Apomorphine is a potent inhibitor of ferroptosis independent of dopaminergic receptors

Apomorphine is a potent inhibitor of ferroptosis independent of dopaminergic receptors | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Apomorphine is a potent inhibitor of ferroptosis independent of dopaminergic receptors Akihiko Miyauchi, Chika Watanabe, Naoya Yamada, Eriko F. Jimbo, and 10 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-3361719/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 27 Feb, 2024 Read the published version in Scientific Reports → Version 1 posted 10 You are reading this latest preprint version Abstract Originally, apomorphine was a broad-spectrum dopamine agonist with an affinity for all subtypes of the Dopamine D1 receptor to the D5 receptor. We previously identified apomorphine as a potential therapeutic agent for mitochondrial diseases by screening a chemical library of fibroblasts from patients with mitochondrial diseases. In this study, we showed that apomorphine prevented ferroptosis in fibroblasts from various types of mitochondrial diseases as well as in normal controls. Well-known biomarkers of ferroptosis include protein markers such as prostaglandin endoperoxide synthase 2 (PTGS2), a key gene for ferroptosis-related inflammation PTGS2, lipid peroxidation, and reactive oxygen species. Our findings that apomorphine induced significant downregulation of Ptgs2 , and suppressed lipid peroxide to the same extent as other inhibitors of ferroptosis also indicate that apomorphine suppresses ferroptosis. To our knowledge, this is the first study to report that the anti-ferroptosis effect of apomorphine is not related to dopamine receptor agonist action and that apomorphine is a potent inhibitor of ferroptotic cell death independent of dopaminergic receptors. Health sciences/Medical research/Drug development Biological sciences/Drug discovery/Pharmacology/Clinical pharmacology Biological sciences/Drug discovery/Drug delivery Biological sciences/Drug discovery/Pharmaceutics Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Introduction Apomorphine is a known treatment for Parkinson’s disease (PD) and erectile dysfunction 1 , 2 , 3 . It is a broad-spectrum dopamine agonist for all subtypes, ranging from the Dopamine D1 receptor to the D5 receptor. Apomorphine exerts protective effects against oxidative toxicity via activation of the Dopamine D4 receptor (D4R) in a micromolar order 4 , 5 . In our previous study, we screened a chemical library based on drugs already approved for central nervous system (CNS) diseases using fibroblasts from two patients with Leigh syndrome (LS) and two patients with myopathy encephalopathy, lactic acidosis, and stroke-like episodes (MELAS) using an L-buthionine-S, R-sulfoximine (BSO)-induced cell viability assay. BSO has been used to induce cell death by inhibiting glutathione (GSH) biosynthesis, which leads to the overproduction of reactive oxygen species (ROS) 6 . In addition, BSO has been recently reported to trigger ferroptosis in various cancer cells 7 , 8 . While searching for potential therapeutic drugs for mitochondrial diseases, we identified apomorphine as a potential therapeutic drug for LS and MELAS 9 . Our findings showed that apomorphine protected against BSO-induced stress with an EC50 (median effect concentration) of approximately 50 nM and improved the mitochondrial respiratory activity of fibroblasts from patients with LS as well as MELAS. In addition, our data suggested that the cell-protective effect of apomorphine may be different from that of dopamine agonists. Apomorphine is also known to inhibit oxytosis and stress-dependent non-apoptotic cell death by activation of dopamine D4 receptors 4 . Because oxytosis shares some concepts with ferroptosis proposed in recent years, we investigated the effect of apomorphine on ferroptosis and the relationship between this effect and dopamine receptors. Ferroptosis is a common form of regulated cell death that is distinguished from apoptosis and necroptosis and is a form of lipid- and iron-dependent cell death associated with glutathione (GSH) depletion 10 , 11 , 12 . Although the exact mechanism underlying ferroptotic cell death is not fully understood, it includes oxidative damage to cellular structures and dysfunction in membrane stability triggered by polyunsaturated fatty acid peroxidation 11,13 . In recent years, ferroptosis has been widely investigated for its involvement in various diseases, including neurodegenerative diseases, liver toxicity, liver fibrosis, hemochromatosis, and cardiomyopathy 11,14,15 , 16 , 17 . Ferroptosis is regulated by various cellular metabolic pathways, including redox homeostasis, mitochondrial activity, and iron metabolism 11,13 , and it can be induced by GSH depletion using chemicals such as BSO, cystine-glutamate transporter inhibition, or GPX4 10 , 18 . In the present study, we found that apomorphine inhibited ferroptosis in fibroblasts from patients with mitochondrial disease as well as in normal controls, and the cell-protective effect was not related to the agonistic action of dopamine. Results Induction of ferroptosis by GSH depletion by BSO and prevention of BSO-induced ferroptosis by apomorphine in LS patient fibroblasts. BSO reduced GSH production in a concentration-dependent manner 19 . Under these conditions, fibroblasts from LS patients showed enhanced cell death and were more vulnerable to BSO-induced cell stress than control fibroblasts, as previously described (Fig. 1 A, B) 9 . We subsequently performed BSO-induced cell survival assays to investigate whether or not they were suppressed by adding ferrostatin-1 (Fer-1), one of the most common ferroptosis inhibitors 11 , 18 , to LS patient-derived fibroblasts. Fer-1 showed dramatic cell-protective effects under BSO-induced stress, and apomorphine showed similar effects (Fig. 1 C). Protective effect of apomorphine against ferroptosis induced by BSO and RSL3. To evaluate the effects of apomorphine against ferroptosis, we examined the survival of LS fibroblasts in the presence of RSL3 as a common assay for ferroptosis screening, in addition to a BSO assay 18 , 20 . Prior to that, we examined the cell survival rate of control and patient fibroblasts under RSL3-induced stress, an inducer of ferroptosis. Fibroblasts obtained from LS, MELAS, mitochondrial cardiopathy, and Kearns-Sayer syndrome (KSS) patients showed enhanced cell death in response to RSL3-induced stress, but this effect was less pronounced than the response to BSO-induced stress (Supplementary Fig. 1A-F). Subsequently, we compared the cell protective effect of apomorphine with that of Fer-1 and Liproxstatin-1 (Lip-1), well-known specific ferroptosis inhibitors 18 , 21 , 22 . Apomorphine in LS fibroblasts under ferroptosis induced by BSO showed cell-protective effects, but we could not investigate the effects of apomorphine in response to BSO-induced stress in control fibroblasts because BSO did not induce cell death in control fibroblasts (Fig. 2 A). In the case of RSL3-induced stress, the difference in the survival between control and LS fibroblasts was less than that in response to BSO-induced stress; apomorphine showed cell-protective effects in both control and LS fibroblasts. The effects of apomorphine were similar to those of Fer-1 and Lip-1 (Fig. 2 B). We also examined other fibroblasts from different subtypes of mitochondrial diseases in the presence of RSL3 and obtained similar results (Fig. 2 C). We next examined the effects of various inhibitors of cell death pathways other than ferroptosis to rule out the possibility that apomorphine protects cells via other cell death pathways using GSK-872 (necroptosis inhibitor) or Z-VAD-FMK (apoptosis inhibitor). Although we could not examine the effects of these inhibitors in response to BSO-induced stress in control fibroblasts, BSO did not induce cell death in control fibroblasts, and neither GSK-872 nor Z-VAD-FMK showed any cell-protective effects against cell death induced by BSO and RSL3 (Fig. 3 A, B). These findings suggest that apomorphine protects against cell death by ferroptosis, but not by necroptosis or apoptosis, in control fibroblasts and fibroblasts derived from patients with mitochondrial disease. Measurement of intracellular ferroptotic metabolites in our cell lines by a BSO cell lipid oxidation assay. Because the accumulation of lipid ROS is a hallmark of ferroptosis, we monitored the lipid ROS accumulation using C11 BODIPY 581/591 by confocal imaging (Fig. 4 ). The C11 BODIPY 581/591 changes the fluorescence from red to green upon oxidation 23 . Even in the absence of BSO, LS patient-derived fibroblasts showed the oxidized form of C11 BODIPY, which is indicative of lipid ROS (Fig. 4 A_b). Upon BSO treatment, fibroblasts showed an increased lipid ROS accumulation (Fig. 4 A_c). Furthermore, Fer-1, one of the most common inhibitors of ferroptosis, recovered the lipid ROS accumulation. When we examined the effect of apomorphine on the accumulation of lipid ROS in LS patient-derived fibroblasts treated with BSO, apomorphine decreased the lipid ROS accumulation and showed a similar effect to Fer-1 (Fig. 4 A_d,e) 18 . In contrast, control fibroblasts showed almost no change in the degree of the lipid ROS accumulation (Fig. 4 B_a-o). The assessment of the expression of genes related to ferroptosis in apomorphine-treated vs. untreated LS fibroblasts by real-time reverse transcription polymerase chain reaction (RT-PCR) and Western blotting. We assessed the expression of prostaglandin endoperoxide synthase 2 (PTGS2) , a biomarker of ferroptosis that encodes cyclooxygenase-2 (COX-2) 11 , 24 – 26 . Real-time RT-PCR showed that RSL3 upregulated PTGS2 expression in LS fibroblasts and that this PTGS2 upregulation was inhibited by apomorphine and Fer-1 treatment (Fig. 5 A). Western blotting for PTGS2 showed that apomorphine significantly decreased the protein levels of PTGS2 in LS fibroblasts, and Fer-1 had a similar effect (Fig. 5 B). We therefore demonstrated that apomorphine suppressed PTGS2, a key factor linking ferroptosis and inflammation, in LS fibroblasts. The analysis of the expression of genes related to ferroptosis in apomorphine-treated vs. untreated LS fibroblasts by real-time RT-PCR. To examine how apomorphine inhibits ferroptosis, we assessed the following key genes related to ferroptosis: AIFM2 (encoding FSP1), GPX4, ACSL4 , and SLC7A11 (cystine/glutamate transporter) 11 , 24 , 25 , 26 . Real-time RT-PCR showed that apomorphine slightly suppressed the upregulation of SLC7A11 ; however, the differences were not statistically significant. Apomorphine also did not affect the mRNA expression of AIFM2, GPX4 , or ACSL4 (Fig. 6 A-D). The anti-ferrotosis effect of apomorphine is not related to the dopamine receptor agonist action of apomorphine. Our previous screening did not show the cell protective effects of other dopamine agonists (Ropinirole hydrochloride and Pramipexole dihydrochloride; both specific to D2 receptors) (Supplemental Fig. 2). Therefore, to evaluate whether or not the agonistic action of apomorphine was related to its cell-protective effect, we examined the effect of the addition of agonists and/or antagonists of each dopamine receptor on the cell-protective effect of apomorphine. Prior to this, the expression of each dopamine receptor in the cells was examined by RT-PCR. The expression of four types of dopamine receptors—D1R, D2R, D4R, and D5R—was confirmed in LS and control fibroblasts, but the expression of D3R was not detected in our fibroblasts (Fig. 7 A). Therefore, we examined the action of each dopamine receptor on the protection of apomorphine, except for D3R. First, we examined the contribution of D4R to the effects of apomorphine using agonists and antagonists of D4R, based on a previous report 4 . As shown in Fig. 7 B, the D4R agonists did not enhance the effect of apomorphine, and the antagonists did not inhibit this effect. Similarly, antagonists of D1R and D2R did not affect the effect of apomorphine (Fig. 7 C, D). Regarding D5R, as there was no commercially available antagonist for D5R, only the D5R agonist was used, but it did not provide any cell-protective effect (Fig. 7 E). Discussion In the present study, we showed that fibroblasts from mitochondrial diseases were vulnerable to ferroptosis, and apomorphine markedly protected fibroblasts from ferroptosis induced by RSL3 in various types of mitochondrial diseases as well as in normal fibroblasts. The effect of ferroptosis on cell protection was comparable to that of the specific ferroptosis inhibitors Fer-1 and Lip-1. We observed that BSO- or RSL3-induced ferroptosis was not inhibited by inhibitors of apoptosis (Z-VAD-FMK) or necroptosis (GSK-872), and PTGS2 , which is known to be a marker of ferroptosis, was decreased by the addition of apomorphine. In addition, we confirmed that the effects of apomorphine were not related to its agonistic dopamine action. Ferroptosis is an iron-dependent form of cell death caused by the accumulation of lipid hydroperoxides and is distinguished from other forms of cell death, such as apoptosis and necroptosis 12 , 18 . Ferroptosis occurs when GPX4 is directly or indirectly inhibited by GSH depletion, which leads to the accumulation of membrane lipid peroxidation and results in cell death 11 , 13 , 18 . RSL3 is commonly used as a specific inducer of ferroptosis and inactivates GPX4, leading to excessive lipid peroxidation, which causes cell death 18 . In contrast, ferroptosis can be suppressed by lipophilic antioxidants, inhibitors of lipid peroxidation, iron chelators, and polyunsaturated fatty acids 18 . Currently, several biomarkers of ferroptosis exist, including protein markers such as PTGS2 (which encodes COX-2), lipid peroxidation, and ROS 24 , 25 , 26 , 27 . We have already shown that apomorphine suppresses the production of ROS 9 . In the present study, we showed that apomorphine prevented lipid peroxidation and upregulation of the mRNA expression of PTGS2 (Fig. 5 ), which are key features of ferroptosis. Studies on cancer cells have indicated that ferroptosis can directly increase the expression of PTGS2 and cause inflammation by accelerating AA metabolism and promoting the secretion of pro-inflammatory molecules 28 , 29 . The downregulation of PTGS2 may be related to the inhibition of various cytokines and chemokines by apomorphine 9 . Recently, ferroptosis has been suggested to be involved in epilepsy with mitochondrial disease 10 . Our study also supports the hypothesis that fibroblasts from various mitochondrial diseases are vulnerable to ferroptosis. Apomorphine reportedly protects against glutamate-induced oxidative cell death via dopamine receptors, especially D4, in the HT22 cell line 4 . In this assay system, the protective effect was reversed by D4 antagonists but not by D1, D2, or D3 antagonists. A selective D4 agonist also protects neurons from glutamate-induced cell death. However, in our assay system using fibroblasts, the D4 agonist did not exert cell protective effects, and the D4 antagonist did not prevent the cell-protective effect of apomorphine. Furthermore, our data do not support the involvement of subtypes D1, D2, and D5. Therefore, we conclude that the cell-protective effect of apomorphine is not related to dopamine receptors in our system. Dopamine agonistic action