A Powerful Mitochondria-Targeted Iron Chelator Affords High Photoprotection against Solar Ultraviolet A Radiation

Mitochondria are the principal destination for labile iron, making these organelles particularly susceptible to oxidative damage on exposure to ultraviolet A (UVA, 320–400 nm), the oxidizing component of sunlight. The labile iron-mediated oxidative damage caused by UVA to mitochondria leads to necrotic cell death via adenosine triphosphate depletion. Therefore, targeted removal of mitochondrial labile iron via highly specific tools from these organelles may be an effective approach to protect the skin cells against the harmful effects of UVA. In this work, we designed a mitochondria-targeted hexadentate (tricatechol-based) iron chelator linked to mitochondria-homing SS-like peptides. The photoprotective potential of this compound against UVA-induced oxidative damage and cell death was evaluated in cultured primary skin fibroblasts. Our results show that this compound provides unprecedented protection against UVA-induced mitochondrial damage, adenosine triphosphate depletion, and the ensuing necrotic cell death in skin fibroblasts, and this effect is fully related to its potent iron-chelating property in the organelle. This mitochondria-targeted iron chelator has therefore promising potential for skin photoprotection against the deleterious effects of the UVA component of sunlight.


Analytical
The analytical RP-HPLC was carried out on a HP1050 HPLC system equipped with an autosampler, a quaternary pump and a Diode-Array Detector. A Zorbax SB C-18 2.1 mm x 10 cm (particle size 3.5 micron) column was employed. The flow rate was 0.2 mL/min and the eluents were monitored at wavelengths between 210-280 nm. A linear gradient of mobile phase B (acetonitrile containing 0.1% TFA) over mobile phase A (0.1% TFA in water) from 0-90% B in 20 minutes was performed. Data were collected and analyzed using ChemStation software. The semi-prep HPLC purification was performed using a X-terra Prep MS C18 Column (5 micron, 10 x 100 mm) operating a flow rate of 7 mL/min. Isolated fractions were reanalyzed via analytical RP-HPLC as above and identical fractions were pooled and lyophilized. The ESI-MS analyses were performed using a Waters Micromass ZQ mass spectrometer (Manchester, UK). UV-vis and fluorescence measurements were performed on a Perkin Elmer spectrophotometer and a Perkin Elmer spectrofluorometer, respectively. Peptide solutions were prepared at the appropriate concentrations in 0.1 M MOPS buffer, pH 7.4.

Cell biology Flow cytometry
Data were obtained using a Becton Dickinson FACSAria III fitted with laser lines of excitation at 405, 488, 561 and 633 nm. Cells were gated via forward scatter versus side scatter dotplot so as to exclude very small debris in the origin. Data were collected from a minimum of 10'000 cells (events) and analyzed with FACSDiva Software version 8.0.1 (Becton Dickinson, San Jose, CA).
Annexin V /PI labeling: Cell status following the different treatments was examined using the dual staining with Annexin V/PI as described previously (Zhong et al., 2004). Populations were determined as percentages via quadrant analysis of dot-plots of Annexin V-FLUOS versus PI. "Live" cells were defined as Annexin V-negative/PI-negative. Double-positive and Annexin V-negative/PI-positive cells were considered "necrotic" and Annexin V-positive/ PInegative cells as "apoptotic''. TMRM labelling: For the measurement of mitochondrial transmembrane potential (∆ψm), at the times indicated post-UVA irradiation, the conditioned medium was removed, cells rinsed with PBS and incubated for 30 min at 37 °C with 50 nM TMRM in phenol-red free Hank's Balanced Salt Solution (HBSS) supplemented with 10 mM 4-(2-hydroxyethyl)-1piperazineethanesulfonic acid (HEPES). The cells were then collected by trypsinization, resuspended in HBSS buffer and median fluorescence intensities (MFI) scored.

