Study Notes on Cellular Stress and Mitochondrial DNA

Examination of Cellular Stress and its Effects on Mitochondrial DNA

Mitochondria

  • Energy Resource

    • Living organisms require a constant energy resource.

    • Energy is obtained through mitochondria, known as the "powerhouse of the cell".

  • Location

    • Mitochondria are situated in the cytosol (the liquid medium within a cell) of eukaryotic cells, which are characterized by a distinct nucleus and a nuclear membrane.

  • Energy Production

    • Mitochondria are responsible for approximately 90% of the energy production stored in adenosine triphosphate (ATP) molecules.

  • Physiological Functions

    • Mitochondria also influence cellular and other physiological functions through signaling molecules.

Supportive Terms

  • Enzyme Definition

    • An enzyme is a biological macromolecule, often a protein, that acts as a biological catalyst, accelerating chemical reactions without being consumed in the process.

  • ATP Synthase

    • ATP synthase is an enzyme that catalyzes the synthesis of ATP using adenosine diphosphate (ADP) and inorganic phosphate (Pi).

More Supportive Terms

  • Electron Transport Chain (ETC)

    • The ETC consists of a series of protein complexes where electrons are transported from electron donors to electron acceptors. This transfer couples with the movement of protons (H+ ions) across a membrane.

    • Many enzymes in the ETC are embedded within the mitochondrial membrane.

Mitochondrial Ultrastructure

  • Visual Representation

    • Electron microscope images show distinct structures within mitochondria, including:

    • Inner membrane

    • Intermembrane space

    • Outer membrane

    • Matrix

    • Cristae

    • Ribosomes

  • Key Components

    • DNA Cart: Refers to the presence of mitochondrial DNA within mitochondria.

    • FOF1-ATPase functions as an ATP-producing enzyme within the mitochondrial membranes.

mtDNA Vulnerability to Oxidative Stress

  • Phosphorylation Definition

    • Phosphorylation is the chemical addition of a phosphoryl group (PO3-) to an organic molecule.

  • Oxidative Phosphorylation

    • This is the process where ATP synthesis is linked with electron movement through the mitochondrial electron transport chain, involving oxygen consumption and energy generation.

    • Responses to stress, both physical or psychological, require oxidative phosphorylation, resulting in the production of Reactive Oxygen Species (ROS).

Effects of Stress on mtDNA

  • Impact of Physical or Psychological Stress

    • Stress causes oxidative stress and builds up intracellular ROS, which adversely affects the mtDNA due to its proximity to these harmful species.

Comparison of mtDNA and Nuclear DNA

  • Mitochondrial DNA (mtDNA)

    • Each mitochondrion contains its own genome, known as mtDNA.

    • Like nuclear DNA, mtDNA is double-stranded, encodes essential genes, and is maternally inherited.

    • mtDNA replicates independently of nuclear DNA, which distinguishes it significantly.

    • It is smaller than nuclear DNA and encodes fewer genes, containing 37 genes, 13 of which are crucial for mitochondrial respiration.

  • Vulnerability

    • mtDNA is particularly vulnerable to oxidative stress, especially from ROS, which may lead to mutations.

Importance of Mitochondrial DNA in Psychological Context

  • Psychological Interest

    • Psychologists examine mitochondria and mtDNA due to existing psychometric measures of stress (e.g. Beck Depression Inventory, Perceived Stress Scale, Maslach Burnout Inventory) since there are no reliable biological measures of stress.

    • A biological measure may reveal a person's stress levels and available resources for functioning and managing stress.

  • Cell-Free Mitochondrial DNA (ccf-mtDNA)

    • In adaptive crises, mitochondria can exit cells, leading to increased levels of ccf-mtDNA.

    • Elevated ccf-mtDNA levels correlate to a higher risk (4-8 times) of mortality in critically ill hospitalized patients.

    • Elevated ccf-mtDNA levels are also observed in several conditions, including:

    • Cancer

    • Acute systemic inflammatory responses

    • Chronic inflammation

    • Diabetes

    • Sepsis

    • Physical trauma

    • Myocardial infarction.

  • Psychological Disorders

    • ccf-mtDNA levels associate with psychological stress; higher levels found in individuals with major depressive disorders (MDD) and suicidal tendencies.

    • Elevated ccf-mtDNA levels persist in MDD patients unresponsive to antidepressant therapy (selective serotonin reuptake inhibitors - SSRIs).

    • Lower ccf-mtDNA levels have been observed in SSRI responders.

Psychological Stress and Mitochondrial Damage

  • Chronic Psychological Stress

    • Recent studies show that chronic stress damages mitochondria and decreases enzymatic activity of ETC complexes, which leads to:

    • Dysregulation of the ETC

    • Altered oxygen consumption rates

    • Changes in intracellular content

    • Genetic sequence alterations.

