The Mitochondrial Stress, Brain Imaging, and Epigenetics Study
Phase: NA
Conditions: Mitochondrial Diseases
Sponsor: Columbia University

Mitochondrial Stress, Brain Imaging, and 
Epigenetics (MiSBIE)
NCT04831424
October 6, 2023

Protocol Summary Form 
Lay Summary of Proposed Research 
Age-related physical and cognitive decline, as well as the risk of neurological diseases, are 
increased by the effects of psychosocial stress. Psychosocial stress triggers neuroendocrine, 
metabolic, cardiovascular, and inflammatory changes in the body. These changes vary in 
nature and magnitude between individuals, and are associated with long-term disease risk. 
However, the biological determinants of the stress response are not well understood. Why do 
some individuals respond more strongly than others to the same stressor? 
Our preclinical studies in mouse models with dysfunctional mitochondria, a cellular organelle 
which produces energy and signals of adaptation, indicate that mitochondria regulate how 
different organ systems and major stress response axes are activated during psychological 
stress. This project aims to translate this discovery in a population of individuals with varying 
degree of mitochondrial dysfunction, and to test potential neural mechanisms. This study will 
take place at Columbia University Irving Medical Center (CUIMC) and the Mortimer B. 
Zuckerman Mind Brain Behavior Institute (ZMBBI) and will recruit patients with genetic 
mitochondrial disease and healthy controls. 
Each participant will be studied over two days. On Day 1, we propose to monitor 
dynamic changes in in different hormones and molecules in blood (catecholamines, 
inflammatory cytokine interleukin 6) and saliva (cortisol) in response to a standardized 
laboratory “stressor”. Participants will also undergo a medical assessment. On Day 2, we 
propose to examine whether these changes are mediated by inter-individual differences in 
functional connectivity among different brain regions measured by functional magnetic 
resonance imaging (fMRI). 
Participants will also complete a questionnaire package to evaluate psychosocial functioning 
and clinical disease severity as well as undergoing a neuropsychological assessment. This 
interdisciplinary project leverages access to a unique population of patients with genetically 

Protocol Summary Form 
 
 
 
 
defined primary mitochondrial disease within our clinic and nationally. This translational 
project will generate a unique combination of complimentary molecular, physiological, and 
neuroimaging data that will substantially advance our understanding of the mechanisms 
linking mind and body processes. We expect that the proposed studies will thus begin to 
explore the role of mitochondria in stress regulation, from organelle to organism, and may 
reveal clinically and/or behaviorally modifiable pathophysiological mechanisms for 
mitochondrial disease. 
 
 
Background, Significance, and Rationale 
  
The cellular mechanisms by which mitochondrial defects lead to disease are not fully 
understood. Mitochondria are organelles that produce cellular energy required for the 
normal function of cells, organs and physiological systems. They are known as the cell’s 
“powerhouse”. When maternally-transmitted mitochondrial DNA (mtDNA) mutations and 
other genetic defects were discovered to cause debilitating human disease in 1988, it was 
naturally assumed that this could be explained by an energy production deficit. Although it is 
certainly the case that energy production deficiency is a consequence of genetic 
mitochondrial defects, dysfunctional mitochondria also impact other cellular processes, 
including the expression of thousands of genes in different cell types (1-4), and possibly 
cellular aging via premature telomere erosion (5). Evidence indicates that respiratory chain 
alterations can have systemic, cell non-autonomous effects on the functioning complex 
physiological systems (6). As a result, the impact of mitochondrial defects may reach beyond 
the confines of individual cells and affect the regulation of multiple physiological systems, 
thus contributing to the pathogenesis of inherited and acquired mitochondrial disease. 
Physiological systems are normally and regularly activated as part of the normal stress 
response to physical, psychological and social events that are perceived as stressors (7). This 
involves the hypothalamic-pituitary-adrenal (HPA) and sympathetic-adrenal- medullary 
(SAM) axes, and the autonomic nervous system (ANS) which is itself divided into sympathetic 
and parasympathetic divisions. The HPA axis leads to cortisol secretion, the SAM axis leads to 
the secretion of adrenaline and noradrenaline, and the ANS exerts direct control over heart 
rate via vagal input. These neuroendocrine factors in turn regulate the levels of metabolic 
substrates (glucose, lipids, amino acids) (8), and immune system- derived pro- and anti- 

Protocol Summary Form 
 
 
 
 
inflammatory cytokines (9). Together, mobilization of these physiological systems aims to 
maintain a stable (healthy) internal milieu within the body by matching these to 
environmental demands. Achieving stability of certain vital parameters through change in 
other parameters (e.g., hormones, metabolites, cytokines) is termed “allostasis” (10). 
 
During chronic stress, the repeated and prolonged activation of stress response systems can 
lead to imbalance, and “wear-and-tear” among these systems. In this case, certain hormones, 
metabolites, and cytokines become chronically dysregulated (elevated or reduced), 
culminating in a multi-systemic state of physiological dysregulation termed “allostatic load” 
(11). The allostatic load (AL) model underlies the potential adaptive and damaging action of 
stress mediators (12). AL is an integrative perspective that is more precise than the term 
“stress”, since it includes the physiological effects of health-promoting and health- damaging 
behaviors as well as stressful experiences (13, 14). These concepts refine our understanding of 
“stress” used to describe adaptation and maladaptation to stressors. 
 
The chronic cumulative effect of systemic dysregulation can be measured by assessing 
allostatic states or specific dysregulated states of specific systems. There are four postulated 
physiological profiles that represent allostatic states (12, 15): (1) repeatedly activated 
responses, (2) non-habituating responses, (3) prolonged responses, and (4) inadequate 
responses. Detecting allostatic states is possible by investigating repeated measures such as 
stress hormone dynamics during diurnal collection or in response to laboratory-based 
stressor. Collecting detailed biological data over time allows researchers to identify 
prodromal/underlying allostatic states that can then be aggregated into a multi- systemic 
algorithm to calculate allostatic load, and possibly predict disease risk. 
 
An allostatic load index can be quantified by combining a specific set of biomarkers 
representing multisystem physiological functions (16-18). Allostatic load measured in large 
cohorts of individuals can predict later physical function and cognitive decline (16, 19, 20), 
indicating the prognostic value of physiological perturbations. As a predictor of mortality and 
decline in physical functioning in the elderly, the allostatic load index outperforms 
traditionally accepted risk factors such as the metabolic syndrome or individual biomarkers 
(21). Thus, allostatic load represents a research concept to comprehensively quantify multi- 
systemic dysregulation, as well as a potentially useful clinical predictor of disease incidence 
and progression. Allostatic load has never been measured nor studied in the context of 
mitochondrial medicine. An objective of this project is therefore to determine the influence 
of mitochondrial defects on allostatic load. 
 
Work from our team and other laboratories have shown that mitochondria may alter the 
regulation of different allostatic physiological systems, which could promote allostatic load 
and exacerbate disease through systemic effects (8). Mouse models generated to have the 

Protocol Summary Form 
 
 
 
 
same nuclear genome (i.e., conplastic) but different mtDNAs showed differential 
neuroendocrine and behavioral responses to stress (22, 23). A different study with mice 
containing a whole-body heteroplasmic mixture of two normal mtDNAs also resulted in 
greater anxiety responses to behavioral testing (24), suggesting that mitochondrial 
dysfunction causes abnormal stress reactivity. 
 
More recently, we mapped the multisystemic stress response in normal mice compared to 
four strains of mice with whole-body genetic mitochondrial defects (25). Defects consisted 
of either a mtDNA point mutation in either the ND6 or COXI genes, deletion of a 
mitochondrial antioxidant enzyme (NNT, nicotinamide nucleotide transhydrogenase), or 
deletion of the major transporter for ATP/ADP across the inner mitochondrial membrane 
(ANT1, adenine nucleotide translocase 1). In response to a single episode of psychological 
challenge, each mitochondrial defect caused a distinct multi-systemic stress response 
signature involving modulation of the HPA, SAM, inflammatory, metabolic, and 
transcriptional responses (25), demonstrating the pervasive effect of mitochondrial 
functions on multiple physiological systems. 
 