Live cell microscopy
Bright-field images used to document morphological changes following treatments were captured on a Motic inverted microscope (model AE2000) (Motic Deutschland GmbH, Wetzlar, Germany) via a Moticam 580 digital camera and a Plan 10x objective. Subcellular localization of the DNS-labelled peptide (compound 3) was investigated on an Olympus IX51 inverted epifluorescence microscope equipped with a 100 W mercury UV lamp. Cells were imaged with a 40x oil objective (UAPONO340-2). Images were acquired via an Olympus DP72 digital camera controlled by Olympus cell^P Analysis Image Processing software (Soft Imaging System GmbH, Muenster, Germany). Cells showing yellow staining, resulting from superimposition of the chelator peptide-specific green signal and the organelle-specific red signal, were qualitatively considered positive for colocalization. The co-occurence in the images, of green and red fluorescent signals above a threshold level was further confirmed by analysis of intensity profiles collected across cells using cell^P (from Olympus) Image Processing software. The extent of colocalization of the fluorescent compound 3 with mitochondria, lysosomes or ER compartments was also assessed quantitatively by Manders' correlation coefficients (MCCs) M1 and M2 using ImageJ software with the JaCoP plug-in (Bolte and Cordelières, 2006;Manders et al., 1993) from a random selection of image fields as described previously (Abbate et al., 2015a). MCCs provide a measure of how much of the signal intensity of a channel occurs in the same location as the other channel. Thus M1 represents the extent of overlap of compound signal (green) with the organelle signal (red), whereas M2 represents the amount of organelle signal overlapping the compound signal. MCCs values range from zero (uncorrelated distributions of two probes with one another) to one (fully correlated distributions of two probes). MCCs were chosen instead of the Pearson's colocalization coefficient as they are particularly well suited when the fluorescent signals distribute to different types of compartments (Dunn et al., 2011).

MTT assay
Cells grown in 96 well plates were treated as indicated in the figure legend. At the end of the treatment, the media was removed and the cells were incubated for 3 h at 37 °C with 100 L serum-free media containing MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide) at a concentration of 0.5 mg/mL. The MTT solution was then removed and 50 l DMSO was added to the cells in order to dissolve the formazan formed. Absorbance was read at 550 nm using a spectrophotometer VERSAmax TM (Molecular devices, California). The percentage of viable cells was calculated by comparing the absorbance of treated versus non-treated control cells.     In this study, a reverse-phase HPLC analysis was performed using a C18 column and a gradient of acetonitrile over water to perform the chromatographic separation. Panels D, H and L from Figure 2 representing the merged channels are shown. Magnifications (x2) correspond to the indicated dotted squares. Representative pixel intensity profiles were collected across field sections indicated by the white arrows. Green profiles depict fluorescent signals from compound 3, whereas red profiles account for fluorescence from markers for mitochondria, lysosomes or ER compartments. Note the marked overlap between the profiles of compound 3 and mitochondria, contrasting with the lack of correlation between profiles of compound 3 and lysosomes or ER. Scale bar = 10 m.

SUPPLEMENTARY TABLE AND FIGURES
Compound 3 Compound 4

Figure S3: Quantitative analysis of colocalization of compound 3 with subcellular organelles in FEK4 cells.
The extent of colocalization of compound 3 with mitochondria, lysosomes or endoplasmic reticulum (ER) compartments was measured quantitatively using Manders' correlation coefficients (MCCs) M1 and M2 using ImageJ software with the JaCoP plug-in as described in Materials and Methods. M1 represents the fraction of signal from compound 3 colocalizing with the signal from the organelles. The values obtained for M1 are presented as means +/-SD per field, calculated from at least two random fields of 8-10 cells over three independent experiments. Representative pixel intensity profiles were collected across field sections indicated by the white arrows. Green profiles depict fluorescent signals from compound 3, whereas red profiles account for fluorescence from markers for mitochondria, lysosomes or ER compartments. Scale bar = 50 m. * * * * FEK4 cells were treated as indicated and the percentage of live cells (double negative for Annexin V and PI) was determined by flow cytometry using double staining with Annexin V/PI as described in Materials and Methods. Data are represented as means +/-SD from at least three independent experiments. * Significantly different from UVA-irradiated alone (p<0.05).