  • Acute Psychological Stress

    • Two studies recorded significant ccf-mtDNA release in response to controlled acute psychological stress exposures:

    • Hummeel et al. observed a 1.6-fold increase in plasma ccf-mtDNA just 2 minutes after stress via the Trier Social Stress Test (TSST).

    • Trumpff et al. documented a 2-3 fold increase in serum ccf-mtDNA within 30 minutes post-TSST among 53 middle-aged subjects, which was the study intended to replicate with an all-female university population.

mtDNA vs. Nuclear DNA Vulnerability

  • Vulnerability Analysis

    • Under oxidative stress environments, mtDNA is 10 to 200 times more susceptible to mutations than nuclear DNA, particularly detrimental to high-energy cells (myocytes and neurons), due to their ROS vulnerability.

    • Non-functional oxidation leads to systemic bodily harm.

Consequences of Oxidative Damage

  • Oxidative Damage Outcomes

    • Accumulating oxidative damage surpasses repair system capacities, resulting in mtDNA damage, which leads to its release into the circulatory system (producing ccf-mtDNA).

    • ccf-mtDNA concentrations serve as indicators of personal stress levels.

  • Inflammatory Responses

    • Liberated mtDNA can induce pro-inflammatory reactions, activating neutrophils and various immune responses.

Research Rationale

  • Mitochondrial Health and Psychological Disorders

    • Mitochondrial health is crucial in chronic psychological diseases, and investigation into acute stress responses must expand to explore other stress induction methods.

Study Design and Participants

  • Trial Design

    • Single-arm trial replicating Trumpff et al.'s study but utilizing different stress induction methods.

  • Participants Criteria

    • University students undergoing a lab stress protocol involving distressing photographs.

  • Inclusion Criteria

    • Age 20-30, Canadian residents, and in good health (free of asthma, cancer treatment, myocardial infarction, and autoimmune disorders).

  • Exclusion Criteria

    • Pregnant or lactating individuals, smokers, those with current mental health conditions, chronic or acute physical disabilities, individuals on prescribed conflictual medications, recent infections, or vaccination symptoms.

Study Procedures

  • Setting

    • The study runs from Monday to Friday, morning sessions (9 am - 12 pm) to eliminate circadian rhythm effects.

    • Required abstinence from non-prescription medications and strenuous activity 24 hours before the session, and abstinence from alcohol 48 hours before the blood draw.

  • Stress Protocol Workflow

    1. Participant arrival: 9:00-10:00 am.

    2. Resting period: 5 minutes.

    3. Exposure to artificial stress via IAPS photography: 10 minutes.

    4. Recovery phase and additional resting: 25 minutes.

Blood Assessment Procedure

  • First blood draw:

    • To assess initial ccf-mtDNA levels.

  • Second blood draw:

    • Conducted 60 minutes post-initial blood draw to measure changes in ccf-mtDNA levels and assess secondary mood states using the Profile of Mood States (POMS).

Serum DNA Extraction and Quantification

  • Isolation Protocol

    1. Blood collected in Serum Clot activator tubes.

    2. Blood allowed to clot for 30 minutes at room temperature.

    3. Serum spun (1,000 X G for 5 minutes at room temperature).

    4. 80% serum transferred to a new tube, then spun (2,000 X G for 10 minutes at 4°C).

    5. Further serum extraction and storage at -80°C.

  • Quantification and Analysis

    • Blood assay quantification and analysis performed via PCR at an external laboratory.

Centrifugation Considerations

  • Using serum instead of plasma is critical since platelets are a significant source of mitochondria and ccf-mtDNA.

  • The use of clotting agents in serum preparation is necessary for ensuring accurate measurements of non-membrane encapsulated mtDNA characteristics.

Research Hypotheses

  • Main Hypothesis

    • Participants are expected to show significant increases in ccf-mtDNA levels after exposure to laboratory stressors, ideally observed within 30 to 45 minutes post-exposure.

  • Analytic Plan

    • Demographics and psychological baseline measures will be summarized using descriptive statistics, followed by evaluations of group differences pre- and post-stressor blood draws.

Results

  • Findings

    • The study analyzed both ccf-mtDNA and nDNA ratios, revealing significant pre-post differences in nDNA but insignificant results for ccf-mtDNA, suggesting a decrease in nDNA contrasted against stable ccf-mtDNA levels.

Discussion

  • Clinical Implications

    • Observed increases of ccf-nDNA in cancer detection studies have promoted the evaluation of ccf-nDNA as a prospective disease biomarker.

    • Growing evidence shows stress-induced nDNA volatility could be a novel pathway for assessing stress-related injuries, particularly where nDNA crosses cellular barriers to become extracellular ccf-nDNA, indicating potential for new diagnostic approaches.