causes several common side effects, especially digestive symptoms, such as nausea, vomiting, loss of appetite, and constipation 30 . Our research presents a promising avenue for the potential development of apomorphine derivatives that offer cellular protection without the undesirable effects associated with dopamine agonists. The limitation of the present study was that we were unable to identify the mechanism underlying the protection from ferroptosis, as we only performed an RT-PCR assay of key inhibitory genes for ferroptosis. Elucidation of the binding proteins that explain anti-ferroptosis and their downstream signaling cascades is the next research question that should be explored. In conclusion, we found that fibroblasts from patients with mitochondrial diseases were vulnerable to ferroptosis, which is inhibited by apomorphine in this study. The cell-protective effect of apomorphine has long been believed to be a result of D4R agonistic action, but we first revealed that at least part of its anti-ferroptosis effect is not related to the dopamine receptor agonist action. Our study suggests that ferroptosis may be a potential therapeutic target for mitochondrial disease and also provides hope for the creation of new drugs that maintain their cell-protective effect without the dopamine agonist effect, to avoid the adverse effects of dopamine. Materials and methods Subjects. This study was approved by the Ethics Committee of Jichi Medical University (Approval Number: J21-014) and all methods of this study were performed in accordance with the relevant guidelines and regulations. Fibroblasts were obtained from six patients at Jichi Medical University, Kanagawa Children’s Medical Center, and Saitama Medical University. The collection was conducted under the approval of the Ethics Committee of each institution, with approval numbers J21-014, H2021-094, and 11303-06, respectively. Written informed consent was obtained from the parents of each patient. And we used fibroblasts from healthy individuals as controls (normal human dermal fibroblasts purchased from PromoCell Company [#C-12300; Heidelberg, Germany]). Our four patients were diagnosed with LS and MELAS, as previously described 9 . Two of the patients had genetically identified cases of LS, including one with an m.10158 T > C, p(S34P) mutation in MT-ND3 (Case 1; LS ND 3 ) and one with a c.55 C > T, p(P19S) mutation in NDUFA1 (Case 2; LS NDUFA 1 ) 31 , 32 . Both ND3 and NDUFA1 are subunits of Complex I in the mitochondrial respiratory chain. Fibroblasts were also obtained from two patients with MELAS, including one with an m.3243 A > G mutation in tRNA-Leu (Case 3; MELAS tRNA−Leu ) and one with an m.5541 C > T mutation in tRNA-Trp (Case 4; MELAS tRNA−Trp ) 33 . We additionally obtained fibroblasts from patients with two other types of mitochondrial diseases: mitochondrial cardiomyopathy (Case 5; MC ND 5 ) and Kearns-Sayer syndrome (KSS) (Case 6; KSS large deletion ). One patient with mitochondrial cardiomyopathy who presented with HCM and died at 4 months old had an m.13513G > A mutation in the ND5 subunit of complex I ( MT-ND5 m.13513G > A), with 78.87% heteroplasmy. The patient with KSS was genetically identified as having a large deletion (m.8290–13802) of mitochondrial DNA (Table 1 ). Fibroblasts with fewer than 20 passages from patients and controls were used in the experiments. The heteroplasmic rate was analyzed by deep sequencing of mutated regions. Table 1 Fibroblast cell lines from patients with mitochondrial disease. Case Cell ID Disease Age Gene mutation Protein Mutation rate (%) 1 LS ND 3 Leigh 0 m.10158 T > C, p.(S34P) ND3 90 2 LS NDUFA 1 Leigh 5 c.55 C > T, p.(P19S) NDUFA1 Nuclear gene 3 MELAS tRNA−Leu MELAS 14 m.3243 A > G (tRNA-Leu) 21 4 MELAS tRNA−Trp MELAS 23 m.5541 C > T (tRNA-Trp) 49 5 MC ND 5 mitochondrial cardiomyopathy 1M m.13513 G > A p.(D393N) ND5 79 6 KSS large deletion KSS 1 m.8290-13802del − − Promo1 Control 0 − − − LS: Leigh syndrome, MELAS: myopathy encephalopathy, lactic acidosis, and stroke-like episodes. Respiratory chain activities from fibroblasts: LS ND 3 - Complex Ⅰ 9.8%, II 93.9%, III 94.5%, IV 47.6%, and CS 100.9%. LS NDUFA 1 - Complex Ⅰ 27.6%, Ⅱ 104.3%, Ⅲ 70.1%, Ⅳ 77.7%, CS 77.4%. MELAS tRNA−Leu - Not available. MELAS tRNA−Trp - Complex I 48%, II 103%, III 65%, IV 18%, MC ND 5 – not available. KSS large deletion – not available. Cell culture and growth conditions . Fibroblasts from patients were cultured in 1.0 g/L low-glucose Dulbecco’s Modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum (FBS), 100 units/mL penicillin, and 100 µg/mL streptomycin. Cells were incubated at 37°C in 5% CO 2 . Reagents. l-Butionine (S, R)-sulfoximine (BSO, No. B690270), a glutathione synthesis inhibitor, was purchased from Wako Pure Chemical Industries (Tokyo, Japan). RSL3 (No. S8155) was purchased from Selleck Chemicals (Houston, TX, USA). Fer-1 (No. SML0583) and Lip-1 (No. SML1414) were obtained from Sigma-Aldrich (St. Louis, MO, USA). Z-VAD-FMK (No. 3188-v) and GSK-872 (No. HY-101872) were purchased from Peptide Institute Inc. (Osaka, Japan) and MedChemExpress (Shanghai, China), respectively. All reagents were dissolved in dimethyl sulfoxide (DMSO). Cell viability assays. The BSO assay was performed as previously described 9 . We performed several cell viability assays to investigate ferroptosis. Ferroptotic cell death was induced in fibroblasts by ferroptosis inducers (RSL3 at 50–100 nM). In these cell viability experiments, fibroblasts were cultured to semi-confluence and plated at 5,000 cells per well in a 96-well culture plate in normal medium. After incubation for 24 h, apomorphine and several compounds were added as positive controls (Fer-1, or Lip-1) and cultured in the assay medium. All compounds were applied at a concentration of 1 µM unless otherwise indicated. After incubating the cell plates for 24 h at 37°C (95% humidity and 5% CO 2 ), a cell viability assay was performed using Cell Count Reagent SF (Nacalai Tesque, Kyoto, Japan). Fluorescence intensity was measured using a Benchmark Plus microplate reader (Bio-Rad, Hercules, CA, USA) according to the manufacturer’s instructions. The assessment of lipid peroxidation. Lipid peroxidation was examined using the fluorescent dye C11-BODIPY581/591 (No. D3861; Thermo Fisher Scientific, Waltham, MA, USA) 23 . Fibroblasts were cultured to semi-confluence and then plated at 7,000 cells per well in a 96-well culture plate in normal medium. After incubation for 24 h, BSO and several other compounds (e.g. apomorphine and Fer-1) were added to each well. After incubation for 24 h, the cells were labeled with 5 µM C11-BODIPY581/591 for 30 min in the assay medium. The nuclei were stained with Hoechst 33342. Representative images were obtained using a Keyence All-in-One Fluorescence BZ-X810 microscope (Keyence Co., Itasca, IL, USA). RNA extraction and real-time RT-PCR. Total RNA was extracted from fibroblasts using an RNeasy® Mini Kit (QIAGEN, Valencia, CA, USA) according to the manufacturer’s instructions 34 . Total RNA was reverse transcribed to cDNA, followed by amplification by PCR with a Superscript VILO cDNA synthesis kit at 60°C (Invitrogen; Thermo Fisher Scientific). Real-time RT-PCR was performed using the SYBR Green system with a primer set that amplified a fragment of the target genes to measure the mRNA expression. The following primers were used: PTGS2 (Forward primer 5’-GCCTGAATGTGCCATAA-GACTGAC-3’, Reverse primer 5’-AAACCCACAGTG-CTTGACACACA − 3’), AIFM2 (Forward primer 5’-ATGGTTCGGCTGACCAAGAG-3’, Reverse primer 5’-GCCACCACATCATTGGCATC-3’), GPX4 (Forward primer 5’-GCCTTCCCGTGTAACCAGT-3’, Reverse primer 5’-GCGAACTCTTTGATCT-CTTCG-3’), ACSL4 (Forward primer 5’-CCCTGAAGGATTTGAGATTCACA-3’, Reverse primer 5’-CCTTAGGT-CGGCCAGTAGAAC-3’), SLC7A11 (Forward primer 5’-ATGCAGTGGCAGTGA-CCTTT-3’, Reverse primer 5’-GGCAACAAAGATCG-GAACTG-3’), and GAPDH (Forward primer 5’-CTTTGTCAAGCTCATTTCCTGG − 3’, Reverse primer 5’-TCTTC-CTCTTGTGCTCTTGC − 3’). The reactions were performed in triplicate. Gene expression was normalized to that of GAPDH , and the data were analyzed in the Excel software program (Microsoft, Redmond, WA, USA) using the ΔΔCt method. Western blotting. For whole-cell extracts, cells were lysed in TNE buffer (20 mM Tris-HCL at pH 7.4, 150 mM NaCl, 1 mM EDTA at pH7.4). Protein concentrations were determined using the Qubit® Protein Assay Kit (Invitrogen; Thermo Fisher Scientific). Whole-cell lysates were mixed with an equal volume of 2× sodium dodecyl sulfate (SDS) sample buffer and boiled. Western blotting was performed using 20 µg of total protein, and immunoprecipitation was performed using XV PANTERA GEL (NXV-361HP; DRC Co., Ltd, Tokyo, Japan). Total cell proteins were separated by 10% SDS-polyacrylamide gel electrophoresis (PAGE) and transferred to a PVDF membrane by electrotransfer. The membrane was blocked for 1 h at room temperature (RT) using 5% skimmed milk/phosphate-buffered saline with Tween (PBST) and then incubated with the following primary antibodies: rabbit monoclonal anti-PTGS2 (Cox2(D5H5)XP #12282; Cell Signaling, Danvers, MA, USA) at 1:1000 and mouse monoclonal anti-beta-actin (A1978; Sigma-Aldrich) at 1:5000 in PBST overnight at 4°C. After washing with PBST 3 times for 10 min each, the membrane was incubated with the following secondary antibodies: anti-rabbit IgG horseradish peroxidase (HRP; Cell Signaling) at 1:3000 and anti-mouse IgG HRP (Santa Cruz Biotechnology, Santa Cruz, CA, USA) at 1:2000 for 1 h at RT. After washing with PBST 3 times for 15 min each, the membrane was incubated with Hyper HRP Substrate (TAKARA BIO INC., Ohtsu, Japan) for 2 min. Finally, chemiluminescence from the membrane was imaged using Amersham Imager 680 (GE Healthcare UK Ltd., Little Chalfont, UK). Protein intensities were measured using the ImageJ software program. Relative protein levels or protein abundances were normalized to those in the control group. Detection of dopamine gene receptor (DRD1-DRD5) expression on control and patient-derived fibroblasts by RT-PCR. RNA was extracted from isolated control and LS patient fibroblasts (LS ND 3 ) using an RNeasy Mini Kit (QIAGEN) according to the manufacturer’s protocol. Commercial human Adult Normal Tissue: Brain: Frontal Lobe (BioChain, Newark, CA, USA) was used as the Positive Control. Total RNA (1500–2000 ng) was reverse-transcribed into first-strand cDNA using the Superscript VILO cDNA Synthesis kit (Invitrogen; Thermo Fisher Scientific). RT-PCR was performed using intron-spanning primers (Supplemental Table 1). The D1R/D2R/D5R cycle conditions were as follows: 94°C for 2 min for polymerase activation, followed by 45 cycles at 98°C for 10 s, 62°C for 30 s, 68°C for 1 min. The D3R cycle conditions were as follows: 94°C for 2 min for polymerase activation, followed by 45 cycles of 98°C for 10 s, 62°C for 15 s, 72°C for 1 min. The D4R cycle conditions were as follows: 94°C for 2 min for polymerase activation, followed by 5 cycles of 98°C for 10 s, 68°C for 1 min, followed by 5 cycles of 98°C for 10 s, 66°C for 1 min, followed by 5 cycles of 98°C for 10 s, 64°C for 1 min, followed by 25 cycles of 98°C for 10 s, 62°C for 1 min, 68°C for 7 min. The products were resolved on a 2% agarose gel containing ethidium bromide and photographed. Interactions between apomorphine and dopamine receptor agonists or antagonists: Effects on cell viability. Cell viability assays to examine the influence of dopamine receptor agonists or antagonists on the effects of apomorphine were performed under RSL-induced oxidative stress. SKF 83566 cells (no. HY-103430A; MedChemExpress) was used as the D1R antagonist 35 , sulpiride (No. S4655; Selleck Chemicals) as the D2R antagonist 36 , PD168077 (No. HY-21098A; MedChemExpress) as the D4R agonist 37 ; L745870 (No. HY-14325; MedChemExpress) as the D4R antagonist 38 , and SKF38393 (No. S7993; Selleck Chemicals) as the D5R agonist 39 . The concentrations used were determined based on the EC50 and Ki values 35 36 37 38 39 . A cell viability assay was performed as previously described 9 . In brief, fibroblasts were cultured in 1.0 g/L low-glucose DMEM with 10% FBS at 37°C in 5% CO 2 until they reached semi-confluence. Fibroblasts were seeded at 5000 cells per well in a 96-well plate. After 24 h of incubation, we divided the cells into 3 groups: a DR agonist- or antagonist-treated group, a DR agonist- or antagonist-treated group at a concentration 10 times the EC50 or Ki values, and a DR agonist- or antagonist-treated group at a concentration 100 times the EC50 or Ki values. Apomorphine was added at a final concentration of 1 µM. After 24 h of incubation, the cell survival rate was checked using Cell Count Reagent SF (Nacalai Tesque). The cell viability of Apomorphin-treated group without RSL3 was used as 100% to compare other group’s cell viabilities. Statistical analyses . The results are expressed as the mean ± standard deviation. Comparisons between multiple-group means were performed using a one-way analysis of variance with Bonferroni’s post-hoc test. Statistical significance was set at P < 0.05. Statistical analyses were performed using the GraphPad Prism software program (GraphPad Software Inc., La Jolla, CA, USA). Declarations Data availability. The datasets and raw data used and/or analyzed during the current study are available from the corresponding author on reasonable request. Acknowledgements We thank the patients and their families as well as all of the staff working, especially Miyuki Watanabe, Tomomi Oyama, and Narumi Omika at Jichi Children Medical Center Tochigi and Jichi Medical University Hospital. Author contributions A.M., T.Y., and H.O. developed the strategy for the research; methodology, C.W., E.F.J., and N.Y.; performed the research, A.M., A.N., M.K., and N.O.; analyzed the data, A.M. and S.A.; resources, N.Y., Y.K., A.O., M.T.; writing—original draft preparation, A.M.; writing—review and editing, N.O. and H.O.. Funding This research was supported by a grant from the Project for Health Research on Infants, Children, Adolescents, and Young Adults from the Agency of Medical Research and Development, and a grant from the Japan Agency for Medical Research and Development (grant number im0210625h0001, 17ek0109270s0301, 22ek0109511h0002) to H.O. and JSPS KAKENHI to H.O. (JP21H03648). 