Novel Fluorescent Mitochondria-Targeted Probe MitoCLox Reports Lipid Peroxidation in Response to Oxidative Stress In Vivo

Abstract

A new mitochondria-targeted probe MitoCLox was designed as a starting compound for a series of probes sensitive to cardiolipin (CL) peroxidation. Fluorescence microscopy reported selective accumulation of MitoCLox in mitochondria of diverse living cell cultures and its oxidation under stress conditions, particularly those known to cause a selective cardiolipin oxidation. Ratiometric fluorescence measurements using flow cytometry showed a remarkable dependence of the MitoCLox dynamic range on the oxidation of the sample. Specifically, MitoCLox oxidation was induced by low doses of hydrogen peroxide or organic hydroperoxide. The mitochondria-targeted antioxidant 10-(6′-plastoquinonyl)decyltriphenyl-phosphonium (SkQ1), which was shown earlier to selectively protect cardiolipin from oxidation, prevented hydrogen peroxide-induced MitoCLox oxidation in the cells. Concurrent tracing of MitoCLox oxidation and membrane potential changes in response to hydrogen peroxide addition showed that the oxidation of MitoCLox started without a delay and was complete during the first hour, whereas the membrane potential started to decay after 40 minutes of incubation. Hence, MitoCLox could be used for splitting the cell response to oxidative stress into separate steps. Application of MitoCLox revealed heterogeneity of the mitochondrial population; in living endothelial cells, a fraction of small, rounded mitochondria with an increased level of lipid peroxidation were detected near the nucleus. In addition, the MitoCLox staining revealed a specific fraction of cells with an increased level of oxidized lipids also in the culture of human myoblasts. The fraction of such cells increased in high-density cultures. These specific conditions correspond to the initiation of spontaneous myogenesis in vitro, which indicates that oxidation may precede the onset of myogenic differentiation. These data point to a possible participation of oxidized CL in cell signalling and differentiation.

Therapeutic Approaches to Treat Mitochondrial Diseases: “One-Size-Fits-All” and “Precision Medicine” Strategies

“…The antioxidant activities of mitochondria-targeted cationic plastoquinone derivatives (SKQs) are accomplished in two different ways: (I) by preventing peroxidation of cardiolipin [ 274 ] (mediated by quinol moieties) and (II) by fatty acid cycling, resulting in mild uncoupling that inhibits the formation of ROS in mitochondrial State IV (mediated by cation moieties) [ 275 ]. SKQ1 can effectively mitigate the oxidation induced either by hydrogen peroxide or by organic hydroperoxide in vitro [ 276 ]. SKQ1 has mainly been tested in several pathological cellular and pre-clinical models in which ROS-mediated mitochondrial dysfunction and cell death play a crucial role, such as Alzheimer’s Disease [ 277 , 278 ], multiple sclerosis [ 279 ], and Parkinson’s Disease [ 280 ].…”

Propagation of Mitochondria-Derived Reactive Oxygen Species within the Dipodascus magnusii Cells

“…Progression of the mitochondrial oxidative stress was independently verified by measuring mitochondrial lipid peroxidation using the novel mitochondria-addressed dye MitoCLox [ 91 , 92 ] ( Figure S1 ). Notably, in conformity with the data presented in Figure 3 and Figure 4 , an increment in mitochondrial lipid peroxidation occurred predominantly before the development of the generalized oxidative stress.…”

“…Mitochondria lipid peroxidation was measured with the novel mitochondria-targeted dye MitoCLox [ 91 , 92 ]. D. magnusii cells were washed in 50 mM PBS, pH 5.5, incubated with 100 nM MitoCLox for 30 min, then washed in a fresh portion of growth medium and incubated with 800 nM SkQ1 for 1 h. Cells were then washed with a fresh portion of growth medium and incubated with 750 µM t -BHP.…”

The biology of human hair greying

“…In situ analyses of HF melanocytes and their progenitor cell populations in organ‐cultured early‐stage greying HFs (Langan et al ., 2015) can be used to assess ( i ) mitochondrial position and morphology through dyes like MitoTracker (Harwig et al ., 2018); ( ii ) mitochondrial membrane potential through TMRM/TMRE and JC1 (Wolf et al ., 2019); ( iii ) mitochondrial enzymatic activity for respiratory chain complexes I, II, III and IV by enzyme histochemistry (Vidali et al ., 2014; Simard et al ., 2018); ( iv ) mitophagy [immunostaining for PTEN‐induced kinase 1(PINK1) and PINK2 combined with light chain 3B (LC3B)/sequestosome 1 (SQSTM1)] (Gökerküçük, Tramier & Bertolin, 2020); ( v ) mitochondrial fission and fusion, through quantification of major players of mitochondrial fusion [mitofusin‐1 (MFN1), MFN2 and optic atrophy 1 (OPA1)] and fission [dynamin‐related protein 1 (DRP1)] (Miret‐Casals et al ., 2018); ( vi ) mitochondrial biogenesis markers like the transcription factor proliferator‐activated receptor gamma coactivator 1‐alpha (PGC1α) and mitochondrial mass markers porin/voltage‐dependent anion‐selective channels (VDAC) and translocase of outer mitochondrial membrane 20 (TOMM20) (Vidali et al ., 2014; Fu, Liu & Yin, 2019); ( vii ) lipid peroxidation dynamics following oxidative stress using MitoCLox (Lyamzaev et al ., 2020); ( viii ) mtDNA copy number (copies of mitochondrial genome per cell) (Malik et al ., 2011); ( ix ) oxidative damage marker 8‐hydroxy‐2′‐deoxyguanosine (8‐OHdG) (Gherardini et al ., 2019), and mtDNA mutations/deletions (Bodo et al . 2007; Lu et al .…”