In humans, limited evidence indicates that allostatic systems are likewise influenced by 
primary mtDNA defects. As in our preclinical study, HPA axis function was found to be blunted 
in individuals with NNT mutations associated with familial hypocortisolemia. In patients with 
ANT1 mutations, resting noradrenaline levels were significantly elevated, indicative of 
hyperactive SAM axis activity (27). In response to exercise, a physical stressor used to discern 
allostatic states, patients with mtDNA 3243A>G (m.3243A>G) mutation and single large- 
scale deletions also show increased adrenaline/adrenaline responses (28), and exaggerated 
cardiovascular responses to exercise (29), indicative of abnormal autonomic regulation. 
Accordingly, heart rate variability (HRV) may also be altered in patients with the m.3243A>G 
mutation, indicative of both sympathetic and parasympathetic changes (30). Collectively, this 
body of evidence is consistent with our pre-clinical data, indicating robust effects of 
mitochondrial functions on the major stress response systems in mouse and human. However, 
no study to date has evaluated AL, stress reactivity, and stress recovery to 
psychological/physical stress in patients with mitochondrial disease. 
 
The reason why this is relevant to health, and to mitochondrial disease in particular, is because 
psychological and social stressors increase disease risk (31). The underlying mechanisms likely 
involve stress-induced release of mediators from the nervous, endocrine, immune and 
metabolic systems (8, 12). In support of this model, research has demonstrated that several 
psychological and social factors are associated with elevated allostatic load index (17, 32). For 
example, elevated allostatic load is correlated with adverse social contexts (e.g., social 
support, social hierarchy, and adverse life events) (33, 34), workplace stress and burnout 
symptoms (35-37), sleep disturbances (38), and psychiatric symptoms (39, 40), to name a few. 

Protocol Summary Form 
 
 
 
 
The allostatic load model can be used to link psychosocial contexts to biological mechanisms 
and health outcomes. For example, mothers caring for children with life- threatening 
conditions manifest elevated allostatic load that is further associated with psychiatric 
symptoms (41) and hippocampal atrophy within the brain (42). 
 
Combining psychosocial measures in allostatic load research provides a more global 
understanding of inter-individual differences in stress (patho)physiology. With the objective 
to delineate specific profiles of disease trajectories, integrating psychosocial measures (i.e., 
questionnaires) with biological parameters (blood-, saliva-, hair-, and urine-based 
biomarkers) and clinical data (i.e., functional capacity, mutation load) enables the 
triangulation of methods. Scientifically, this combination of converging methods is 
especially important when investigating the function of stress response  axes and 
hormones such as the HPA axis and cortisol that may exhibit hypo- or hyper-activity in 
association with diverse psychiatric  conditions, disease states, and ultimately allostatic 
load (43). 
 
Evaluating biological disturbances in the context of psychosocial factors can enhance overall 
understanding of existing dysregulations of neuroendocrine systems, and to isolate the role 
of mitochondrial dysfunction independent from potentially confounding variables. A meta- 
analysis of 62 studies concluded that while the cortisol awakening response is positively 
associated with workplace stress and general life stress, it is negatively associated with 
symptoms of burnout, fatigue, and exhaustion (44). Furthermore, hypocortisolism is present 
in approximately 20-25% of patients suffering from stress-related diseases like burnout, 
chronic fatigue syndrome, fibromyalgia, post-traumatic stress disorder, and atypical 
depression (43). Taken together, testing the effects of mitochondrial dysfunction 
(independent variable) on specific biomarkers/AL (dependent variable) while accounting for 
continuous measures of depressive, burnout, and other symptoms (covariates) will add 
strength to our study findings. 
 
The inclusion of questionnaires may provide insights that have yet to be applied in 
mitochondrial biology. By studying genetic, biochemical, and physiological measurements 
together, this project will use validated questionnaires for assessing domains of psychological 
and social functioning previously shown to correlate with allostatic load and specific 
biomarkers. The goals of these measurements are two- fold: i) To account for potential 
confounding variables; and ii) To understand the determinants of vulnerability and resilience 
in patients by exploring psychosocial factors/chronic stressors which may interact with 
mitochondrial dysfunction to impact physiological dysregulation, and eventually disease 
progression. 

Protocol Summary Form 
 
 
 
 
Multi-systemic dysregulation can arise from peripheral alterations of specific organ functions, 
such as the adrenal glands, the liver, peripheral nerve function, or the cardiovascular system. 
Alternatively, maladaptive neuroendocrine, metabolic, and epigenetic profiles may be driven 
by abnormal central nervous system functions, including resting brain connectivity and 
activation patterns during stressful situations. Thus, the brain is understood to play a central 
role in stress- related pathogenesis (45). To date, however, neither brain connectivity nor 
activation has been examined in patients with mitochondrial disease. The proposed study will 
be the first study to assess the pathways linking mitochondrial dysfunction to stress 
pathophysiology, and to identify candidate neural regulatory mechanism to explore further. 
This would provide us with unique insights into the long-standing question of how 
psychosocial stress ‘gets under our skin and skull’. 
 
To study the potential role of the brain as a driver of abnormal stress reactivity, we 
therefore plan to use functional magnetic resonance imaging (fMRI) and multivariate 
analyses to test a mediation model where abnormal stress perception caused by 
mitochondrial dysfunction occurs at the level of the brain and drives abnormal allostatic 
and epigenetic responses peripherally. This study will thus begin to untangle the role of 
central nervous system vs peripheral physiological processes in mitochondrial disease, 
allowing us to explore a previously unrecognized sub- cellular mitochondrial 
pathophysiological mechanism. 
 
Deciphering the role of mitochondrial dysfunction and the mechanism(s) by which it alters 
stress physiology and is transduced into allostatic load is not only directly relevant to 
mitochondrial disease, but also to a greater spectrum of neurodegenerative disorders where 
both life stress (46, 47) and mitochondrial dysfunction (48, 49) have been implicated, including 
Alzheimer disease, Parkinson disease, schizophrenia, and depression. In addition, this will be 
the first study to examine the relations between mitochondria, epigenetic changes, and brain 
activation. At the end of this study, we will have identified candidate molecular, 
neuroendocrine, and neural mechanisms that can be further investigated in clinical 
populations and mechanistically explored further in animal models. 
 

Protocol Summary Form 
 
 
 
 
Specific Aims and Hypotheses 
  
To translate our preclinical findings and test these hypotheses with the same level of 
genetic specificity in humans, we leverage our access to a population of patients with 
molecularly defined m.3243A>G mtDNA mutation and individuals with single large scale 
mtDNA deletions in our clinical service for mitochondrial disease at CUIMC. To ascertain if 
the observed associations are linked to symptom types, we will also recruit individuals with 
the m.3243A>G mutation who have experienced stroke-like episodes or seizures. These 
symptoms characterize a clinical subtype known as MELAS (mitochondrial encephalopathy, 
lactic acidosis, and stroke-like episodes). 
 
Aim 1: Determine the effect mtDNA m.3243A>G and single mtDNA deletion on 
chronic and reactive stress biomarkers. 
Longitudinal studies of stress biomarkers demonstrate that cumulative dysregulation of the 
HPA (hypothalamic-pituitary-adrenal) and SAM (sympathetic- adrenal- medullary) axes, 
cardiovascular, metabolic, and pro-inflammatory systems predispose to chronic physical and 
cognitive decline that overlap with symptoms of mitochondrial diseases. We hypothesize 
that patients with mtDNA defects (m.3243A>G) will manifest elevated allostatic load and 
stress reactivity compared to healthy controls. This will be assessed on a first hospital visit 
(Day 1) with repeated measures of blood (catecholamines, inflammatory cytokine IL-6), saliva 
(cortisol), and cardiovascular monitoring (heart rate variability, blood pressure) in response 
to a validated laboratory stress protocol, the socially evaluated cold pressor test (SECPT), 
which elicits strong multi-systemic reactivity. Results from this aim will establish for the first 
time the effect of inherited mtDNA defects on the nature and magnitude of stress responses 
in humans, and evaluate their association with patients’ symptoms. 
 