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Curr Drug Targets 19 , 1599-1611, doi:10.2174/1389450118666171117124340 (2018). Kouga, T. et al. Japanese Leigh syndrome case treated with EPI-743. Brain Dev 40 , 145-149, doi:10.1016/j.braindev.2017.08.005 (2018). Miyauchi, A. et al. Leigh syndrome with spinal cord involvement due to a hemizygous NDUFA1 mutation. Brain Dev 40 , 498-502, doi:10.1016/j.braindev.2018.02.007 (2018). Hatakeyama, H., Katayama, A., Komaki, H., Nishino, I. & Goto, Y. Molecular pathomechanisms and cell-type-specific disease phenotypes of MELAS caused by mutant mitochondrial tRNA(Trp). Acta Neuropathol Commun 3 , 52, doi:10.1186/s40478-015-0227-x (2015). Novoradovskaya, N. et al. Universal Reference RNA as a standard for microarray experiments. BMC Genomics 5 , 20, doi:10.1186/1471-2164-5-20 (2004). Stouffer, M. A. et al. SKF-83566, a D1-dopamine receptor antagonist, inhibits the dopamine transporter. J Neurochem 118 , 714-720, doi:10.1111/j.1471-4159.2011.07357.x (2011). Huang, F. et al. Retinal Dopamine D2 Receptors Participate in the Development of Myopia in Mice. Invest Ophthalmol Vis Sci 63 , 24, doi:10.1167/iovs.63.1.24 (2022). Glase, S. A. et al. Substituted [(4-phenylpiperazinyl)-methyl]benzamides: selective dopamine D4 agonists. J Med Chem 40 , 1771-1772, doi:10.1021/jm970021c (1997). Patel, S. et al. Biological profile of L-745,870, a selective antagonist with high affinity for the dopamine D4 receptor. J Pharmacol Exp Ther 283 , 636-647 (1997). Seeman, P. & Van Tol, H. H. Dopamine receptor pharmacology. Trends Pharmacol Sci 15 , 264-270, doi:10.1016/0165-6147(94)90323-9 (1994). Additional Declarations No competing interests reported. Supplementary Files Supplementalfigure12.tiff Supplemental Figure 1. The vulnerability of patient-derived fibroblasts to RSL3-induced ferroptosis. The cell survival rates of patient fibroblasts (A-F; each left side) and controls (A-F; each right side) under RSL3-induced stress. After 24 h of treatment with 100 nM RSL3, with or without Apo, we checked the cell survival rate and compared the findings with those of RSL3-untreated cells. Fibroblasts from patients with LS and MELAS were more sensitive to RSL3 exposure than normal fibroblasts (A-D). In addition, fibroblasts from patients with other mitochondrial diseases, mitochondrial cardiomyopathy, mitochondrial liver disease, and KSS were also more vulnerable than normal fibroblasts (E, F). Apo, apomorphine; Fer-1: Ferrostatin-1, KSS: Kearns‒Sayre Syndrome. Data are expressed as the mean ± SD *P < 0.05, ** P < 0.01, ***P < 0.001. n.s., no statistical significance. Supplemental Figure 2. The cell viability assay of fibroblasts from patients with LS in the presence of RSL3 with two dopamine agonists (ropinirole hydrochloride and pramipexole dihydrochloride) was based on our previous screening. Data are expressed as the mean ± SD, ***P < 0.001. n.s., no statistical significance. Supplementalfigure3.tiff Supplemental Figure 3. Full-lengths dot blot of Fig. 5. SupplementaltableRTPCRprimerinformation.xlsx Cite Share Download PDF Status: Published Journal Publication published 27 Feb, 2024 Read the published version in Scientific Reports → Version 1 posted Editorial decision: Revision requested 03 Jan, 2024 Reviews received at journal 31 Oct, 2023 Reviews received at journal 14 Oct, 2023 Reviewers agreed at journal 13 Oct, 2023 Reviewers agreed at journal 13 Oct, 2023 Reviewers invited by journal 26 Sep, 2023 Editor assigned by journal 26 Sep, 2023 Editor invited by journal 26 Sep, 2023 Submission checks completed at journal 26 Sep, 2023 First submitted to journal 16 Sep, 2023 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-3361719","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":236034321,"identity":"011d1ee0-980f-4db2-8e5b-3e57d5697680","order_by":0,"name":"Akihiko Miyauchi","email":"","orcid":"","institution":"Jichi Medical University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Akihiko","middleName":"","lastName":"Miyauchi","suffix":""},{"id":236034322,"identity":"a56875fc-9621-49e6-9798-f9ae9678c979","order_by":1,"name":"Chika Watanabe","email":"","orcid":"","institution":"Jichi Medical University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Chika","middleName":"","lastName":"Watanabe","suffix":""},{"id":236034323,"identity":"2f726e19-f42d-44b7-a928-08621322dadd","order_by":2,"name":"Naoya Yamada","email":"","orcid":"","institution":"Jichi Medical University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Naoya","middleName":"","lastName":"Yamada","suffix":""},{"id":236034324,"identity":"228fa896-a12f-45a0-ba4d-fe81c7487133","order_by":3,"name":"Eriko F. Jimbo","email":"","orcid":"","institution":"Jichi Medical University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Eriko","middleName":"F.","lastName":"Jimbo","suffix":""},{"id":236034325,"identity":"4d03154a-f6d1-446a-aa25-9ef06c05e3ba","order_by":4,"name":"Mizuki Kobayashi","email":"","orcid":"","institution":"Jichi Medical University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Mizuki","middleName":"","lastName":"Kobayashi","suffix":""},{"id":236034326,"identity":"a1c48cc9-f56a-43ee-bb5e-988112be2ecf","order_by":5,"name":"Natsumi Ohishi","email":"","orcid":"","institution":"Jichi Medical University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Natsumi","middleName":"","lastName":"Ohishi","suffix":""},{"id":236034327,"identity":"6fbdcc00-b78d-417b-8bb6-54b0b0ee2743","order_by":6,"name":"Atsuko Nagayoshi","email":"","orcid":"","institution":"Jichi Medical University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Atsuko","middleName":"","lastName":"Nagayoshi","suffix":""},{"id":236034328,"identity":"6f882134-8382-467a-acc9-98309a9932a3","order_by":7,"name":"Shiho Aoki","email":"","orcid":"","institution":"Jichi Medical University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Shiho","middleName":"","lastName":"Aoki","suffix":""},{"id":236034329,"identity":"46a7c134-9081-41cd-9858-e99ac76e7e4d","order_by":8,"name":"Yoshihito Kishita","email":"","orcid":"","institution":"Juntendo University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Yoshihito","middleName":"","lastName":"Kishita","suffix":""},{"id":236034330,"identity":"92ef25c0-e64b-406d-9f80-dfcdb290fc79","order_by":9,"name":"Akira Ohtake","email":"","orcid":"","institution":"Saitama Medical University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Akira","middleName":"","lastName":"Ohtake","suffix":""},{"id":236034331,"identity":"f2a134bd-3f99-40bd-a1b3-e7fb69a0260a","order_by":10,"name":"Nobuhiko Ohno","email":"","orcid":"","institution":"Jichi Medical University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Nobuhiko","middleName":"","lastName":"Ohno","suffix":""},{"id":236034332,"identity":"72efe512-e9db-41ef-bf8c-04456e3f6da2","order_by":11,"name":"Masafumi Takahashi","email":"","orcid":"","institution":"Jichi Medical University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Masafumi","middleName":"","lastName":"Takahashi","suffix":""},{"id":236034333,"identity":"3cd2a343-78fc-4487-b960-78c7298aa0ab","order_by":12,"name":"Takanori Yamagata","email":"","orcid":"","institution":"Jichi Medical University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Takanori","middleName":"","lastName":"Yamagata","suffix":""},{"id":236034334,"identity":"5cb07e37-3f3f-416b-9ce3-83a248824aeb","order_by":13,"name":"Hitoshi Osaka","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAz0lEQVRIiWNgGAWjYBACAyBmbGCwYWZj4GE4wADhEaUljXQth4EUD5EOM2dvf/hxRs15dj72swcPMNTYMTDPJmCNZc8ZY8kNx24zs/HkJRxgOJbMwDjnAAGH3chhkHzABtQiwWNwgIHtAAPjjARCWtIf/3zw7xxUyz+itCSYSW5sOwDRwthGjJYzZ8wsZ/YlA/2SY3AgsS+Zh7Bfjrc/vtnzzS5Zvv2M8YcP3+zkDAmFGAwkg0mgk3gMZxCng8EOzpKXIFLLKBgFo2AUjBgAAFm5Q76TA0ADAAAAAElFTkSuQmCC","orcid":"","institution":"Jichi Medical University","correspondingAuthor":true,"submittingAuthor":false,"prefix":"","firstName":"Hitoshi","middleName":"","lastName":"Osaka","suffix":""}],"badges":[],"createdAt":"2023-09-16 16:44:13","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-3361719/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-3361719/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1038/s41598-024-55293-1","type":"published","date":"2024-02-27T15:01:19+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":44037606,"identity":"660172f5-f094-40ee-bc63-7c285ccdbf07","added_by":"auto","created_at":"2023-10-03 18:08:23","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":511779,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eThe cell viability assay for fibroblast cells from LS patients (LS\u003c/strong\u003e\u003csup\u003e\u003cstrong\u003eND3\u003c/strong\u003e\u003c/sup\u003e\u003cstrong\u003e) under BSO-induced stress.