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EFFECT OF OMAVELOXOLONE ON MITOCHONDRIAL DYNAMICS UNDER OXIDATIVE STRESS IN CELLS WITH PARKINSON'S DISEASE ASSOCIATED MUTATION
K.A. Kritskaya*, E.I. Fedotova, D.P. Laryushkin, A.V. Berezhnov*
Institute of Cell Biophysics, Russian Academy of Sciences, Federal Research Center "Pushchino Scientific Center for Biological Research of the Russian Academy of Sciences", 3 Institutskaya St., Pushchino, 142290, Russia.
* Corresponding authors: [email protected] (KAK), [email protected] (AVB)
Abstract. This study investigates the effects of omaveloxolone, a Keapl inhibitor, on mitochondrial network (MN) dynamics and cell survival under oxidative stress in control fibroblasts and fibroblasts from a Parkinson's disease patient with a PINK1 mutation. The PINKl-mutant fibroblasts showed increased sensitivity to hydrogen peroxide-induced stress. Omaveloxolone pre-treatment improved cell viability under stress conditions in both cell types. Under normal conditions, PINKl-mutant fibroblasts exhibited lower MN connectivity compared to control cells. Oxidative stress reduced MN density in both cell types, while omaveloxolone treatment normalized MN connectivity in PINKl-mutant cells and maintained higher MN connectivity under stress. Similar effects were observed for mitochondrial branch length. Omaveloxolone (50 nM) also increased mitophagy in both cell types under normal conditions. Our findings demonstrate that omaveloxolone exerts protective effects by maintaining mitochondrial dynamics and activating mi-tophagy. It enhances mitophagy under normal conditions and supports MN structure under oxidative stress, improving cell viability in both control and PINKl-mutant fibroblasts. These results highlight omaveloxolone's potential as a therapeutic agent for protecting cells in diseases associated with impaired mitochondrial dynamics, particularly Parkinson's disease linked to PINKl mutations.
Keywords: mitochondrial network dynamics, omaveloxolone, oxidative stress, Parkinson's disease.
List of Abbreviations
MN - mitochondrial network
PINK1 - PTEN-induced kinase 1
Introduction
Parkinson's disease is the second most prevalent neurodegenerative disorder worldwide. It is hypothesized that one of the causes of Parkinson's disease development is the disruption of mitochondrial dynamics. Mitochondrial dynamics is a set of processes including mito-chondrial fission/fusion, transport, and mitophagy. Mitochondrial dynamics is essential to maintain the functional state of "healthy" mitochondria and timely utilization of damaged mitochondria, which can become sources of increased ROS production (Chen et al, 2023). A mutation in the gene encoding the PINK1 (PTEN-induced kinase 1) protein, which is involved in PINKl/Parkin-dependent mitophagy, is associated with hereditary Parkinson's disease (Gandhi et al, 2009). Although it is believed that neurons are primarily affected in Parkinson's disease, we have shown that fibro-blasts from patients with established Parkin-
son's disease and various mutations related to mitochondrial dynamics, including the gene encoding PINKl, also exhibit altered mitochondrial network (MN) morphology and increased rates of cytosolic and mitochondrial ROS production (Kritskaya et al, 2024).
It is thought that increased ROS production can lead to disruption of mitochondrial dynamics and, in turn, an imbalance in mitochondrial fission/fusion processes and mitophagy can lead to further development of oxidative stress (Dasuri et al, 20l3). A promising approach to break this positive feedback loop is to target the cell's internal defense pathways, for example, through activation of the transcription factor Nrf2, which is responsible for the synthesis of several antioxidant enzymes (Calkins et al., 2005). Omaveloxolone is a promising activator of the Nrf2 pathway, that has demonstrated its safety and efficacy in a wide range of pathologies, and is currently participating in a clinical trial for the treatment of Friedreich's ataxia (Abeti et al, 20l8). It is worth noting that some studies suggest that omaveloxolone, as well as possibly other Nrf2 activators, may have a pro-
tective effect not only by directly affecting the Nrf2 pathway but also through influencing cell mitochondrial dynamics (Dinkova-Kostova & Abramov, 2015), however, convincing evidence for this is still lacking. One way to assess mitochondrial dynamics in a cell is to evaluate the morphological parameters of the MN using confocal imaging of cells stained with mito-chondrial probes and automated image analysis.