Aim 2: Assess DNA methylation signatures and cell aging in patients with the 
m.3243A>G mutation and single mtDNA deletion. 
Mitochondrial signaling involves the production of diffusible signals that alter the epigenetic 
machinery and the expression of most genes in the human genome; and stress alters DNA 
methylation in brain remodelling and stress reactivity genes. We hypothesize that patients 
with mitochondrial disease will show abnormal baseline DNA methylation signatures, and 

Protocol Summary Form 
 
 
 
 
exaggerated epigenetic responses to stress. We will isolate sub-types of peripheral blood 
mononuclear cells at baseline to map genome- wide cytosine methylation levels. Pre- and 
post-stress blood samples will also be collected to track stress reactive differences by 
pyrosequencing of target genes linked to stress regulation and brain remodelling. Results 
from this aim will complement those of Aim 1 by defining stress reactivity at the sub-cellular, 
molecular levels. 
 
Aim 3: Map brain-wide neural connectivity patterns in m.3243A>G and single mtDNA 
deletion patients, and determine if this mediate abnormal peripheral stress reactivity. 
Combining measurements of peripheral stress physiology with functional neuroimaging 
(fMRI-BOLD), our team and others have shown that neuroendocrine, inflammatory, and 
cardiovascular reactivity to stress can be predicted from discrete 
 
activation patterns among specific brain regions in healthy adults. Furthermore, the brain is 
an organ primarily affected in mitochondrial diseases and functional connectivity is sensitive 
to energetic defects, but the nature and impact of these alterations in human mitochondrial 
disease is unknown. We hypothesize that patients with mitochondrial defects will exhibit 
abnormal patterns of resting state and task- elicited neural connectivity, including the 
executive network, which will statistically mediate alterations in physiological stress 
reactivity. On a second visit (Day 2), we will perform structural and functional whole-brain 
neuroimaging of default mode network and tasks selected to match the stress reactivity on 
Day 1. 
 
Taken together, this translational study will establish how neural connectivity is altered in 
mitochondrial disease, and begin to untangle neural pathways that may translate 
mitochondrial dysfunction into abnormal physiological dysregulation. This study will be the 
first to test the role of mitochondria in mediating mind-body processes. The resulting papers 
will inform future work in various fields including clinical mitochondrial medicine and 
neurology, psycho-neuro-endocrinology, psycho- neuro- immunology, and psychosomatic 
medicine. 
 
Aim 4: To establish the within-person and technical variability in key study outcomes, 
two healthy volunteers will “repeat” visits to obtain blood samples up to 10 timepoints 
over the study period. 
To establish the within-person and technical variability in key study outcomes, we will 

Protocol Summary Form 
 
 
 
 
perform “repeat” visits on two healthy volunteers. This will provide essential quantitative 
information on some novel mitochondrial parameters that are measured in the MiSBIE study 
(cell bioenergetics, cell-free mitochondrial DNA, metabolic markers), including how they may 
vary within a person over time, relative to between- person differences. These difficult to 
obtain data can be used to quantify intra- individual variation over months-to-years and 
provide an empirical basis to evaluate the magnitude of differences among the primary study 
outcomes between the MiSBIE study groups. 
 
Description of Subject Population 
  
Sample #1 
 
Sample subject population: 
 
Subject Population 
Number of completers 
required to accomplish 
study aims 
Projected number of 
subjects who will be 
enrolled to obtain required 
number of completers 
Age range of subject 
population 
Individuals with the 
m.3243A>G mitochondrial 
DNA mutation 
30 
33 (accounting for 10% 
attrition or unusable data) 
18 to 60 
 
Description of subject population: 
 
We will investigate individuals carrying the m.3243A>G tRNALeu(UUR) mutation which is the most 
frequent mtDNA point mutation, and presents in the most severe case as MELAS (Mitochondrial 
Encephalopathy, Lactic Acidosis, and Stroke-like episodes) (50, 51) as well as non-MELAS carrier 
relatives. From this cohort and the existing patient registry, individuals who have previously 
had genetic diagnostic testing confirming the presence of the m.3243A>G will be identified and 
invited to participate in the study. Maternally related individuals with a first-degree relative 
who is a known carrier of the mutation are considered “obligate carriers” and will also be 
included. 
 
Individuals without a confirmed genetic test who are otherwise eligible and who have family 
members with 3243A>G mitochondrial DNA mutation and/or suspect they have the mutation 
will be invited to provide a buccal or urine sample to test for mitochondrial mutations. The 
genetic counselor or physician will review the specific consent form for genetic testing 

Protocol Summary Form 
 
 
[Genetic Testing Consent Form MiSBIE], and sample collection instructions remotely with 
these participants or at the Columbia University Irving Medical Center research site. 
 
Sample #2 
 
Sample subject population: 
 
Subject Population 
Number of completers 
required to accomplish study 
aims 
Projected number of 
subjects who will be 
enrolled to obtain required 
number of completers 
Age range of subject 
population 
Individuals with single large-
scale mitochondrial DNA 
deletions 
30 
33 (accounting for 10% 
attrition or unusable data) 
18 to 60 
 
Description of subject population: 
 
We will investigate individuals carrying a single large-scale mtDNA deletions that causes 
CPEO (chronic progressive ophthalmoplegia) and Kearns-Sayre syndrome (KSS) (52). Dr. 
Hirano follows >20 patients with CPEO/KSS, and leads a national consortium (NAMDC – 
North American mitochondrial disease consortium) where >70 patients with CPEO/KSS have 
consented to be contacted for research. From this group, patients who have previously had 
diagnostic testing confirming the presence of mtDNA single deletion will be identified and 
invited to participate in the study. 
 
Individuals without a confirmed genetic test who are otherwise eligible and who have 
family members with single large-scale mitochondrial DA deletions and/or suspect they 
have the deletion will be invited to provide a buccal or urine sample to test for 
mitochondrial mutations. The genetic counselor or physician will review the specific 
consent form for genetic testing [Genetic Testing Consent Form MiSBIE], and sample 
collection instructions remotely with these participants or at the Columbia University Irving 
Medical Center research site. 
 
Sample #3 
 
Sample subject population: 
 

Protocol Summary Form 
 
 
Subject Population 
Number of completers 
required to accomplish 
study aims 
Projected number of 
subjects who will be 
enrolled to obtain required 
number of completers 
Age range of subject 
population 
Controls 
100 
118 (accounting for 10% 
attrition or unusable data 
n=10 and pilot testing n=8) 
18 to 60 
 
Description of subject population: 
 
We will study healthy individuals without a diagnosis of mitochondrial disease recruited from 
the community. These participants will be age-, sex- and physical activity level- matched to 
participants from Groups 1 and 2. 
 
