Psychobiological regulation and molecular characterization of cell-free mitochondrial DNA in humans

PROJECT SUMMARY Mitochondrial signal transduction is an emerging biological pathway linking adverse psychosocial exposures to disease risk. However, we lack scalable, sensitive, and biologically meaningful markers to explore the role of mitochondrial biology in the stress-disease cascade. We have identified blood cell-free mitochondrial DNA (cf-

mtDNA) as a stress-inducible marker, discovered the existence of cf-mtDNA in saliva, and developed a scalable laboratory platform (MitoQuicLy) to quantify cf-mtDNA from thousands of samples in various biofluids. Building on these discoveries, in Aim 1 we will deploy MitoQuicLy to profile cf-mtDNA dynamics across >5,800 blood and

saliva samples from three laboratory socio-evaluative stress studies. Building on the existing deep phenotyping across these cohorts, these new measurements of cf-mtDNA dynamics, including built-in validation across cohorts, will provide robust estimates of cf-mtDNA reactivity effect sizes, sex differences, baseline psychosocial

and mental health correlates including anxiety and depressive symptoms, and cortisol – a putative driver of cf- mtDNA dynamics. Two studies include experimental or quasi-experimental manipulations of systemic energy metabolism and mitochondrial health, providing an opportunity to examine the influence of mitochondrial

energetics on cf-mtDNA reactivity. In Aim 2, we will generate the first comprehensive human psychosocial stress proteome using aptamer-based technology proteomics to quantify ~6,100 circulating proteins. With this dataset, we will define when and how much known proteins increase, decrease, or are unchanged following socio-

evaluative stress, providing a high-resolution map of systemic molecular recalibrations. This will show how the human brain-body system as a whole responds to acute mental stress, potentially providing new cues about the interplay of metabolic, immune, endocrine, digestive, and other systems under stress. Using this exciting new

dataset, we will identify functionally relevant families of proteins i) that precede and statistically predict peak cf- mtDNA reactivity, ii) that are co-released and follow the same quantitative dynamic trajectory as cf-mtDNA, and iii) that follow the peak of cf-mtDNA reactivity and represent potential systemic consequences of cf-mtDNA

signaling. In Aim 3 we apply a combination of sensitive molecular and biochemical approaches to define the molecular nature of cf-mtDNA in humans. Building on recent work demonstrating that cf-mtDNA does not circulate as naked DNA fragments but rather as encapsulated cargo in extracellular vesicles, we will profile the

size, density, morphology, molecular composition, and functional characteristics of cf-mtDNA subtypes. To gain further insight into the distinct forms of human cf-mtDNA, their potential origin, and functional physiological significance in the stress-disease cascade, we will deploy these approaches to well-controlled baseline samples,

as well as post-stress samples at cf-mtDNA peak reactivity. These large-scale and molecular experiments will generate foundational knowledge to develop new mitochondrial markers for stress and mental health research.