The aim of the present work is to investigate the effect of omaveloxolone on MN morphology and mitophagy levels in control fibroblasts and fibroblasts with a mutation in the PINK1 protein gene under normal conditions and under stress induced by hydrogen peroxide.
Materials and Methods
Fibroblast Culture
All cell cultures were kindly provided by Prof. Andrey Y. Abramov (UCL Institute of Neurology, London, UK). The studies were conducted on human fibroblast lines. Fibroblasts were obtained from a donor (52-year-old female) with hereditary Parkinson's disease and an established mutation (homozygous p.Try90Leufsx12 in PINK1) and from healthy control donors (49-year-old female and 56-year-old male). Fibroblasts were cultured under standard conditions in 25 cm2 culture flasks in complete medium containing DMEM (Sigma-Aldrich, US), 10% fetal bovine serum (Sigma-Aldrich), 2 mM glutamine (Gibco, US), and 1 mM sodium pyruvate (Gibco) at 37°C, 5% CO2, and 100% humidity. Cells were seeded on glass coverslips for experiments. Cells no older than passage 18 were used in experiments. All work with cell cultures was carried out in accordance with legal requirements and approved by the Ethics Committee of the Institute of Cell Biophysics of the Russian Academy of Sciences (Permit No. 4 dated March 14, 2022; Permit No. 3 dated March 12, 2023).
Omaveloxolone (RTA 408, AbMole BioScience) at a concentration of 50 nM was added to the cells in the culture medium for 24 hours prior to exposure to hydrogen peroxide. In the case of experiments assessing MN parameters hydrogen peroxide at a concentration of
150 ^M was added to the cells in the culture medium for 30 min and then washed 3 times with Hanks' solution (PanEco, Russia). In the case of experiments assessing of cell viability, hydrogen peroxide at a concentration of 250 ^M was added to the cells in the culture medium for 1.5 h.
Assessment of Mitochondrial Network Morphology
Assessment of MN parameters was carried out using automatic image analysis based on approaches described previously (Kritskaya et al, 2024; Valente et al, 2017). The fluorescent dye TMRM was used to visualize the MN. Cells were incubated with 25 nM TMRM (Tetramethylrhodamine, Thermo Fisher Scientific, US) in HBSS (PanEco) for 40 min at room temperature. MN visualization was performed using a confocal microscope Leica TCS SP5 (Leica Microsystems, Wetzlar, Germany) equipped with a 63x oil immersion objective. The «mitochondrial branch length» parameter was assessed using self-developed Fiji (https://imagej.net/software/fiji/down-loads) plugin for automatic batch processing of MN (Kritskaya et al, 2024). Briefly, the obtained confocal micrographs of MN underwent preliminary processing in Fiji (noise removal, local contrast enhancement, Gaussian blur), after which a binarized image of the MN was created using a threshold function for further analysis. We also evaluated the MN connectivity. This parameter indicates the average number of junctions in the MN per cell. A change in mitochondrial connectivity may indicate cell adaptation to decreased substrate supply, a violation of bioenergetics and mitochondrial fission/fusion. To assess MN connectivity based on the «graph density» characteristic, binarized images of MN were analyzed using the Python programming language in Jupyter Notebook and the NetworkX library. Object contours were extracted using the findContours algorithm. The resulting contours were considered as vertices of the MN graph.