\u003c/strong\u003e While the fibroblasts of control subjects were able to maintain almost full viability (\u003cstrong\u003eA\u003c/strong\u003e), those from patients with LS showed enhanced BSO dose-dependent cell death (\u003cstrong\u003eB\u003c/strong\u003e). Under the conditions of 100 uM BSO treatment, we evaluated the effects of Fer-1, which is one of the most common ferroptosis inhibitors and Apo. Fer-1 showed dramatic cell-protective effects, similar to those of Apo (\u003cstrong\u003eC\u003c/strong\u003e). Apo: apomorphine, Fer-1: Ferrostatin-1. Data (n = 6) are expressed as the mean ± SD ***P \u0026lt; 0.001. n.s., no statistical significance.\u003c/p\u003e","description":"","filename":"1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-3361719/v1/328ae0d5e48cd634d6a641b6.jpg"},{"id":44037604,"identity":"7462f0d7-2d9b-446d-bd92-5046aa525271","added_by":"auto","created_at":"2023-10-03 18:08:23","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":1783330,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eThe anti-ferroptosis effect of apomorphine. \u003c/strong\u003e(\u003cstrong\u003eA, B\u003c/strong\u003e) The cell viability assay of fibroblasts from LS patients (LS\u003csup\u003eND3\u003c/sup\u003e) (left panel) and control (right panel) in the presence of 100 uM BSO and RSL3. (\u003cstrong\u003eC\u003c/strong\u003e) The cell viability assay of fibroblasts from patients with LS (LS\u003csup\u003eNDUFA1\u003c/sup\u003e), MELAS (MELAS\u003csup\u003etRNA-Leu\u003c/sup\u003e, MELAS\u003csup\u003etRNA-Trp\u003c/sup\u003e), mitochondrial cardiomyopathy (MC\u003csup\u003eND5\u003c/sup\u003e), and KSS (KSS\u003csup\u003elarge deletion\u003c/sup\u003e). The cell-protective effects of apomorphine were also shown in comparison to Fer-1, and Lip-1 in the presence of RSL3. Apo: apomorphine, Fer-1: Ferrostatin-1. Data (n = 6) are expressed as the mean ± SD *P \u0026lt; 0.05, ** P \u0026lt; 0.01, ***P \u0026lt; 0.001. n.s. indicates no significance.\u003c/p\u003e","description":"","filename":"2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-3361719/v1/74736fe288b233238312e89e.jpg"},{"id":44038322,"identity":"ad07094f-d97b-4adb-af41-32722572f0fa","added_by":"auto","created_at":"2023-10-03 18:16:23","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":1157607,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eCell viabilities by apoptosis and necroptosis inhibitors.\u003c/strong\u003e The effects of apoptosis and necroptosis inhibitors under conditions of 100 uM BSO- (\u003cstrong\u003eA\u003c/strong\u003e), and RSL3- (\u003cstrong\u003eB\u003c/strong\u003e) induced stress for fibroblast cells from LS patients (LS\u003csup\u003eND3\u003c/sup\u003e) (left) and controls (right). Apo: apomorphine, Fer-1: Ferrostatin-1. Data (n = 3) are expressed as the mean ± SD *P \u0026lt; 0.05, ** P \u0026lt; 0.01, ***P \u0026lt; 0.001. n.s. indicates no significance.\u003c/p\u003e","description":"","filename":"3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-3361719/v1/1e40cf27001d0d5bcb012a6e.jpg"},{"id":44037610,"identity":"3e52a77c-4c89-4799-a18c-fcd6ae1922b9","added_by":"auto","created_at":"2023-10-03 18:08:23","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":5647074,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eResults of a C11 BODIPY581/591 assay in fibroblasts from patients and controls. \u003c/strong\u003eMeasurement of intracellular ferroptotic metabolites by the BSO cell lipid oxidation assay. (\u003cstrong\u003eA\u003c/strong\u003e) Fibroblasts from LS patients (LS\u003csup\u003eND3\u003c/sup\u003e) were treated with BSO (100 μM) for 24 h in the presence or absence of Apo (1 μM) and Fer-1 (1 μM). (\u003cstrong\u003eB\u003c/strong\u003e) Control fibroblasts were treated with BSO (100 μM) for 24 h in the presence or absence of Apo (1 μM) and Fer-1 (1 μM). Lipid peroxidation was assessed by C11 BODIPY581/591 staining. Representative images of C11 BODIPY581/591 staining in LS\u003csup\u003eND3\u003c/sup\u003e cells and control cells; Oxidized form (\u003cstrong\u003ea-e\u003c/strong\u003e), Reduced form (\u003cstrong\u003ef-j\u003c/strong\u003e), and merge (\u003cstrong\u003ek-o\u003c/strong\u003e). Apo: apomorphine, Fer-1: Ferrostatin-1.\u003c/p\u003e","description":"","filename":"4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-3361719/v1/ab2894ad290bd96f55be5e12.jpg"},{"id":44037607,"identity":"b76f120e-7eed-4ca0-bb26-1564a07c6cbd","added_by":"auto","created_at":"2023-10-03 18:08:23","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":708506,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eAn expression analysis of prostaglandin-endoperoxide synthase 2 (PTGS2).\u003c/strong\u003e\u003cem\u003e \u0026nbsp;\u003c/em\u003e(\u003cstrong\u003eA\u003c/strong\u003e) \u003cem\u003ePTGS2\u003c/em\u003e mRNA levels in LS\u003csup\u003eND3\u003c/sup\u003e cells treated with RSL3 for 24 h in the presence or absence of Apo and Fer-1 were assessed using real-time RT-PCR. (\u003cstrong\u003eB\u003c/strong\u003e) PTGS2 protein levels in LS\u003csup\u003eND3\u003c/sup\u003e cells treated with RSL3 for 24 h in the presence or absence of Apo and Fer-1 were assessed by Western blotting. Full-length gels and blots are included in a Supplementary (Supplemental Fig. 3) as full-length dot blots. Apo: apomorphine, Fer-1: Ferrostatin-1. Data are expressed as the mean ± SD; ***P \u0026lt; 0.001.\u003c/p\u003e","description":"","filename":"5.jpg","url":"https://assets-eu.researchsquare.com/files/rs-3361719/v1/88973f6bd61a3c59086359e7.jpg"},{"id":44037608,"identity":"a2e0c40a-622c-4ded-8aea-1442dd104084","added_by":"auto","created_at":"2023-10-03 18:08:23","extension":"jpg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":600900,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003emRNA levels of various ferroptosis marker genes. \u003c/strong\u003e(\u003cstrong\u003eA\u003c/strong\u003e) \u003cem\u003eAIFM2\u003c/em\u003e (encoding FSP1), (\u003cstrong\u003eB\u003c/strong\u003e) \u003cem\u003eGPX4, \u003c/em\u003e(\u003cstrong\u003eC\u003c/strong\u003e) \u003cem\u003eACSL4\u003c/em\u003e, and (\u003cstrong\u003eD\u003c/strong\u003e) \u003cem\u003eSCL7A11 \u003c/em\u003e(Cystine/glutamate transporter) mRNA levels in LS\u003csup\u003eND3\u003c/sup\u003e cells treated with RSL3 for 24 h in the presence or absence of Apo and Fer-1 were assessed by real-time RT-PCR. Apo: apomorphine, Fer-1: Ferrostatin-1. Data are expressed as the mean ± SD *P \u0026lt; 0.05, ** P \u0026lt; 0.01, ***P \u0026lt; 0.001. n.s. indicates no significance.\u003c/p\u003e","description":"","filename":"6.jpg","url":"https://assets-eu.researchsquare.com/files/rs-3361719/v1/a31fe8101ad225c2d369dbf4.jpg"},{"id":44037609,"identity":"c1c2459d-3774-42dd-a13f-29c54d5750e8","added_by":"auto","created_at":"2023-10-03 18:08:23","extension":"jpg","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":3617085,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eThe expression of dopamine receptors (D1R-D5R) and interactions between apomorphine and dopamine receptor agonists or antagonists\u003c/strong\u003e. (\u003cstrong\u003eA\u003c/strong\u003e) Agarose gel electrophoresis of the RT-PCR product of dopamine receptors. Results are shown from left to right in the order of D11R(size 184 bp), D2R (size 353 bp), D3R (size 137 bp), D4R (size 115 bp), and D5R (size 149 bp). In each column, the left lane shows the band of the frontal lobe as a positive control, the middle shows the band of control cells, and the right shows the band of LS cells. (\u003cstrong\u003eB-E\u003c/strong\u003e) The results of an RSL3-induced stressed cell viability assay to determine the influence of each dopamine receptor agonist or antagonist on the effects of apomorphine. The cell viability of the Apomorphine-treated group without RSL3 was used as 100% to compare other group’s cell viabilities (n = 3). (\u003cstrong\u003eB\u003c/strong\u003e) D4R agonist: PD168077 (Ki=8.7 nM) and D4R antagonist: L745870 (Ki=0.43 nM), (\u003cstrong\u003eC\u003c/strong\u003e) D1R antagonist: SKF683566 (Ki=0.56 nM), (\u003cstrong\u003eD\u003c/strong\u003e) D2R antagonist: Sulpiride (Ki=0.015 μM), (\u003cstrong\u003eE\u003c/strong\u003e) D5R agonist: SKF38393 (Ki=0.5 nM).