Low micromolar concentrations of the superoxide probe MitoSOX uncouple neural mitochondria and inhibit complex IV

Abstract

MitoSOX Red is a fluorescent probe used for the detection of mitochondrial reactive oxygen species by live cell imaging. The lipophilic, positively charged triphenylphosphonium moiety within MitoSOX concentrates the superoxide-sensitive dihydroethidium conjugate within the mitochondrial matrix. Here we investigated whether common MitoSOX imaging protocols influence mitochondrial bioenergetic function in primary rat cortical neurons and microglial cell lines. MitoSOX dose-dependently uncoupled neuronal respiration, whether present continuously in the assay medium or washed following a ten minute loading protocol. Concentrations of 5-10 μM MitoSOX caused severe loss of ATP synthesis-linked respiration. Redistribution of MitoSOX to the cytoplasm and nucleus occurred concomitant to mitochondrial uncoupling. MitoSOX also dose-dependently decreased the maximal respiration rate and this impairment could not be rescued by delivery of a complex IV specific substrate, revealing complex IV inhibition. As in neurons, loading microglial cells with MitoSOX at low micromolar concentrations resulted in uncoupled mitochondria with reduced respiratory capacity whereas submicromolar MitoSOX had no adverse effects. The MitoSOX parent compound dihydroethidium also caused mitochondrial uncoupling and respiratory inhibition at low micromolar concentrations. However, these effects were abrogated by pre-incubating dihydroethidium with cation exchange beads to remove positively charged oxidation products, which would otherwise by sequestered by polarized mitochondria. Collectively, our results suggest that the matrix accumulation of MitoSOX or dihydroethidium oxidation products causes mitochondrial uncoupling and inhibition of complex IV. Because MitoSOX is inherently capable of causing severe mitochondrial dysfunction with the potential to alter superoxide production, its use therefore requires careful optimization in imaging protocols.

MitoSOX-Based Flow Cytometry for Detecting Mitochondrial ROS

“…This is in line with early observations that high concentrations (e.g., 5 μM) of MitoSOX may adversely affect the mitochondrial electron transport chain and lead to diffusion of MitoSOX from the mitochondrial compartment into the cytosol [3]. While the optimal concentrations of MitoSOX in detecting mitochondrial ROS remain to be further determined, 1 μM of MitoSOX appears to be able to more reliably detect the relative differences in mitochondrial ROS formation between control and mitochondrial DNA-deficient cells.…”

“…However, concerns have been raised regarding the potential diffusion of MitoSOX from the mitochondrial compartment to the cytosol when the probe is used at 5 μM, thereby making the assay non-selective for detecting mitochondrial superoxide/ROS [3]. In addition, MitoSOX at the conventionally used concentrations (e.g., 5 μM) also disrupted the mitochondrial electron transport chain, adding to the complexity in data interpretation [3]. Likewise, the LDCL assay has been criticized for the potential of lucigenin to undergo redox cycling at high concentrations, especially in cell-free and physiologically irrelevant artificial systems [4].…”

“…It is possible that increased MitoSOX concentrations may lead to the increased accumulation of the probe in the cytosol [3], and its subsequent extra-mitochondrial oxidation, thereby preventing the exclusive detection of ROS production inside the mitochondria. When determining the relative difference in mitochondrial ROS production between control and mitochondrial DNA-deficient cells, it is essential to use a MitoSOX concentration that selectively detects ROS generated inside the mitochondria.…”

Mitochondria-Targeted Triphenylphosphonium-Based Compounds: Syntheses, Mechanisms of Action, and Therapeutic and Diagnostic Applications

“…Recent results suggest that MitoSOX affects mitochondrial bioenergetic function due to the mitochondrial uncoupling and inhibition of complex IV. 183,261 Thus, in order to obtain useful and reliable information using MitoSOX, its effects must be tested on the cellular respiratory function, and the superoxide-specific product, 2-OH-Mito-E + , and other products, must be monitored. Before using MitoSOX as a probe for detecting superoxide, it is important to determine the optimal concentration at which the probe does not affect the bioenergetic function in a given biological system.…”

Methods for imaging mammalian mitochondrial morphology: A prospective on MitoGraph

“…Aside from monitoring membrane potential, dyes have been designed to report on mitochondrial-derived superoxide [106,129]. One such dye, MitoSox, is a frequently used superoxide indicator, but at higher concentrations leads to minor off target nuclear staining [130,131] and inhibition of mitochondrial function [131–133]. Furthermore, MitoSox fluorescence is not a reliable indicator of the amount of mitochondrial superoxide for two reasons.…”