Measurement of Mitochondria and Lyso-some Colocalization Level
To assess the level of mitophagy, the degree of colocalization of mitochondria and lyso-
somes was investigated according to the method described earlier (Berezhnov et al, 2016; Fedotova et al, 2022). Cells were treated with fluorescent dyes: 0.3 ^M MitoTracker Green (Thermo Fisher Scientific) and 0.1 ^M LysoTracker Red (Thermo Fisher Scientific). Staining was performed for 40 min in serumfree medium in a CO2 incubator: mitochondria were stained before exposure to putative mi-tophagy inducers, and lysosomes - immediately after. A Leica TCS SP5 confocal microscope with a 63x objective was used to obtain images. An argon laser (488 nm) and a heliumneon laser (543 nm) were used as excitation sources. The fluorescent signal was recorded in the ranges of 525 ± 25 nm and 665 ± 35 nm. For quantitative assessment of mitophagy, a Fiji macro was used, developed to assess the percentage of image area occupied by mitochondria and lysosomes, and to calculate the proportion of mitochondria colocalized with lysosomes (Berezhnov et al., 2016; Fedotova et al., 2022).
Cell Viability
Cell viability was assessed using a double staining method with 5 ^M Hoechst 33342 (Thermo Fisher Scientific) and 20 ^M Propid-ium Iodide (Thermo Fisher Scientific) in HBSS for 45 min at room temperature. Propidium Iodide can only stain cells with impaired membrane permeability during necrosis and late stages of apoptosis, while Hoechst 33342 stains DNA in all cells. Since dead cells detach from the substrate and may not be included in the count when visualized using microscopy, the density of the cell culture in the control before various treatments was also taken into account to calculate viability.
Statistical Analysis
Statistical tests were conducted using the R programming language in R-studio (https://posit.co/download/rstudio-desktop/). OriginLab 2024 Pro was used to create graphs. The data were tested for normality using QQ-plots and the Shapiro-Wilk normality test, and parametric tests were subsequently used. Since no statistical differences were found in
the studied parameters between control fibroblast lines, they were combined for simplicity of interpretation. Box plots show median and interquartile range, with individual data points represented as dots. Statistical significance was determined using one-way ANOVA with Tukey's multiple comparison test. The Bonferroni correction was applied to adjust the significance level for multiple comparisons. Statistical significance for cell lines before and after treatment with omaveloxolone was determined using an unpaired Student's t-test (p-value < 0.05).
Results
MN connectivity is a parameter indicating how closely mitochondria are connected within the network. Changes in MN connectivity occur under various types of stress, adaptation to substrate starvation, etc. MN connectivity in the studied cells was determined based on analysis of MN graph density in control fibroblasts and PINK1 fibroblasts (Fig. 1A). Figure 1B presents data on MN connectivity for control and mutant fibro-blasts. The graph shows that the initial MN connectivity in PINK1 cells is lower than in control fibroblasts (Fig. 1B).
Interestingly, the action of omaveloxolone (50 nM, 24 h) does not affect MN connectivity in control cells but significantly increases this parameter in mutant fibroblasts (Fig. 1B). When exposed to hydrogen peroxide, MN connectivity in control cells decreases significantly, while in fibroblasts with the PINK1 gene mutation, this parameter remains at a consistently low level (Fig. 1B). Under oxidative stress conditions induced by H2O2 and with pre-treatment with omaveloxolone, MN connectivity decreased to a lesser extent in control fibroblasts, while in PINK1 fibroblasts, this parameter remained even higher than in these fibroblasts without any treatment (Fig. 1B).
In addition to MN connectivity, an important indicator of MN state is the length of mitochondria within the network. The addition of omaveloxolone alone does not have a significant effect on the length of mitochondria in the MN in either control cells or PINK1 fibroblasts (Fig. 1C).
