dans l’effet Warburg et procure ainsi un avantage prolifératif et protecteur dans l’OIR. La présente étude propose SIRT3 comme nouvelle cible thérapeutique potentielle dans la rétinopathie du prématuré, une maladie dont les complications désastreuses persistent tout au long de la vie. / Proliferative ischemic retinopathies such as proliferative diabetic retinopathy and retinopathy of prematurity (ROP) are the leading causes of blindness in working age and pediatric populations in industrialized countries. These pathologies are characterized by an initial microvascular degeneration followed by a disproportionate compensatory but pathological hyper-vascularization mounted by the hypoxic and energy deficient retina in an attempt to reinstate metabolic equilibrium. Sirtuins are an important family of protein involved in metabolism and stress response. Sirtuin 3 (SIRT3) in particular is a major mitochondrial deacetylase central to energy metabolism and the regulation of many oxidative pathways. For the first time, our results show that a lack of SIRT3 decreases the severity of vascular lesions in the oxygen-induced retinopathy (OIR) mouse model. Deficiency in SIRT3 not only stimulates angiogenesis, but also increases glycolysis, possibly through indirect activation of the gene family 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase (PFKFB). We suggest that a lack of SIRT3 is involved in the Warburg effect and therefore confers a proliferative advantage that is protective in OIR. The present study puts forward SIRT3 as a new potential therapeutic target for ROP, a disease leading to life-long vision complications.