Sample #4 
 
Sample subject population: 
 
Subject Population 
Number of 
completers 
required to 
accomplish study 
aims 
Projected number of 
subjects who will be 
enrolled to obtain 
required number of 
completers 
Age range of 
subject 
population 
Individuals with the m.3243A>G mitochondrial 
DNA mutation with MELAS symptoms. This is 
the same diagnostic group as Group 1, with the 
exception that patients who have experienced 
stroke-like episodes or seizures will not be 
excluded 
30 
33 (accounting for 
10% attrition or 
unusable data) 
18 to 60 

Protocol Summary Form 
 
 
Description of subject population: 
 
We will investigate individuals carrying the m.3243A>G tRNALeu(UUR) mutation who have 
experienced stroke-like episodes, seizures, or both which characterize MELAS (Mitochondrial 
Encephalopathy, Lactic Acidosis, and Stroke-like episodes) (50, 51). From this cohort and the 
existing patient registry, individuals who have previously had genetic diagnostic testing 
confirming the presence of the m.3243A>G will be identified and invited to participate in the 
study. 
Individuals without a confirmed genetic test who are otherwise eligible and who have family 
members with 3243A>G mitochondrial DNA mutation with MELAS and/or suspect they have 
the mutation with MELAS will be invited to provide a buccal or urine sample to test for 
mitochondrial mutations. The genetic counselor or physician will review the specific consent 
form for genetic testing [Genetic Testing Consent Form MiSBIE], and sample collection 
instructions remotely with these participants or at the Columbia University Irving Medical 
Center research site. 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 

Protocol Summary Form 
 
 
Inclusion/Exclusion Criteria 
 
 
 
Groups 1 (m.3243A>G) & 2 (single mtDNA deletion) 
 
CRITERION 
METHOD OF ASCERTAINMENT 
Inclusion: 
 
1. Patients between the age of 18-60. 
Patient clinical records 
Phone screen or clinical evaluation 
2. Willing to provide saliva samples and have 
venous catheter installed for blood collection 
during the hospital visit 
Phone screen or clinical evaluation 
3. Willing to provide informed consent and 
capacity to consent 
Self report 
4. Use of effective method of birth control for 
women of childbearing capacity 
Phone screen or clinical evaluation 
5. Harbours a mtDNA mutation. Either the 
m.3243A>G point mutation, or a single large scale 
mtDNA deletion. 
Patient clinical records, or family history 
involving them as “obligate carrier” of the 
mtDNA defect 
6. English Speaking 
Self report 
Phone screen or clinical evaluation 
7. Confirmatory genetic test or willing to undergo 
genetic testing if one is not available. 
 
Exclusion: 
 
1. Patients with cognitive deficit incapable of 
providing informed consent will not be included 
TICS scores > 30, administered via phone 
screen or clinical evaluation 
2. Neoplastic disease 
Patient clinical records, and self-report 

Protocol Summary Form 
 
 
 
 
3. Symptoms of flu or other seasonal infection 
four weeks preceding hospital visit, as this would 
influence immune system parameters 
Phone screen or clinical evaluation 
4. Strokes & seizures. 
Patient clinical records, and self-report 
5. Raynaud’s syndrome (Rayneau phenomenon) 
Phone screen or clinical evaluation 
6. Involvement in any therapeutic trials listed on 
clinicaltrials.gov, including exercise 
Phone screen or clinical evaluation 
7. Clinical use of steroid therapy, which would 
impact the HPA-axis and other physiological 
systems (e.g., oral dexamethasone, prednisone, 
or similar) 
Patient clinical records 
Phone screen or clinical evaluation 
 
 
Group 3 (Controls) 
 
CRITERION 
METHOD OF ASCERTAINMENT 
Inclusion: 
 
1. Individuals between the age of 18- 60. 
Patient clinical records 
Phone screen or clinical evaluation 
2. Willing to provide saliva samples and have 
venous catheter installed for blood collection 
during the hospital visit 
Phone screen or clinical evaluation 
3. Willing to provide informed consent and 
capacity to consent 
Self report 
4. Use of effective method of birth control for 
women of childbearing capacity 
Phone screen or clinical evaluation 

Protocol Summary Form 
5. English Speaking
Self report, Phone screen or clinical 
evaluation 
Exclusion: 
1. Individuals with cognitive deficit incapable
of providing informed consent will not be
included
TICS scores > 30, administered via phone 
screen or clinical evaluation 
2. Symptoms of flu or other seasonal
infection four weeks preceding hospital visit,
as this would influence immune system
parameters
Phone screen or clinical evaluation 
3. Raynaud’s syndrome (Rayneau
phenomenon)
Phone screen or clinical evaluation 
4. Involvement in any therapeutic trials
listed on clinicaltrials.gov, including
exercise
Phone screen or clinical evaluation 
5. Metal inside or outside the body or
claustrophobia prohibitive to MRI testing
Phone screen or clinical evaluation 
6. Diagnosed with mitochondrial disease
m.3243A>G, or large scale mtDNA deletion
Phone screen or clinical evaluation 
Group 4 (m.3243A>G with MELAS symptoms 
CRITERION 
METHOD OF ASCERTAINMENT 
Inclusion: 
1. Patients between the age of 18- 60.
Patient clinical records 
Phone screen or clinical evaluation 

Protocol Summary Form 
 
 
 
 
2.Willing to provide saliva samples and 
have venous catheter installed for 
blood collection during the outpatient 
visit 
Phone screen or clinical evaluation 
3. Willing to provide informed consent 
and capacity to consent 
Self-report and clinician assessment 
4. Use of effective method of birth control for 
women of childbearing capacity 
Phone screen or clinical evaluation 
5. Carries the m.3243A>G point mutation and 
has had at least one stroke-like episode, 
seizure, or both. 
Patient clinical records, or family history 
involving them as “obligate carrier” of 
the mtDNA defect 
6. English Speaking 
Self-report 
Phone screen or clinical evaluation 
7. Confirmatory genetic test or willing to 
undergo genetic testing if one is not 
available. 
Medical Records 
Genetic Test 
Exclusion: 
 
1. Patients with cognitive deficit incapable 
of providing informed consent will not be 
included 
TICS scores > 30, administered via phone 
screen or clinical evaluation 
2. Neoplastic disease 
Patient clinical records, and self-report 
3. Symptoms of flu or other seasonal infection 
four weeks preceding hospital visit, as this 
would influence immune system parameters 
Phone screen or clinical evaluation 
4. Raynaud syndrome (Raynaud phenomenon) 
Phone screen or clinical evaluation 

Protocol Summary Form 
 
 
 
 
5. Involvement in any therapeutic trials listed on 
clinicaltrials.gov, including exercise 
Phone screen or clinical evaluation 
6. Clinical use of steroid therapy, which would 
impact the HPA-axis and other physiological 
systems (e.g., oral dexamethasone, prednisone, 
or similar) 
Patient clinical records 
Phone screen or clinical evaluation 
 
 
 
Study Procedures 
  
We will only recruit and see one participant per week, as per the standard MiSBIE protocol. 
This low scheduling frequency eliminates any possible exposure between participants. 
 
The study will unfold in two visits, distributed over two consecutive days. For review 
purposes, the study “Data Collection Form” (Appendix #5) in addition to a Study diagram 
summarizing study procedures in chronological order (Figure 1), are uploaded with this 
protocol. Breaks have been built into the study protocol, and participants are free to request 
breaks at any time. The study procedures comprise eight parts: 
 
Participants who have family member with 3243A>G mitochondrial DNA mutation or 
deletion and/or who suspect they have the mutation or deletion will be asked to confirm 
through genetic testing prior to participation. Kris Engelstad will approach these patients 
with information regarding genetic testing. These participants will provide a buccal cell or 
urine sample to test for mitochondrial variants. This genetic testing procedures (consent, 
sample obtain, genetic consult) will occur online over video chat or at the research site. The 

Protocol Summary Form 
 
 
 
 
genetic counselor or physician will walk you through taking the at- home or at site sample 
which we will use for genetic testing. The sample may take up to 5 minutes to collect. 
Participants will then mail samples using a prepaid label through USPS if they are not on the 
Columbia University Medical Campus. 
 
Samples will be processed at the Personalized Genome Laboratory (PGL) in pathology at 
Columbia University – this is a CLIA approved laboratory for genetic analysis. Samples will not 
be coded and will be stored according to standard CLIA approved mandate. The sample will 
be destroyed when a verification of genetic mutation or none is complete. Name and date 
of birth will be collected as mandated for genetic testing and verification of genetic 
mutation. Only the PGM laboratory will have access to these samples during processing. The 
results of this verification of genetic mutation will be shared with the genetic counselor, Kris 
Engelstad, PGM lab, Principal Investigator and Study Coordinator. 
 