\u003c/p\u003e","description":"","filename":"7.jpg","url":"https://assets-eu.researchsquare.com/files/rs-3361719/v1/f99c61502f768addd4e928b7.jpg"},{"id":51958247,"identity":"677c8dc7-ae14-4f31-b550-830c2d2846ea","added_by":"auto","created_at":"2024-03-04 15:14:43","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1293928,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-3361719/v1/6891c105-5207-4d5f-8d36-4b2c00bc61fc.pdf"},{"id":44037634,"identity":"0189d985-cd66-45b2-b774-cd0a5d42a1d6","added_by":"auto","created_at":"2023-10-03 18:08:27","extension":"tiff","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":62619206,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSupplemental Figure 1. The vulnerability of patient-derived fibroblasts to RSL3-induced ferroptosis. \u003c/strong\u003eThe cell survival rates of patient fibroblasts (\u003cstrong\u003eA-F\u003c/strong\u003e; each left side) and controls (\u003cstrong\u003eA-F\u003c/strong\u003e; each right side) under RSL3-induced stress. After 24 h of treatment with 100 nM RSL3, with or without Apo, we checked the cell survival rate and compared the findings with those of RSL3-untreated cells. Fibroblasts from patients with LS and MELAS were more sensitive to RSL3 exposure than normal fibroblasts (\u003cstrong\u003eA-D\u003c/strong\u003e). In addition, fibroblasts from patients with other mitochondrial diseases, mitochondrial cardiomyopathy, mitochondrial liver disease, and KSS were also more vulnerable than normal fibroblasts (\u003cstrong\u003eE, F\u003c/strong\u003e). Apo, apomorphine; Fer-1: Ferrostatin-1, KSS: Kearns‒Sayre Syndrome. Data are expressed as the mean ± SD *P \u0026lt; 0.05, ** P \u0026lt; 0.01, ***P \u0026lt; 0.001. n.s., no statistical significance.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSupplemental Figure 2. \u003c/strong\u003eThe cell viability assay of fibroblasts from patients with LS in the presence of RSL3 with two\u003cstrong\u003e \u003c/strong\u003edopamine agonists (ropinirole hydrochloride and pramipexole dihydrochloride) was based on our previous screening. Data are expressed as the mean ± SD, ***P \u0026lt; 0.001. n.s., no statistical significance.\u003c/p\u003e","description":"","filename":"Supplementalfigure12.tiff","url":"https://assets-eu.researchsquare.com/files/rs-3361719/v1/351b43f6f1fc65de84c24e0a.tiff"},{"id":44037612,"identity":"c2a4d0fa-e10d-4faf-8d8d-4bde0534d69a","added_by":"auto","created_at":"2023-10-03 18:08:24","extension":"tiff","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":25174366,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSupplemental Figure 3. \u003c/strong\u003eFull-lengths dot blot of Fig. 5.\u003c/p\u003e","description":"","filename":"Supplementalfigure3.tiff","url":"https://assets-eu.researchsquare.com/files/rs-3361719/v1/fd742657ff73b079130327db.tiff"},{"id":44037603,"identity":"03949ca4-5274-4b79-bcdb-d41b9b3d1019","added_by":"auto","created_at":"2023-10-03 18:08:22","extension":"xlsx","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":10780,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaltableRTPCRprimerinformation.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-3361719/v1/044e6379cef81c8b06ef12ad.xlsx"}],"financialInterests":"No competing interests reported.","formattedTitle":"\u003cp\u003e\u003cstrong\u003eApomorphine is a potent inhibitor of ferroptosis independent of dopaminergic receptors\u003c/strong\u003e\u003c/p\u003e","fulltext":[{"header":"Introduction","content":"\u003cp\u003eApomorphine is a\u0026nbsp;known treatment for Parkinson\u0026rsquo;s disease (PD) and erectile dysfunction\u003csup\u003e1\u003c/sup\u003e\u003csup\u003e,\u003c/sup\u003e\u003csup\u003e2\u003c/sup\u003e\u003csup\u003e,\u003c/sup\u003e\u003csup\u003e3\u003c/sup\u003e. It is a broad-spectrum dopamine agonist for all subtypes, ranging from the Dopamine D1 receptor to\u0026nbsp;the D5 receptor. Apomorphine exerts protective effects against oxidative toxicity via activation of the Dopamine D4 receptor (D4R) in\u0026nbsp;a micromolar order\u003csup\u003e4\u003c/sup\u003e\u003csup\u003e,\u003c/sup\u003e\u003csup\u003e5\u003c/sup\u003e.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eIn our previous study, we screened a chemical library based on drugs already approved for central nervous system (CNS) diseases using fibroblasts from two patients with Leigh syndrome (LS) and two patients with myopathy encephalopathy, lactic acidosis, and stroke-like episodes (MELAS)\u0026nbsp;using an L-buthionine-S, R-sulfoximine (BSO)-induced cell viability assay. BSO has been used to induce cell death by inhibiting glutathione (GSH) biosynthesis, which leads to the overproduction of reactive oxygen species (ROS)\u003csup\u003e6\u003c/sup\u003e. In addition, BSO has been recently reported to trigger ferroptosis in various cancer cells\u003csup\u003e7\u003c/sup\u003e\u003csup\u003e,\u003c/sup\u003e\u003csup\u003e8\u003c/sup\u003e.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eWhile searching for potential therapeutic drugs for mitochondrial diseases, we identified apomorphine as a potential therapeutic drug for LS and MELAS\u003csup\u003e9\u003c/sup\u003e. Our findings showed that apomorphine protected against BSO-induced stress with\u0026nbsp;an EC50\u0026nbsp;(median effect concentration) of approximately 50 nM and improved the mitochondrial respiratory activity of fibroblasts from patients with LS as well as MELAS. In addition, our data suggested that the cell-protective effect of apomorphine may be different from\u0026nbsp;that of dopamine agonists.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eApomorphine is also known to inhibit oxytosis and stress-dependent non-apoptotic cell death by activation of dopamine D4 receptors\u0026nbsp;\u003csup\u003e4\u003c/sup\u003e. Because oxytosis shares some concepts with ferroptosis proposed in recent years, we investigated the effect of apomorphine on ferroptosis and the relationship between this effect and dopamine receptors. Ferroptosis is a common form of regulated cell death that is distinguished from apoptosis and necroptosis and\u0026nbsp;is\u0026nbsp;a form of lipid- and iron-dependent cell death associated with glutathione (GSH) depletion\u003csup\u003e10\u003c/sup\u003e\u003csup\u003e,\u003c/sup\u003e\u003csup\u003e11\u003c/sup\u003e\u003csup\u003e,\u003c/sup\u003e\u003csup\u003e12\u003c/sup\u003e. Although the exact mechanism underlying ferroptotic cell death is not fully understood, it includes oxidative damage to cellular structures and dysfunction in membrane stability triggered by polyunsaturated fatty acid peroxidation\u003csup\u003e11,13\u003c/sup\u003e. In recent years, ferroptosis has been widely investigated for its involvement in various diseases, including neurodegenerative diseases, liver toxicity, liver fibrosis, hemochromatosis, and cardiomyopathy\u003csup\u003e11,14,15\u003c/sup\u003e\u003csup\u003e,\u003c/sup\u003e\u003csup\u003e16\u003c/sup\u003e\u003csup\u003e,\u003c/sup\u003e\u003csup\u003e17\u003c/sup\u003e. Ferroptosis is regulated by various cellular metabolic pathways, including redox homeostasis, mitochondrial activity, and iron metabolism\u003csup\u003e11,13\u003c/sup\u003e, and it can be induced by GSH depletion using chemicals such as BSO, cystine-glutamate transporter inhibition, or GPX4\u003csup\u003e10\u003c/sup\u003e\u003csup\u003e,\u003c/sup\u003e\u003csup\u003e18\u003c/sup\u003e.