CRABS-ROC, A respirometry protocol for overcoming substrate limitations, reveals excess brain mitochondrial Complex I...

Mitochondrial bioenergetic competency in cells is frequently assessed by the Mito Stress Test protocol, which includes uncoupler addition for evaluating respiratory capacity. The uncoupled oxygen consumption rate (OCR) is usually defined as maximal respiration, with little consideration of whether the measured rate is restricted by substrate supply. In this study, we show that the uncoupled OCR is substrate-limited in rat primary cortical neurons and isolated mouse forebrain synaptosomes. We use a different respirometry protocol we name CRABS-ROC (Complex Respirometry Assay Bypassing Substrate-Restricted Oxygen Consumption) that enables evaluation of individual electron transport chain (ETC) complex capacities using saturating levels of substrates to bypass this restriction. Optimization of the cytochrome c concentration was critical for ETC complex capacity assessment. Applying CRABS-ROC to primary cortical neurons reveals >2-fold excess Complex I capacity beyond the uncoupled OCR of cells metabolizing glucose and pyruvate. Furthermore, we demonstrate that CRABS-ROC can expose a Complex I deficit in isolated harlequin mutant brain mitochondria that display wild-type levels of Complex I-substrate-linked respiration despite having about half the normal level of Complex I. Thus, CRABS-ROC should be broadly useful for studies on mitochondrial function because it can both reveal excess ETC capacity and unmask ETC alterations that may be missed by the most widely used methods.