The study team will reach out to participants with their genetic results. If they test positive 
for the m.3243A>G mutation or a single large-scale deletion, they will be eligible to 
continue in MiSBIE. 
 
Genetic testing results will be reported to the participant in person or via telemedicine by 
the investigator or the genetic counselor. The post-test consult includes: 1) A review of 
mitochondrial genetics regarding the mutation, 2) Potential clinical symptoms, 3) Treatment 
options/or lack of options, and 3) Brief discussion about family planning options (if female). If 
the participant is upset by the results and requests additional counseling they will be referred 
to a genetic counselor at Columbia University. Additionally, should they wish genetic 
counseling near to their home, the study team will provide them with contact for 
appropriate local genetic counseling services. 
 
DAY 1 
 
(1) Recruitment 
(2) Initial Blood Draw and Biological Samples Collection 
(3) Medical Examination 
(4) Breaks 
(5) Stress Reactivity-Recovery Paradigm 
(6) Urine Collection 
DAY 2 
(7) Metabolic Rate Analysis 

Protocol Summary Form 
 
 
 
 
(8) Questionnaires and Neuropsychological Assessment 
(9) Brain Imaging 
(10) Take Home Protocol 
(11) Study Monitor 
 
 
DAY 1 
(1) Recruitment: 
Patients 
with 
mitochondrial 
disease 
and 
healthy 
age/sex/race/physical activity level-matched controls will be recruited. As part 
of 
existing 
clinical 
care, 
some 
patients 
will 
have 
undergone 
(a) 
neuropsychological assessment, (b) metabolic assessment and (c) structural 
magnetic resonance imaging (MRI) as part of the diagnostic services of the 
Clinical Research Center for Muscular Dystrophy and Related Diseases 
Laboratory. From this patient pool, those molecularly identified to carry mtDNA 
defects (m.3243A>G, single deletion) will be provided with a study brochure and 
invited to sign a form agreeing to be contacted for future research. Potential 
participants will be contacted directly by the clinical coordinator or study 
coordinator who will provide information concerning the MiSBIE study and 
assess inclusion/exclusion criteria over the phone or in person. Should potential 
participants be interested in the study, they will be forwarded consent forms for 
their review. 
 
We will screen participants for COVID symptoms at the following timepoints: 
during the initial screening call: the Monday night before the participant 
arrives, the Tuesday morning of study activities and the Wednesday morning of 
study activities. We will take the participant’s temperature before starting 
study activities. We have a no-touch thermometer used for other MiSBIE 
procedures and have the required expertise to perform this measure. 

Protocol Summary Form 
 
 
 
 
Figure 1. Diagram illustrating study design and workflow. 

Protocol Summary Form 
 
 
 
 
For participants who agree to participate in the study, the a 2-day visit at the 
Columbia University Irving Medical Center will be scheduled and 
transportation/lodging arranged. Outside participants will be housed at the Edge 
Hotel (Washington Heights, 514 W 168th street), and given detailed instructions to 
follow prior to their arrival. 
 
(2) Initial Blood Draw and Biological Samples Collection: On the first outpatient visit 
(Day 1), a fasting blood draw to index allostatic load and cell isolation for 
mitochondrial function measurements and DNA/RNA analyses. Participants will first 
arrive at the Division of Behavioral Medicine, Presbyterian Hospital, 15th floor at 
9:00am and will be greeted by Catherine Kelly, the MiSBIE study coordinator. 
Participants will have been instructed not to eat or drink beverages other than water 
starting at midnight the previous day. 
Height, weight, waist circumference, hip circumference, and blood pressure will be 
collected before blood draw. Weight will be assessed on an electrical impedance 
scale (TANITA) to simultaneously estimate body composition (% body fat). Two 
readings will be taken, and the average of both used for analyses. 
 
Participants will next be relocated to the neighboring Psychophysiology Laboratory 
(Presbyterian Hospital, 16th floor). As per CUIMC’s workspace assessment, this 
space can comfortably accommodate 1 researcher and 1 patient. Furthermore, we 
will keep a window open to facilitate airflow, and designate one door to the lab as 
entrance and one as exit to ensure safe traffic flow. 
The study nurse will insert a venous catheter in the antecubital vein to draw blood 
amounting to 100 mL. This includes one EDTA coated tube (2mL), two Serum 
Separation Tubes (5mL), and five Buffered Sodium Citrate tubes (2.7mL) for 
complete blood count on whole blood and two for plasma (8mL), two non- coated 
tubes for serum (8mL) and five Acid Citrate Dextrose tubes (8.5mL) for cells to be 
used to assess mitochondrial function. Allostatic load biomarker analyses will be 
performed on plasma and serum. 
 
Cheek swab: A cheek swab (MasterAmp™ Buccal Swab Brush) of the internal 
mucosa from both cheeks will be performed to collect DNA. This procedure takes 
approximately 5 seconds and is painless. Participant will pull down their face mask 
for the duration of this cheek swab. Collected material will enable the measurement 
of mtDNA copy number, DNA methylation, and mtDNA mutation heteroplasmy in a 

Protocol Summary Form 
 
 
 
 
somatic tissue. 
 
Breakfast: Following blood draw and anthropometric measurements, participants 
will break their fast with a standardized continental breakfast in accord with any 
 
dietary restrictions (gluten intolerance, etc.) ascertained beforehand on a 
standardized menu. Participant will eat alone with face covering removed. 
 
Participants will be suggested to go for a bathroom break before the next part. 
Clip of hair: A small clip of hair is collected from the back of the head to measure 
cumulative levels of steroid hormones (cortisol, sex hormones, endocannabinoids). 
Two small bundles of approximately 100-200 hairs each (a few millimeters) are 
clipped near the scalp, just below the cranial bone. The instructions are attached 
with this protocol (Appendix #1.2). Analyses will be performed by our collaborator 
Dr. Clemens Kirshbaum (Technische Universität Dresden, Germany). 
 
Spirometry: A spirometry test will be used to assess lung function of the 
participants. The participant will be asked to inhale as much air as they can and 
exhale into the spirometer as hard as possible and as long as possible. 
 
(3) Medical Examination: 
A medical assessment of clinical status and symptomatology will be performed by 
Dr. Shufang Li using three validated clinical forms: 
 
Measure 
Description 
Time 
(minutes) 
Columbia Neurological Score (CNS) 
 
Newcastle Mitochondrial Disease 
Assessment Scale (NMDAS) 
 
 
 
 
 
NAMDC Case Report Form (CRF 
Assessment of central nervous system 30 
symptoms 
Semi-quantitative assessment of 
40 
different domains of functioning 
Current Function (Section I) 
Functional capacity and daily living 
System Specific Involvement (Section 
II) Current Clinical Assessment 
(Section III) Clinical assessment of 
current symptoms 
Detailed assessment of clinical 
30 
history, comorbidities, symptoms 
checklist 

Protocol Summary Form 
 
 
 
 
 
Karnofsky Performance Scale Index 
comorbidities, symptoms checklist 
Classifies functional impairment 
 
1 
Total Time: 101 
 
 
 
(4) Breaks: Given the lengthy nature of the protocol and that some participants may 
experience fatigue, breaks have been scheduled at designated times along the protocol: 
a. Breakfast 
b. After the Medical assessment (Day 1 
c. Lunch Break on Day 1 (30 minutes) 
d. Following the Stress Reactivity–Recovery Paradigm (10 minutes) 
e. After the neuropsychological assessment (30 minutes) 
f. Lunch Break on Day 2 (30 minutes) 
g. After the MRI scan (15 minutes) 
 
 
 
