Mitochondrial functions modulate neuroendocrine, PNAS PLUS metabolic, inflammatory, and transcriptional responses to acute psychological stress Martin Picarda,b,1, Meagan J. McManusa,b, Jason D. Grayc, Carla Nascac, Cynthia Moffatd, Piotr K. Kopinskia,b, Erin L. Seifertd, Bruce S. McEwenc, and Douglas C. Wallacea,b,2 aCenter for Mitochondrial and Epigenomic Medicine, The Children’s Hospital of Philadelphia, Philadelphia, PA 19104; bDepartment of Pathology and Laboratory Medicine, University of Pennsylvania, Philadelphia, PA 19104; cLaboratory for Neuroendocrinology, The Rockefeller University, New York, NY 10065; and dMitoCare Center, Department of Pathology, Anatomy and Cell Biology, Thomas Jefferson University, Philadelphia, PA 19107 Contributed by Douglas C. Wallace, September 22, 2015 (sent for review June 16, 2015; reviewed by José Antonio Enríquez and Susan Lutgendorf) The experience of psychological stress triggers neuroendocrine, where it fuels most energy-dependent cellular reactions. During inflammatory, metabolic, and transcriptional perturbations that ul- electron flow through the respiratory chain, reactive oxygen species timately predispose to disease. However, the subcellular determi- (ROS) are generated, leading to oxidative stress when antioxidant nants of this integrated, multisystemic stress response have not defenses are insufficient (11). Mitochondrial ROS are detoxified by been defined. Central to stress adaptation is cellular energetics, a specialized intramitochondrial antioxidant system maintained involving mitochondrial energy production and oxidative stress. by the redox-regulating enzyme nicotinamide nucleotide trans- We therefore hypothesized that abnormal mitochondrial functions hydrogenase (NNT) (12). The collective process of mitochondrial would differentially modulate the organism’s multisystemic re- energy homeostasis therefore involves energy transformation by sponse to psychological stress. By mutating or deleting mitochon- the partially mtDNA-encoded respiratory chain complexes, mito- drial genes encoded in the mtDNA [NADH dehydrogenase 6 (ND6) chondrial energy export by ANTs, and the maintenance of redox and cytochrome c oxidase subunit I (COI)] or nuclear DNA [adenine balance by NNT. nucleotide translocator 1 (ANT1) and nicotinamide nucleotide trans- Three major reasons justify the hypothesis that mitochondrial GENETICS hydrogenase (NNT)], we selectively impaired mitochondrial respira- functions would modulate the stress response. First, mitochon- tory chain function, energy exchange, and mitochondrial redox dria are the major source of cellular ATP. Requirements for balance in mice. The resulting impact on physiological reactivity ATP are increased to face any stress-induced cellular perturba- and recovery from restraint stress were then characterized. We tion, including ion pumping across membranes for action po- show that mitochondrial dysfunctions altered the hypothalamic– tentials, synaptic transmission, gene transcription, protein and pituitary–adrenal axis, sympathetic adrenal–medullary activation hormone biosynthesis, secretion, and other functions (e.g., ref. and catecholamine levels, the inflammatory cytokine IL-6, circulating 13). Stressors therefore necessarily engage mitochondrial energy metabolites, and hippocampal gene expression responses to stress. production across multiple organ systems (14), with the nervous Each mitochondrial defect generated a distinct whole-body stress- COGNITIVE SCIENCES PSYCHOLOGICAL AND response signature. These results demonstrate the role of mitochon- Significance drial energetics and redox balance as modulators of key patho- physiological perturbations previously linked to disease. This In humans and animals, stress triggers multisystemic physiological work establishes mitochondria as stress-response modulators, responses that vary in nature and magnitude. The organism’s with implications for understanding the mechanisms of stress response to stress, rather than actual stressors, leads to allostatic pathophysiology and mitochondrial diseases. load that predisposes to disease. This study in mice demonstrates that a specific cellular component that sustains life via energy stress reactivity | mitochondria | HPA axis | catecholamines | hippocampus transformation and intracellular signaling—the mitochondrion— influences the organism’s integrated response to psychological epeated exposure to psychological stress can predispose to stress. Each component of the stress response assessed was Rdisease (1, 2). The underlying mechanisms involve dysre- modified by at least one mitochondrial defect. When analyzed gulation of peripheral stress response elements including the collectively, stress-induced