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Fig. 1. Effect of omaveloxolone on mitochondrial network (MN) morphology under oxidative stress conditions in control fibroblasts and fibroblasts with a mutation in the PINK1 protein gene associated with Parkinson's disease. A) Confocal images of control fibroblasts and fibroblasts with PINK1 mutation: under normal conditions, under stress conditions (incubation with 150 ^M H2O2 for 30 min); after incubation with omaveloxolone (50 nM, 24 h) and combined action of omaveloxolone first (24 h) followed by H2O2-induced stress (150 ^M, 30 min), stained with the potential-sensitive mitochondrial probe TMRM (red pseudocolor) and Hoechst 33342, staining the cell nucleus (blue pseudocolor). Scale bar 10 ^m. B) MN connectivity (MN graph density) and C) mitochondrial branch length within the MN in control fibroblasts and fibroblasts with PINK1 protein gene mutation: under conditions without treatment ("0"), under stress conditions (incubation with 150 ^M H2O2 for 30 min); after incubation with omaveloxolone (50 nM, 24 h) and combined action of omaveloxolone first (24 h) followed by H2O2-induced stress (150 ^M, 30 min). Box plots show median and interquartile range, and dots represent individual data points. Statistical significance was determined using one-way ANOVA with Tukey's multiple comparison test. The Bonferroni correction was used to adjust the significance level for multiple comparisons (adjusted p-value = 0.0167). n = 6 cell coverslips in 3 independent experiments
Fig. 2. Effect of omaveloxolone on mitophagy in control fibroblasts and fibroblasts with PINK1 protein gene mutation under normal conditions and on the viability of these cells under oxidative stress. A) Confocal images of control fibroblasts and fibroblasts with PINK1 protein gene mutation, stained with fluorescent probes specific to mitochondria (MitoTracker Green) and lysosomes (LysoTracker Red) without treatment and after incubation with 50 nM omaveloxolone for 24 h. Scale bar 10 ^m. B) Percentage of mitochondria and lysosome colocalization in fibroblasts without treatment and after incubation with 50 nM omaveloxolone for 24 h. C) Percentage of viable cells relative to the initial level under oxidative stress induced by the addition of 250 цМ H2O2 for 1.5 h and pre-incubation with omaveloxolone (50 nM, 24 h) followed by the addition of hydrogen peroxide. Statistical significance for cell lines before and after the addition of omaveloxolone was determined using a paired Student's t-test (p-value < 0.05). n = 6 cell coverslips in 3 independent experiments
When hydrogen peroxide is added, the length of mitochondria in the MN significantly decreases in both control cells and cells with the PINK1 gene mutation (Fig. 1C), which may indicate activation of the mitochondrial fission process in response to stress caused by H2O2. However, it is worth noting that with pre-incubation of cells with omaveloxolone and subsequent exposure to H2O2, the length of mitochondria within the MN does not decrease as significantly in control and mutant fibroblasts (compared to exposure to hydrogen peroxide H2O2 alone) (Fig. 1C).
To determine the level of mitophagy, approaches based on assessing the degree of col-
ocalization of mitochondria and lysosomes are currently used (Berezhnov et al, 2016; Fedotova et al, 2022). We investigated the effect of omaveloxolone on the level of mitophagy (Fig. 2AB), as well as the potential protective effect of this substance on control and mutant fibroblasts under stress conditions (Fig. 2C) induced by H2O2.
It is important to emphasize that under normal conditions, the level of mitophagy does not significantly differ between control fibroblasts and those with the PINK1 gene mutation, which is likely due to the activation of an alternative mitophagy pathway in these cells (Komilova et
al., 2022), although there is a tendency towards decreased colocalization of mitochondria and lysosomes in PINK1 fibroblasts. Incubation of cells with 50 nM omaveloxolone for 24 h increases the level of mitophagy in both control cells and fibroblasts with the PINK1 gene mutation (by 3.6 and 3.1 times, respectively) (Fig. 2B).
Cell viability under stress conditions was assessed by the double staining method. Under stress conditions induced by the addition of H2O2 (250 pM, 1.5 h), the number of viable cells significantly decreases in the PINK1 fibroblast culture (to 43.5 ± 9.1%) and in the control fibroblast culture (to 80.3 ± 8.3%) compared to the initial number (Fig. 2C). Preincubation of fibroblasts with 50 nM omave-loxolone for 24 h increased the percentage of viable cells under stress conditions in both the control culture (to 89.8 ± 3.1%) and in the mutant fibroblast culture (to 76.8 ± 8.6%) (Fig. 2C).
Discussion
In this study, we investigated the effects of omaveloxolone, an activator of Nrf2 pathway, on mitochondrial dynamics and cell survival under oxidative stress conditions in both control fibroblasts and those carrying a PINK1 mutation associated with Parkinson's disease. Our findings reveal significant impact on MN connectivity, branch length, and the level of mi-tophagy, suggesting multiple mechanisms through which Nrf2 activation influences mito-chondrial dynamics.