The study procedures have been scheduled with ample time to account for 
unanticipated delays and breaks. During the consent procedures, the study coordinator 
will explicitly tell participants that he/she can request breaks at any time. The study 
coordinator will continuously interact with the participant, be available to answer any 
question, and to organize breaks as needed. During breaks, participant will also have the 
option of utilizing either a reclining chair to rest in a horizontal position. 
(5) Stress Reactivity–Recovery Paradigm: After the lunch, participants will have an 
IV line placed and be instrumented with physiological data acquisition equipment. 
Participants will be exposed to a standardized socio-evaluative laboratory challenge (58, 
59). Participants will be asked to perform a simulated public speaking task, consisting of 
2 min of preparation for a speech defending themselves against an alleged transgression 
followed by 3 min of videotaped speech delivery, as previously described (58, 59) 
(please find the protocol attached in Appendix #6). Throughout, we will collect repeated 
measures of blood (cortisol, ACTH, catecholamines, inflammatory cytokines), saliva 
(cortisol), and physiological monitoring (blood pressure, heart rate variability, galvanic 
skin response, and respiration). Research assistant Sophia Tepler and Vincenzo Lauriola 
will monitor physiological data collection during the session and equip participants. This 
stress paradigm session consists of a five-part protocol: 
 
(A) Participants will be equipped with: 

Protocol Summary Form 
 
 
 
 
a. A 3-lead ECG to monitor of heart rate 
b. A finger cuff blood pressure sensor to monitor blood pressure 
c. Skin conductance surface device to monitor sympathetic nervous system activity 
(sweating) 
d. Two elastic breathing bands on the chest and abdomen. 
 
(B) Participants will rest for 30 minutes in a calm room and asked to refrain from any other 
medium (e.g., smartphones) that could be a source of distraction. This will avoid to 
introduce variability in inter-individual responses. This will allow them to acclimate to 
their surroundings and to the equipment (e.g., ECG electrodes, finger cuff). 
 
(C) Participants will be exposed to a socio-evaluative stress (58, 59) that induces significant 
psychophysiological responses among multiple systems. Psychosocial stress is induced 
by simulating videotaping and that these video recordings will be analyzed for speech 
performance by comparing their performance with those of other participants. Eight 
(10) mL of blood for plasma (5ml) and serum (5ml) will be collected via the venous 
catheter at eight defined time-points relative to the onset of the SECPT (-5, +5, +10, +20, 
+30, +60, +90 and +120 minutes), for a total of 80 mL of blood. Time points are 
illustrated in Figure 2. 
 
 
 
Figure 2. Expected kinetics of stress biomarkers after the socio-evaluative stress. BP = blood pressure; CORT = 
cortisol; E = epinephrine; HR = heart rate; IL-6 = interleukin-6; NE = norepinephrine. 
 
Affect ratings will be collected at each time point on an iPad to measure subjective 
feelings of positive and negative affect (calmness and nervousness, see attached in 
appendix #1.3). 

Protocol Summary Form 
 
 
 
 
In addition, 1 mL of saliva will be collected with salivettes (Starsted) at each time point. A 
sterile cotton swab will be introduced in the participant’s mouth, soaks with saliva for a 
period of approximately 2-3 minutes, and is placed back into the tube for subsequent 
saliva extraction by centrifugation. Oral (sub- lingual) and epidermal (anterior part of 
neck, back of the dominant hand) temperature will be measured non-invasively at the 
same time points using a no-touch infrared thermometer (AccuMed AT2102 or HTD8808) 
to detect thermal changes during stress reactivity and recovery. 
At the last time point (+120 minutes), an additional 8.5 mL of blood will be collected to 
isolate PBMCs to assess effects of stress on epigenetic modifications of DNA and gene 
expression on sub-populations of cells and one EDTA coated tube (2ml) for complete 
blood count on whole blood, which totals 10.5ml. 
 
Total blood volume drawn including the fasting morning collection and stress reactivity is 
approximately 190.5 mL (100 mL baseline for AL, mitochondrial function, and DNA/RNA) 
+ (10 x 8 = 80 mL for stress reactivity biomarkers) + (10.5 mL for DNA/RNA)]. This volume 
does not exceed the prescribed limit for adult individuals above 110lb of body weight 
(200mL), and it is less than half the volume of a standard blood donation (approximately 
500 mL). After the 120min time point, the venous catheter will be removed. 
 
(D) 
Deep breathing: Five minutes later, participants will be asked to perform a paced 
deep breathing exercise with visual feedback lasting two minutes while heart rate is 
recorded. This will enable assessment of autonomic regulation through breathing. 
 
(E) 
Standing: Sit-to-stand posture changes elicit changes in heart rate and blood 
pressure. Participants will be asked to go from the sitting position and stand for 5 minutes. 
Then sit down again. 
 
(F) 
Functional capacity: Participants will then be asked to stand as many times as they 
can, as quickly as possible over the course of 30 seconds. This simple 30 second sit-to- 
stand test is used to measure functional capacity. 
 
Respiratory rate, heart rate, blood pressure, and skin conductance will be continuously 
recorded. 
 
If the participant’s gait is unsteady or if participants are known to have ataxia or other 
movement abnormalities, an additional study personnel will be in the room to spot the 
participant during this task. 
 
(G) 
Cold pressor test: Participants will be asked to immerse their right hand up to and 

Protocol Summary Form 
 
 
 
 
including the wrist into a bucket filled with ice cold water (4±0.5°C) for 90 seconds (60). 
This will be followed by a 10-minute recovery period after which a saliva sample will be 
collected. 
 
(H) 
Metabolic Rate Analysis: Participants will then be asked to breathe normally into 
the ReeVue, a device that measures basal metabolic rate, for 10 minutes. The device 
analyzes oxygen consumption and uses standard algorithms to convert oxygen 
consumption to metabolic rate or energy consumption. 
The ReeVue breathing hose is specifically designed with a one-way valve such that 
participants breathe in from the room and out through the device, thus avoiding exposure 
to any particles from the device. All participants will use a new, disposable breathing tube 
and nose clip during data collection. 
 
(I) Participants will (a) be de instrumented of equipment. 
 
For most of the afternoon protocol, only 1 researcher will be in the lab space with the 
participant. The only exception to this is during periods where the nurse will collect a 
blood sample at the same time as the study coordinator is collecting a saliva sample. This 
timing is imperative to the study design of this protocol. 
 
The participant will be stationary and instrumented with physiological equipment for the 
course of the afternoon. 
 
Time estimation task: After being de-instrumented, participants will take part in a time 
estimation task. For the first part of this task, the coordinator sets a buzzer for a certain 
number of seconds, and the participant must estimate how much time has passed. For 
the second part of the task, the participant must say when a certain amount of time has 
passed. Each of the two parts consists of 10 brief trials of 10, 30, or 60 seconds. In total, 
this task takes about 20 minutes. 
 
Early MiSBIE participants who did not complete this task during their visit will be 
recontacted to complete the task over password-protected video call. Calls will take 
places between 3PM-5PM to replicate timing during normal study procedures. 
 
(6) 
Urine collection: At the end of the Day 1 visit, participants will be provided with a 
designated container to collect overnight urine. The container contains a small amount of 
acetic acid to preserve catecholamines. Participants are provided with a disposable plastic 
cup(male) or a urine hat(female) and instructed to urinate in the cups, and then poor their 

Protocol Summary Form 
 
 
 
 
content into the larger container to avoid any potential skin contact with the preservative. 
The target period will be from 8pm to 8am (12 hours) between Day 1 and 2. The container 
will be provided into a large sealed “Ziplock” bag and in an opaque tissue bag for 
transportation. This prevents undesirable odors and minimize psychological discomfort 
or stigmatization that could be associated with this task. An instructions sheet (attached 
for review in appendix #1.4) will be provided to all participants along with the container 
at the end of Day 1. Urine will be processed and biomarkers analyzed on site. 
 