neuroendocrine, inflammatory, met- – – hypothalamic pituitary adrenal (HPA) axis and glucocorticoids (3), abolic, and transcriptional responses coalesced into unique sig- – the sympathetic adrenal medullary (SAM) axis and catechol- natures that distinguish groups based on their mitochondrial “ ” amines (4), systemic inflammation (5), and the diabetic-like state genotype. This work shows that mitochondria can regulate of excess circulating glucose and lipids (i.e., metabolic oversupply) complex whole-body physiological responses, impacting stress promoted by stress hormones (6, 7). In addition, stress leads to perception at the cellular and organismal levels. neuronal remodeling, which involves changes in brain gene ex- pression, particularly within the hippocampus (8, 9). However, the Author contributions: M.P., M.J.M., and D.C.W. designed research; M.P., M.J.M., C.M., and subcellular factors that modify these systemic responses to stress E.L.S. performed research; M.P., M.J.M., J.D.G., C.N., C.M., E.L.S., B.S.M., and D.C.W. ana- have not been defined. The objective of this study was to determine lyzed data; and M.P., M.J.M., P.K.K., and D.C.W. wrote the paper. if mitochondria mediate physiological stress responses in mice. Reviewers: J.A.E., Centro Nacional de Investigaciónes Cardiovasculares Carlos III; and S.L., Mitochondria are symbiotic organelles that contain their own University of Iowa. genetic material, the mtDNA, which encodes essential subunits The authors declare no conflict of interest. of the respiratory chain complexes I, III, IV, and V. At complex Freely available online through the PNAS open access option. I, electrons derived from energetic substrates (glucose and lipids) 1Present address: Division of Behavioral Medicine, Department of Psychiatry, Department of Neurology, and Columbia Translational Neuroscience Initiative, Columbia University enter the respiratory chain and travel to complex IV, where they College of Physicians and Surgeons, Columbia University Medical Center, New York, are combined with oxygen to produce energy in the form of ATP NY 10032. required for life (10). ATP generated inside mitochondria then is 2To whom correspondence should be addressed. Email: [email protected]. exported across the inner mitochondrial membrane into the cyto- This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. plasm by adenine nucleotide translocators 1 and 2 (ANT1 and 2), 1073/pnas.1515733112/-/DCSupplemental. www.pnas.org/cgi/doi/10.1073/pnas.1515733112 PNAS Early Edition | 1of10 Downloaded by guest on September 26, 2021 system appearing particularly vulnerable to defects in mito- the dynamics of stress responses. A second cohort of mice was ex- chondrial energy production (15). posed to 60 min of uninterrupted restraint stress. The study design is Second, mitochondria generate reactive metabolic intermedi- detailed in Fig. S1. ates and ROS, which collectively constitute signals of adaptation, altering chromatin regulation and gene expression within the nu- HPA Axis Activation. All strains had low levels of corticosterone cleus (16–19). Mitochondria therefore could contribute to reported (CORT) at baseline when unperturbed, indicating no basal dif- stress-induced epigenetic and transcriptional changes within the ferences among genotypes in HPA axis activity. Following hippocampus (8, 9, 20). 30-min stress exposure, mice with normal mitochondria (WT) Third, stress-induced physiological responses such as SAM showed a robust rise in CORT (Fig. 2A). Introducing an mtDNA hyperactivation (21), peripheral insulin resistance leading to hy- mutation in ND6 but not COI exaggerated this effect (Fig. 2A). perglycemia and metabolic syndrome (22), and inflammation (23, Likewise, deleting the ADP/ATP translocator ANT1 caused more 24) can be triggered autonomously by mitochondrial dysfunction severe activation of the HPA axis, with CORT peaking at alone. Therefore, mitochondria are functionally positioned to 170% of control. In contrast, removal of the mitochondrial redox- modulate the major stress–response axes. regulator gene NNT blunted the CORT increase to only 56% of To study the impact of mitochondrial functions on the stress control (Fig. 2A). response in vivo, we genetically manipulated two mtDNA-encoded To examine these strain differences further, a second cohort of respiratory chain subunits, NADH dehydrogenase 6 (ND6)and mice was exposed to restraint stress for 60 min, and blood was cytochrome c oxidase subunit I (COI), and two nuclear DNA collected at the end of stress. Both mtDNA mutations showed an (nDNA) genes, ANT1 and NNT (Fig. 1). We then evaluated the exaggerated stress-induced CORT elevation (Fig. 2B). However, impact of these selective mitochondrial defects upon the stress 60-min levels