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CRABS-ROC, A respirometry protocol for overcoming substrate limitations, reveals excess brain mitochondrial Complex I
Naibo Zhang1, Brian A Roelofs2, Evan A Bordt1
1Department of Anesthesiology and Center for Shock, Trauma and Anesthesiology Research, 685 W. Baltimore St., MSTF 5-34, Baltimore, MD, 21201, USA; Program in Neuroscience, University of Maryland School of Medicine, 685 W. Baltimore St., MSTF 5-34, Baltimore, MD, 21201, USA.
The Mito Stress Test may underestimate maximal cell respiration due to substrate limitations. A new CRABS-ROC assay overcomes this, revealing excess electron transport chain capacity and hidden deficits in mitochondrial function.
Area of Science:
- Mitochondrial bioenergetics
- Cellular respiration
- Neuroscience
Background:
- Mitochondrial bioenergetic competency is crucial for cellular function.
- The Mito Stress Test is a common method to assess this, but may be limited by substrate availability.
- Uncoupled oxygen consumption rate (OCR) is often equated with maximal respiration without accounting for substrate supply.
Purpose of the Study:
- To develop a novel respirometry protocol to accurately assess individual electron transport chain (ETC) complex capacities.
- To investigate if substrate supply limits uncoupled OCR in neuronal cells.
- To evaluate the utility of the new protocol in identifying excess ETC capacity and masked mitochondrial deficits.
Main Methods:
- Development of the Complex Respirometry Assay Bypassing Substrate-Restricted Oxygen Consumption (CRABS-ROC) protocol.
- Utilizing saturating substrate levels to bypass substrate-restriction limitations.
- Optimization of cytochrome c concentration for accurate ETC complex capacity assessment.
- Application of CRABS-ROC to primary cortical neurons and isolated brain mitochondria.
Main Results:
- Uncoupled OCR was found to be substrate-limited in rat primary cortical neurons and mouse forebrain synaptosomes.
- CRABS-ROC revealed over 2-fold excess Complex I capacity in primary cortical neurons compared to standard assays.
- The assay identified a Complex I deficit in harlequin mutant mitochondria that was not apparent with standard methods.
Conclusions:
- The CRABS-ROC protocol accurately assesses individual ETC complex capacities by overcoming substrate limitations.
- This method can uncover significant excess ETC capacity and reveal mitochondrial alterations missed by conventional techniques.
- CRABS-ROC offers a valuable tool for comprehensive studies of mitochondrial function.