DAY 2 
The urine container will be received, and compliance assessed. 
 
(7) Metabolic Rate Analysis: Participants will be asked to breathe normally into the ReeVue 
for 10 minutes. This will give us information about the participant’s resting metabolic rate. 
 
(8) Questionnaires and Neuropsychological Assessment: The participant will be asked to 
complete a few questionnaires on paper at the hotel both at the day of arrival and at the 
end of Day 1 (Package #1 and #2). After the breakfast and between the +60 and +120 
minute time points of the stress reactivity protocol, participants will complete a small 
questionnaire package (listed in the “Assessment Instruments” section, Package #3 and 
#4) on an electronic device (iPad) via an encrypted online system (RedCap) to assess 
psychosocial variables. Two RedCap projects will be created to manage data: one for 
screening of potential participants, and one for enrolled participants. RedCap will be used 
throughout the study to store and export data for analyses. 
For participants who have visual processing difficulties, questions can be read aloud to the 
participant by either the coordinator or caregiver. 
 
On Day 2, participants will also complete another set of questionnaires listed in the 
“Assessment Instruments” section (Package #5) via RedCap. The participant will be alone 
while completing the questionnaire package. 
Some of the questions included in these questionnaires address suicidal ideation. To 
assess participant safety, we will monitor participant responses to these questions. If the 
participant indicates 1 (I have thoughts of killing myself but would not carry them out) on 
Beck Depression Inventory (BDI) (Day 2 Part 2) plus a moderate BDI score (17 or over) OR 
Participant rates a 2(I would like to kill myself on BDI) the study coordinator will follow a 
series of questions to assess risk. If the participant indicates they have a plan, contact Dr. 
Peter Shapiro, if he is unreachable contact Dr. Luis Periera. If the participant indicates, 
they have the means and/or a timeframe inmind, this indicates serious risk and the study 
coordinator will remain with the participant at all times and contact Dr. Peter Shapiro. 

Protocol Summary Form 
 
 
 
 
In addition, a compliance form will be submitted to the participants at the beginning of Day 
1 and Day 2 to ensure they followed the instructions (please see attached in appendix 8 
and 9). 
 
Neuropsychological functioning will be assessed by a trained study coordinator using the 
following instruments: 
 
Measure 
Description 
Time (minutes 
) 
Wechsler Abbreviated Test of 
Intelligence (WASI-II) (54) 
Estimate of current intellectual 
function 
20 
Vocabulary 
 
 
Matrix Reasoning 
 
 
Neuropsychological 
Assessment Battery (NAB) (55) 
 
10 
Digits Forward and Backward 
Working memory 
 
Shape Learning 
Visual learning and memory 
 
Numbers and Letters 
Selective and divided attention 
 
Repeatable Battery for the 
Assessment of 
Neuropsychological Status 
(RBANS) (56) 
 
20 
Line Orientation 
Visual spatial skills 
 
List Learning 
Verbal learning and memory 
 
Picture Naming 
Word generation 
 
Coding 
Processing speed 
 
List Recall 
Delayed Memory 
 
List Recognition 
Delayed Recognition 
 
Delis-Kaplan Test of Executive 
Functions (D- KEFS) (57) 
 
38 
Trail Making 
Working memory, cognitive 
flexibility and speed 
 
Verbal Fluency 
Language production 
 
Color-Word 
Inhibition 
 

Protocol Summary Form 
 
 
 
 
Sorting 
Mental flexibility and 
conceptualization 
 
Test of Premorbid Functioning 
(TOPF) 
Estimate of premorbid 
intellectual function 
2 
 
 
90 
 
 
(9) 
Brain Imaging: On the second outpatient hospital visit (Day 2) that will take place at 
the Mortimer B. Zuckerman Mind Brain Behavior Institute (ZMBBI), we will collect fMRI- 
BOLD data during psychological tasks and rest. ZMBBI now sanitizes the MRI machine before 
and after each participant. Specifically, we will administer three tasks to assess function of 
the putative threat/salience network (61, 62), or brain appraisal system (63), a neural 
network showing stress-related activity that includes the amygdala, anterior cingulate 
cortex, and hippocampus. 
 
Magnetic resonance imaging will be used with a protocol consisting of one structural scan 
of high–resolution T1–weighted images, and fMRI blood oxygen level-dependent imaging 
(BOLD) will be employed over task runs consisting of T2*weighted gradient–echo images. 
Scanning will take place on a GE MR750 3 Tesla scanner at ZMBBI. These techniques were 
selected as they are non- invasive and widely used, ideally suited to evaluate functional 
connectivity, and they will provide measurements built on the previous literature. 
 
Participants will be escorted to ZMBBI on the second day of the study, on the first floor. 
Upon arrival, participants will be ushered by the study coordinator to a private waiting room 
in the MRI suite on the basement floor of the ZMBBI building. The urine container will be 
received, and compliance assessed. 
 
Five minutes after arriving, 1 mL of saliva will be collected with a salivette, as per the 
previous day. Affect ratings (4 questions) will also be collected, as per the previous day. 
 
For female participants, a pregnancy test will be performed because of potential unknown 
long-term effects of MRI on fetal development. Participants who refuse to take a pregnancy 
test prior to the start of the MRI scan for any reason they will forgo their participation in the 
MRI. 
The scanning protocol will last 1.5 hours, including all scanning sequences and set up of the 
participant in the scanner suite. The participant will be positioned on the MRI table, using a 
plastic head-holder to decrease head movement during the scan. Participants will be given 
foam earplugs to reduce the noise of the MRI as well as a MRI-compatible hand held 

Protocol Summary Form 
 
 
 
 
response pad. 
A three-lead ECG will be connected to participants to monitor heart rate responses in 
conjunction with brain activation. 
 
The task instructions will be projected from a liquid crystal display projector zoomed to a 
screen inside the MR suite or viewed through dual channel binocular goggles. Functional 
fMRI-BOLD images will be acquired. 
 
To account for the accessibility needs of patients with visual processing issues or 
ophthalmoplegia, instructions and text during scanning will be read aloud by the 
coordinator. 
During each pause between acquisitions, the participant is asked to rate their physical 
discomfort on a scale of 0-10. If the score rises above 5, the coordinator pauses the session 
and asks if the participant wishes to continue or if any adjustments can be made (i.e. change 
in position, an additional blanket for warmth, a sitting break). 
As on Day 1, at three time points during the MRI imaging session saliva samples will be 
collected, along with affect ratings (5 questions). 
 
These fMRI tasks will be administered in the following order: 
 
Saliva sample and affect rating #10 
 
(A) 
Localizer (1 run, 1 min) 
Localizing of brain and defining region of interest. 
 
(B) 
Field map (1 run, 1 min) 
Acquisition of field map for multiband correction. 
 
(C) 
T2 scan (1 run, 12 min) 
T2-weighted images for detection of potential lesions and ventricles. This scan will be 
collected as participants are instructed to “rest and allow their minds to wonder and not to 
think of anything in particular”. Participants are also instructed not to fall asleep. 
 
(D) 
T1 structural scan (1 run, 5 min) 
T1-weighted images for anatomical co-registration. This scan will be collected as 
participants are instructed to “rest and allow their minds to wonder and not to think of 
anything in particular”. 
 
(E) 
Multisensory (1 run, 5 min) 

Protocol Summary Form 
 
 
 
 
Participants will be trained and later exposed to the multisensory task. This taskconsists 
of visual (checker board), auditory stimulation (constant pitch sound), button push on the 
button box. 
 
(F) 
Resting State Run #1 (1 run, 11 min) 
This scan will be collected as participants are instructed to “rest and allow their minds to 
wonder and not to think of anything in particular”. Participants are reminded not to fall 
asleep. 
 
