A mitochondria-targeted derivative of ascorbate: MitoC.

Mitochondrial oxidative damage contributes to a wide range of pathologies. One therapeutic strategy to treat these disorders is targeting antioxidants to mitochondria by conjugation to the lipophilic triphenylphosphonium (TPP) cation. To date only hydrophobic antioxidants have been targeted to mitochondria; however, extending this approach to hydrophilic antioxidants offers new therapeutic and research opportunities. Here we report the development and characterization of MitoC, a mitochondria-targeted version of the hydrophilic antioxidant ascorbate. We show that MitoC can be taken up by mitochondria, despite the polarity and acidity of ascorbate, by using a sufficiently hydrophobic link to the TPP moiety. MitoC reacts with a range of reactive species, and within mitochondria is rapidly recycled back to the active ascorbate moiety by the glutathione and thioredoxin systems. Because of this accumulation and recycling MitoC is an effective antioxidant against mitochondrial lipid peroxidation and also decreases aconitase inactivation by superoxide. These findings show that the incorporation of TPP function can be used to target polar and acidic compounds to mitochondria, opening up the delivery of a wide range of bioactive compounds. Furthermore, MitoC has therapeutic potential as a new mitochondria-targeted antioxidant, and is a useful tool to explore the role(s) of ascorbate within mitochondria.

Using exomarkers to assess mitochondrial reactive species in vivo.

BACKGROUND: The ability to measure the concentrations of small damaging and signalling molecules such as reactive oxygen species (ROS) in vivo is essential to understanding their biological roles. While a range of methods can be applied to in vitro systems, measuring the levels and relative changes in reactive species in vivo is challenging. SCOPE OF REVIEW: One approach towards achieving this goal is the use of exomarkers. In this, exogenous probe compounds are administered to the intact organism and are then transformed by the reactive molecules in vivo to produce a diagnostic exomarker. The exomarker and the precursor probe can be analysed ex vivo to infer the identity and amounts of the reactive species present in vivo. This is akin to the measurement of biomarkers produced by the interaction of reactive species with endogenous biomolecules. MAJOR CONCLUSIONS AND GENERAL SIGNIFICANCE: Our laboratories have developed mitochondria-targeted probes that generate exomarkers that can be analysed ex vivo by mass spectrometry to assess levels of reactive species within mitochondria in vivo. We have used one of these compounds, MitoB, to infer the levels of mitochondrial hydrogen peroxide within flies and mice. Here we describe the development of MitoB and expand on this example to discuss how better probes and exomarkers can be developed. This article is part of a Special Issue entitled Current methods to study reactive oxygen species - pros and cons and biophysics of membrane proteins. Guest Editor: Christine Winterbourn.

Mitochondrial accumulation of a lipophilic cation conjugated to an ionisable group depends on membrane potential, pH gradient and pK(a): implications for the design of mitochondrial probes and therapies.

Mitochondria play key roles in a broad range of biomedical situations, consequently there is a need to direct bioactive compounds to mitochondria as both therapies and probes. A successful approach has been to target compounds to mitochondria by conjugation to lipophilic cations, such as triphenylphosphonium (TPP), which utilize the large mitochondrial membrane potential (Δψ(m), negative inside) to drive accumulation. This has proven effective both in vitro and in vivo for a range of bioactive compounds and probes. However so far only neutral appendages have been targeted to mitochondria in this way. Many bioactive functional moieties that we would like to send to mitochondria contain ionisable groups with pK (a) in the range that creates an assortment of charged species under physiological conditions. To see if such ionisable compounds can also be taken up by mitochondria, we determined the general requirements for the accumulation within mitochondria of a TPP cation conjugated to a carboxylic acid or an amine. Both were taken up by energised mitochondria in response to the protonmotive force. A lipophilic TPP cation attached to a carboxylic acid was accumulated to a greater extent than a simple TPP cation due to the interaction of the weakly acidic group with the pH gradient (ΔpH). In contrast, a lipophilic TPP cation attached to an amine was accumulated less than the simple cation due to exclusion of the weakly basic group by the ΔpH. From these data we derived a simple equation that describes the uptake of lipophilic cations containing ionisable groups as a function of Δψ(m), ΔpH and pK(a). These findings may facilitate the rational design of additional mitochondrial targeted probes and therapies.

A ratiometric fluorescent probe for assessing mitochondrial phospholipid peroxidation within living cells.

Mitochondrial oxidative damage contributes to a wide range of pathologies, and lipid peroxidation of the mitochondrial inner membrane is a major component of this disruption. However, despite its importance, there are no methods to assess mitochondrial lipid peroxidation within cells specifically. To address this unmet need we have developed a ratiometric, fluorescent, mitochondria-targeted lipid peroxidation probe, MitoPerOx. This compound is derived from the C11-BODIPY(581/591) probe, which contains a boron dipyromethane difluoride (BODIPY) fluorophore conjugated via a dienyl link to a phenyl group. In response to lipid peroxidation the fluorescence emission maximum shifts from ∼590 to ∼520nm. To target this probe to the matrix-facing surface of the mitochondrial inner membrane we attached a triphenylphosphonium lipophilic cation, which leads to its selective uptake into mitochondria in cells, driven by the mitochondrial membrane potential. Here we report on the development and characterization of MitoPerOx. We found that MitoPerOx was taken up very rapidly into mitochondria within cells, where it responded to changes in mitochondrial lipid peroxidation that could be measured by fluorimetry, confocal microscopy, and epifluorescence live cell imaging. Importantly, the peroxidation-sensitive change in fluorescence at 520nm relative to that at 590nm enabled the use of the probe as a ratiometric fluorescent probe, greatly facilitating assessment of mitochondrial lipid peroxidation in cells.