Omaveloxolone induces Nrf2 translocation to the nucleus by inhibiting Keap1-Cul3-dependent ubiquitination of Nrf2, leading to up-regulation of antioxidant genes (Dinkova-Kos-tova & Abramov, 2015; Madsen et al, 2020). Our results demonstrated that pre-incubation with omaveloxolone increased the percentage of viable cells under stress conditions in both control and PINK1-mutant fibroblasts, indicating a pronounced antioxidant effect. This improved redox balance likely contributes to the observed effects on mitochondrial dynamics, given the sensitivity of fission/fusion machinery to oxidative stress.
Under normal conditions, PINK1-mutant fi-broblasts exhibited lower MN connectivity compared to control cells, consistent with previous studies on PINK1's role in maintaining normal mitochondrial dynamics (Cui et al., 2010). Notably, omaveloxolone treatment normalized MN connectivity in PINK1-mutant cells without affecting control cells, suggesting its ability to correct impaired mitochondrial dynamics. Under H2O2-induced oxidative stress, omaveloxolone maintained higher MN connectivity in both cell types, possibly by promoting a more balanced fission/fusion equilibrium.
Moderate activation of mitophagy is one of the promising directions for cell protection in various neurodegenerative diseases (Geor-gakopoulos et al., 2017; Masaldan et al, 2022). We observed increased mitophagy in both control and PINK1-mutant fibroblasts following omaveloxolone treatment. This suggests that Nrf2 activation enhances mitochondrial quality control, potentially through upregulation of mi-tophagy-related genes, improved autophagic flux (Robledinos-Antón et al., 2019). Resting fibroblasts have a low basal level of mitophagy. In this regard, it is difficult to detect differences in this parameter between mutant and control cells. However, under conditions of cellular stress with the addition of hydrogen peroxide, it can be noted that mitophagy is impaired in cells carrying mutated PINK1 compared to the control. At the same time, the discovered possibility of mitophagy activation under the action of omaveloxolone in cells with a mutation is of interest and may indicate the activation of an alternative BN^/Nix-dependent mitophagy pathway (a PINK1/Parkin independent pathway).
Exposure to hydrogen peroxide significantly decreased MN connectivity in both cell types, indicating enhanced mitochondrial division under oxidative stress. This aligns with increased activity of Drp1, a protein mediating mitochondrial division under stress conditions and characteristic of Parkinson's disease (Portz & Lee, 2021; Qi et al, 201З). The ability of omavelox-olone to maintain higher MN connectivity under these conditions suggests it may counteract stress-induced mitochondrial fragmentation.
There is some indirect evidence linking Nrf2 and Parkinson's disease: for instance, Nrf2 activity decreases with age, and age is known to be one of the risk factors for Parkinson's disease. On the contrary, the increased nuclear localization of Nrf2 observed in dopaminergic neurons from Parkinson's disease patients suggests a compensatory activation of this pathway (Cuadrado, 2016; Esteras et al, 2016). Omavelox-olone's ability to normalize mitochondrial dynamics and enhance mitophagy in PINK1-mu-tant fibroblasts highlights its potential as a therapeutic agent for Parkinson's disease, especially in cases associated with PINK1 mutations.
Conclusion
Thus, omaveloxolone exhibits a protective effect by maintaining mitochondrial dynamics and activating mitophagy. The observed imp-
rovements in MN connectivity, branch length, and mitophagy, particularly in PINK1-mutant fibroblasts, underscore the potential of Nrf2 activators as therapeutic agents for diseases associated with impaired mitochondrial dynamics, such as Parkinson's disease. Future studies should aim to elucidate the relative contributions of these mechanisms to the overall impact of Nrf2 activation on mitochondrial health and dynamics, particularly in the context of neuro-degenerative diseases.
Acknowledgments
This work was supported by the Federal Research Center, Institute of Cell Biophysics of the Russian Academy of Sciences, within the framework of the task no. 075-00609-24-01 (project no. 1022080100047-5-1.6.4, New Generation Neuroprotective Drugs).
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