Saliva sample and affect rating #13 
 
(G) 
N-back task Run #1 (1 run, 6 min) 
This working memory task (64) consists of 9 blocks presented in ABBA or BAAB order, 
interspersed by 25 s of resting fixation. On each of 360 trials, a word is presented for 2 s 
followed by an inter-stimulus-interval fixation cross for 1 s. Participants are instructed to 
respond as quickly and accurately as possible to each word with a button press indicating, 
“yes, this word matches the word presented n-trials ago” (a target response) or “no, this 
word does not match the one presented n-trials ago” (a nontarget response). The first 3 
trials of each block are not included in the analysis; these trials are always nontargets in the 
3-back block and therefore did not carry the same task demand as the remaining trials. 
 
(H) 
N-back task Run #2 (1 run, 6 min) 
This is the second run of the N-back task. Instructions are the same as in “G”. 
 
(I) 
Socio-evaluative Task (1 run, 6 min) 
The procedure will be the same as in day 1 except subjects will be told that their 
“performance on the first speech task was slightly below average when compared with 
other participants’ speeches” and participants will be asked to “try to be more persuasive 
when delivering this speech”. They will be told that the speech will be delivered after the 
scan session to the expert who evaluated you yesterday. At the end of the task, participants 
will be told that they were randomly selected not to give their speech, as in previous work. 
A pulse oximeter will be hooked up to participants, and heart rate responses in conjunction 
with brain activation will be assessed 
 
(J) 
Cold Pressor Task (1 run, 6 minutes) 
This is a modified task from the cold pressor administered on Day 1. Participants 
will be instrumented with a thermal arm wrap designed specifically for the MRI 
environment and will be instructed as follows: “We will place this arm wrap around 
your hand and arm. It is quite cold and may be uncomfortable, but it will not injure 
you.” The arm wrap is a fabric-based foldable unit fitted with Velcro attachments 

Protocol Summary Form 
 
 
 
 
 
for detachable gel packs, which are kept in a temperature-controlled freezer prior 
to the procedure. Five (5) minutes before the procedure, gel packs are removed 
from the freezer, attached to the arm wrap, and layered with two layers of 
waterproof film paper to adjust immediate contact temperature to safe but 
uncomfortable -2±2 degrees Celsius, as previously described (65, 66). 
 
(K) 
Diffusion Tensor Imaging – DTI (Seq 1-2, 6 min) 
This scan will be collected as participants are instructed to “rest, allow their minds to 
wonder and not to think of anything in particular”. 
 
Saliva sample and affect rating #14 
 
(L) 
Diffusion Tensor Imaging – DTI (Seq 3-6, 15 min 
This scan will be collected as participants are instructed to “rest, allow their minds to 
wonder and not to think of anything in particular”. 
 
Participants will then be de-instrumented (chest electrodes removed) and recover their 
personal belongings that could not be introduced in the MRI room. 
 
We expect all participants to perform all tasks adequately. The total scan time is (1 
+ 1 + 12 + 5 + 5 + 11 + 5 + 5 + 6 + 6 + 6 + 15 min) 90 minutes. The scanner will be booked 
for 90 minutes for each participant to accommodate saliva sample #11, 
instrumentation/de-instrumentation with EKG, time between tasks, and additional setup 
time. 
 
Finally, participants will be debriefed  on  the  nature  of  the  stress  reactivity tasks  
and the reason for deception. The debriefing script has been uploaded to this protocol 
(Appendix #7). Specifically, the social-evaluative component is meant to maximize 
neuroendocrine stress responses (67), and understanding these profiles allows insights 
into physiological stress functions that are predictive of many health problems. 
Participants will be reassured that this task was meant to elicit a physiological stress 
response, not to evaluate them as a person, and that they did very well. Participants will 
be invited to ask the study staff any question they may have. 
 
(10) 
Take Home Protocol: 
(A) Saliva collection: Starting a week from the study visit, participants will be 
instructed to provide saliva samples on three non-consecutive weekdays separated by 
one day. Each participant will receive an iPad with a custom made home logbook 

Protocol Summary Form 
 
 
 
 
application prompting them to provide four 1 mL saliva samples: 
(1) upon awakening, (2) +30 minutes after awakening, (3) +45 minutes after 
awakening, and (4) before bedtime. Participants will then be given a Medication 
Event Monitoring System (MEMS 6 TrackCap Monitor, Aardex Ltd., Switzerland) 
cap container with the salivettes, which time stamps usage of each salivette. In 
addition, the application will record awakening time, sample collection times, 
and affect ratings. These measures will insure compliance and document any 
deviation from the protocol. Instructions for home collection of saliva samples 
are provided in the Home Logbook application and will be reviewed with the 
study coordinator at the end of Day 2. The Home Logbook application content is 
attached with the instruments for review (Appendix #10). 
(B) Fecal sample collection: Along with the saliva samples, participants will be 
provided with a sample collection kit (collection tube, spatula and toilet hat) for fecal 
collection. The container will be provided into a large sealed “Ziplock” bag. Instruction 
for fecal collection will be watched with the study coordinator at the end of Day 2 
(https://www.youtube.com/watch?v=ytr_hmJdHqM) and an instructions sheet 
(attached for review in Appendix #11) will be provided to all participants along with 
the container. 
 
(C) Actigraph: Participant sleep and activity will be monitored over 7 days. Participants 
will wear a wrist actigraph, a device that detects body movement and weighs about 2 
ounces. It is worn comfortably around the wrist, similar to a wristwatch, and will be 
left in place for the entire 7 days. A pre-stamped express courier box (FedEx) will be 
provided to participants to return samples upon completion. 
 
(11) 
Study monitor: Although we do not anticipate any adverse effects of the study 
procedures, at all times during the study protocol a clinician will be on call in the event 
that a participant would become distressed. The identified medical doctors are Dr. Li and 
Dr. Hirano. The Division of Behavioral Medicine, where the laboratory stressor is 
conducted, has on call clinical psychologists and social workers. 
 
To establish the within-person and technical variability in key study outcomes, we will 
perform “repeat” visits on two healthy volunteers. This will provide essential 
information on some novel mitochondrial parameters that are measured in the MiSBIE 
study (cell bioenergetics, cell-free mitochondrial DNA, metabolic markers), including 
how they may vary within a person over time, relative to between-person differences. 
These difficult to obtain data will allow us to quantify intra-individual variation over 
months-to-years, thus providing an empirical basis upon which to evaluate the 
magnitude of differences among the primary study outcomes between study groups. 

Protocol Summary Form 
 
 
 
 
 
For this purpose, we will obtain blood samples from two individuals at multiple points 
over the study period. These repeated (MiRep) visits will not be complete study visits, 
and are therefore not considered as MiSBIE visits for study monitoring purposes. Each 
visit will take no longer than 15 minutes and will be completed by a trained 
phlebotomist or by the study nurse. Consent will be completed prior to the blood draw, 
all study procedures other than the blood draw will be crossed out prior to obtaining a 
signature by the study coordinator. 

Protocol Summary Form 
 
 
 
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 Picard M, Zhang J, Hancock S, Derbeneva O, Golhar R, Golik P, et al. Progressive 
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2. 
Schroeder EA, Raimundo N, Shadel GS. Epigenetic silencing mediates mitochondria 
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3. 
Elstner M, Turnbull DM. Transcriptome analysis in mitochondrial disorders. Brain Res 
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4. 
Hamalainen RH, Manninen T, Koivumaki H, Kislin M, Otonkoski T, Suomalainen 
A. Tissue- and cell-type-specific manifestations of heteroplasmic mtDNA 3243A>G mutation 
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Schinzel R, Dillin A. Endocrine aspects of organelle stress-cell non- autonomous 
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7. 
McEwen BS. Stress, adaptation, and disease. Allostasis and allostatic load. Ann N Y 
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8. 
Picard M, Juster RP, McEwen BS. Mitochondrial allostatic load puts the 'gluc' back in 
glucocorticoids. Nat Rev Endocrinol. 2014;10(5):303-10. 
 
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