SIRT3, the Anti-aging Major Mitochondrial Deacetylase, Is Important for Preventing Pulmonary Fibrosis Renea Jablonski, MD Kamp Lab November 14, 2015

Apoptosis Nobel et al. JCI 2012. King et al. Lancet. 2011.

Damage to mtDNA due to ROS plays a key role in the pathogenesis of IPF MtDNA is more sensitive to damage from exogenous ROS than nuclear DNA Link between ROS, mtDNA damage and apoptosis Mitochondria repair DNA damage primarily via the base excision repair (BER) pathway Key BER enzymes are nuclear-encoded and imported to the mitochondria 8-oxo-guanine DNA glycosylase (OGG1) important BER enzyme Critical link between mitochondrial dysfunction in AEC and IPF (?mtDNA damage) Biochim Biophys Acta. 2013 Jul;1832(7):1028-40. Am J Respir Crit Care Med. 2010 Feb 1;181(3):254-63. Gu et al. Free Rad Biol Med. 2015(83): 149-158. Zhang et al. Shock. 2010(34):55-59. Kazak et al. Nat Rev Mol Cell Biol. 2012(13):659-671.

OGG1 and ACO-2 implicated in repair of oxidative mtDNA damage Aconitase (ACO-2) maintains mtDNA integrity as mtDNA nucleoid Overexpression is protective ACO-2 is vulnerable to oxidative inactivation Mt-hOgg1 blocks decreases in ACO-2 expression and function induced by oxidative stress J Biol Chem. 2000 Dec 1;275(48):37518-23. Free Radic Biol Med. 2009 Sep 15;47(6):750-9.

ROS Mitochondrial DNA Mt-DNA Damage Mt-DNA Damage Mt-Acon TCA cycle hOgg1 ROS Mt-DNA Damage Mt-Acon hOgg1 Mitochondrial DNA Apoptosis High levels of ROS Mt-Acon Redox-active Iron Mt-DNA Damage hOgg1 Low levels of ROS Cell Survival Mitochondrial DNA Repair Mt-Acon hOgg1 TCA cycle

Sirtuins Mammalian family with 7 members Homologous to highly conserved C. elegans silent information regulator 2 gene (SIR2) Mammalian family with 7 members Nuclear: SIRT1, SIRT6, SIRT7 Cytosolic: SIRT2 Mitochondrial: SIRT3, SIRT4, SIRT5 Chronic oxidative stress leading to SIRT3 depletion and alterations in mitochondrial function and biogenesis have been implicated in multiple age-related diseases Guardian of the mitochondrial genome SIRT3 interacts with OGG1 → mtDNA repair Cheng et al Cell Death Dis. 2013 Jul 18;4:e731.

Hypothesis SIRT3 plays a key role in limiting oxidant-induced AEC mitochondrial dysfunction, mtDNA damage and apoptosis via preservation of OGG-1 and ACO-2 expression and function.

SIRT3 is depleted in a time- and dose-dependent manner following oxidative injury SK Jablonski et al. Presented at ATS 2015, Denver, CO.

SIRT3 deficiency enhances mtDNA damage and intrinsic apoptosis in A549 cells following oxidative stress SK Jablonski et al. Presented at ATS 2015, Denver, CO.

SIRT3-EE protects against AEC oxidant-induced mtDNA damage and apoptosis SK Jablonski et al. Presented at ATS 2015, Denver, CO.

Sirt3-deficient mice are more susceptible to asbestos—induced pulmonary fibrosis PC Jablonski et al. Presented at ATS 2015, Denver, CO.

Lung fibrosis in Sirt3-deficient mice is augmented by exposure to bleomycin PC Jablonski et al. Presented at ATS 2015, Denver, CO.

Sirt3 deficient mice develop increased asbestos- and bleomycin-induced lung fibrosis Fibrosis score Lung Collagen Asbestos Bleomycin PC

Asbestos-induced apoptosis (cleaved caspase 3 activation) at the BAD junction is increased in Sirt3-/- mice PC

Asbestos-induced mtDNA damage is amplified in Sirt3-/- mice in vitro and in vivo SK Jablonski et al. Presented at ATS 2015, Denver, CO.

SIRT3 deacetylase targets ACO-2 and MnSOD show increased acetylation following asbestos and H2O2 treatment A549: MLE-12: AT2: SK Jablonski et al. Presented at ATS 2015, Denver, CO.

AEC OGG1 acetylation is augmented by SIRT3 silencing and diminished by SIRT3-EE SK

Acetylation is increased in lung tissue from IPF patients

Summary 1. SIRT3 is decreased in AEC following oxidative insult. 2. SIRT3 deficiency is associated with increased susceptibility to oxidant- induced mtDNA damage and intrinsic apoptosis, while SIRT3-EE protects against these effects. 3. SIRT3 deficient mice are more prone to asbestos- and bleomycin- induced lung fibrosis and this occurs in association with increased AT2 cell apoptosis. 4. Isolated AT2 cells from asbestos-exposed mice have increased mtDNA damage following asbestos exposure and this effect is amplified in Sirt3- KO mice. 5. Lungs from patients with IPF have increased acetylation of MnSOD and OGG1.

Acknowledgements Kamp Lab Budinger Lab Ridge Lab Lam Lab John Varga Seok-Jo Kim Paul Cheresh David Williams Budinger Lab Monica Chi Luisa Morales-Nebreda Ridge Lab Yuan Cheng Erin Hogan Lam Lab Joseph Sennello John Varga David Guis Anjana Yeldandi Annie Pardo Moisés Selman Funding: NIH/NHLBI Training Grant 2T32HL076139-11A1, VA Merit (DK), NIH R01 ES02037-01A1 (DK)