Mitochondrial-targeted fluorescent probes for reactive oxygen species

Abstract

As the primary consumers of oxygen within all aerobic organisms, mitochondria are a major source of cellular reactive oxygen species (ROS) derived from the in vivo chemistry of oxygen metabolism. Mitochondrial ROS have been traditionally implicated in aging and in a variety of pathologies, including cancer, neurodegeneration, and diabetes, but recent studies also link controlled mitochondrial ROS fluxes to cell regulation and signaling events. Progress in the development of mitochondrial-targeted fluorescent small-molecule indicators that detect specific ROS with high selectivity offers a promising approach for interrogating mitochondrial ROS production, trafficking, and downstream biological effects.

Figure 1

A simplified scheme for mitochondrial ROS metabolism. The primary source of ROS in the mitochondria is derived from the one-electron reduction of molecular oxygen (O₂) by the electron transport chain (ETC) to form superoxide (O₂⁻). O₂⁻ can then be converted to hydrogen peroxide (H₂O₂) either spontaneously or catalyzed by superoxide dismutases (SOD). H₂O₂ can also be produced by monoamine oxidases (MAO), which catalyze the oxidative deamination of dietary and neurotransmitter amines. Mitochondrial nitric oxide synthases (NOS) produce nitric oxide (NO) that can potentially react with superoxide to produce peroxynitrite (ONOO⁻). H₂O₂ can either be destroyed by peroxiredoxins (PRX), glutathione peroxidases (GPx), or catalases (Cat), converted to a hydroxyl radical (•OH) by iron or copper-mediated Fenton chemistry, or transformed into hypochlorous acid (HOCl) by myeloperoxidase (MPO) catalysis.

Figure 2

General methods for delivering molecular cargo to the mitochondria. Small molecules can be delivered to the mitochondria through the use of lipophilic cations such as rhodamine dyes (a) and triphenylphosphonium moieties (b), which take advantage of the proton gradient and subsequent electrochemical potential generated within the matrix of mitochondria. Recently, mitochondria-targeted peptides (c) that can contain both natural and unnatural amino acids have been rationally designed and screened as mitochondrial delivery vehicles [45].

Figure 3

Selected mitochondrial-targeted probes for detection of reactive oxygen species (ROS). (a) MitoSOX is a dihydroethidium-based probe bearing a triphenylphosphonium (TPP) targeting moiety. This probe reacts with several ROS, but the product from superoxide oxidation can be distinguished from other potentially formed oxidized products by selective excitation of the 2-hydroxylethidium product. (b) Two examples of mitochondrial-targeted nitrone spin-trap probes that utilize TPP localization groups. (c) MitoAR and MitoHR are rhodamine-like probes that react with highly reactive oxygen species (hROS) including hydroxyl radical (•OH), hypochlorous acid (HOCl), and peroxynitrite (ONOO⁻). (d) MitoPY1 is a boronate-based hybrid rhodamine/fluorescein probe with a TPP targeting group for chemospecific detection of hydrogen peroxide (H₂O₂).

Figure 4

Confocal fluorescence images of live Cos-7 cells with varying levels of mitochondrial H₂O₂ as visualized using MitoPY1. Cos-7 cells incubated with 5 µM MitoPY1 for 60 minutes at 37 °C and imaged with either MitoPY1 (a), MitoTracker Deep Red and Hoechst (overlay, b), MitoPY1 with MitoTracker Deep Red (overlay, c), or in brightfield mode (d). Cos-7 cells incubated with 5 µM MitoPY1 with 300 µM H₂O₂ added for the final 40 minutes and imaged with either MitoPY1 (e), MitoTracker Deep Red and Hoechst (overlay, f), MitoPY1 with MitoTracker Deep Red (overlay, g), or in brightfield mode (h) with a 20 µm scale bar.