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eIn the present study, we found that apomorphine inhibited ferroptosis in fibroblasts from patients with mitochondrial disease as well as in normal controls, and the cell-protective effect was not related to the agonistic action of dopamine.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e \u003cb\u003eInduction of ferroptosis by GSH depletion by BSO and prevention of BSO-induced ferroptosis by apomorphine in LS patient fibroblasts.\u003c/b\u003e BSO reduced GSH production in a concentration-dependent manner\u003csup\u003e\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u003c/sup\u003e. Under these conditions, fibroblasts from LS patients showed enhanced cell death and were more vulnerable to BSO-induced cell stress than control fibroblasts, as previously described (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA, B)\u003csup\u003e\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u003c/sup\u003e. We subsequently performed BSO-induced cell survival assays to investigate whether or not they were suppressed by adding ferrostatin-1 (Fer-1), one of the most common ferroptosis inhibitors\u003csup\u003e\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e,\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003e, to LS patient-derived fibroblasts. Fer-1 showed dramatic cell-protective effects under BSO-induced stress, and apomorphine showed similar effects (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eProtective effect of apomorphine against ferroptosis induced by BSO and RSL3.\u003c/b\u003e To evaluate the effects of apomorphine against ferroptosis, we examined the survival of LS fibroblasts in the presence of RSL3 as a common assay for ferroptosis screening, in addition to a BSO assay\u003csup\u003e\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e,\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u003c/sup\u003e. Prior to that, we examined the cell survival rate of control and patient fibroblasts under RSL3-induced stress, an inducer of ferroptosis. Fibroblasts obtained from LS, MELAS, mitochondrial cardiopathy, and Kearns-Sayer syndrome (KSS) patients showed enhanced cell death in response to RSL3-induced stress, but this effect was less pronounced than the response to BSO-induced stress (Supplementary Fig.\u0026nbsp;1A-F).\u003c/p\u003e \u003cp\u003eSubsequently, we compared the cell protective effect of apomorphine with that of Fer-1 and Liproxstatin-1 (Lip-1), well-known specific ferroptosis inhibitors\u003csup\u003e\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e,\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e,\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u003c/sup\u003e. Apomorphine in LS fibroblasts under ferroptosis induced by BSO showed cell-protective effects, but we could not investigate the effects of apomorphine in response to BSO-induced stress in control fibroblasts because BSO did not induce cell death in control fibroblasts (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA). In the case of RSL3-induced stress, the difference in the survival between control and LS fibroblasts was less than that in response to BSO-induced stress; apomorphine showed cell-protective effects in both control and LS fibroblasts. The effects of apomorphine were similar to those of Fer-1 and Lip-1 (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB). We also examined other fibroblasts from different subtypes of mitochondrial diseases in the presence of RSL3 and obtained similar results (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eWe next examined the effects of various inhibitors of cell death pathways other than ferroptosis to rule out the possibility that apomorphine protects cells via other cell death pathways using GSK-872 (necroptosis inhibitor) or Z-VAD-FMK (apoptosis inhibitor). Although we could not examine the effects of these inhibitors in response to BSO-induced stress in control fibroblasts, BSO did not induce cell death in control fibroblasts, and neither GSK-872 nor Z-VAD-FMK showed any cell-protective effects against cell death induced by BSO and RSL3 (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA, B).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThese findings suggest that apomorphine protects against cell death by ferroptosis, but not by necroptosis or apoptosis, in control fibroblasts and fibroblasts derived from patients with mitochondrial disease.\u003c/p\u003e \u003cp\u003e \u003cb\u003eMeasurement of intracellular ferroptotic metabolites in our cell lines by a BSO cell lipid oxidation assay.\u003c/b\u003e Because the accumulation of lipid ROS is a hallmark of ferroptosis, we monitored the lipid ROS accumulation using C11 BODIPY 581/591 by confocal imaging (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). The C11 BODIPY 581/591 changes the fluorescence from red to green upon oxidation\u003csup\u003e\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u003c/sup\u003e. Even in the absence of BSO, LS patient-derived fibroblasts showed the oxidized form of C11 BODIPY, which is indicative of lipid ROS (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA_b). Upon BSO treatment, fibroblasts showed an increased lipid ROS accumulation (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA_c). Furthermore, Fer-1, one of the most common inhibitors of ferroptosis, recovered the lipid ROS accumulation. When we examined the effect of apomorphine on the accumulation of lipid ROS in LS patient-derived fibroblasts treated with BSO, apomorphine decreased the lipid ROS accumulation and showed a similar effect to Fer-1 (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA_d,e)\u003csup\u003e\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003e. In contrast, control fibroblasts showed almost no change in the degree of the lipid ROS accumulation (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB_a-o).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eThe assessment of the expression of genes related to ferroptosis in apomorphine-treated vs. untreated LS fibroblasts by real-time reverse transcription polymerase chain reaction (RT-PCR) and Western blotting.\u003c/b\u003e We assessed the expression of \u003cem\u003eprostaglandin endoperoxide synthase 2 (PTGS2)\u003c/em\u003e, a biomarker of ferroptosis that encodes cyclooxygenase-2 (COX-2)\u003csup\u003e\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e,\u003cspan additionalcitationids=\"CR25\" citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e\u003c/sup\u003e. Real-time RT-PCR showed that RSL3 upregulated \u003cem\u003ePTGS2\u003c/em\u003e expression in LS fibroblasts and that this \u003cem\u003ePTGS2\u003c/em\u003e upregulation was inhibited by apomorphine and Fer-1 treatment (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA). Western blotting for PTGS2 showed that apomorphine significantly decreased the protein levels of PTGS2 in LS fibroblasts, and Fer-1 had a similar effect (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB). We therefore demonstrated that apomorphine suppressed PTGS2, a key factor linking ferroptosis and inflammation, in LS fibroblasts.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eThe analysis of the expression of genes related to ferroptosis in apomorphine-treated vs. untreated LS fibroblasts by real-time RT-PCR.\u003c/b\u003e To examine how apomorphine inhibits ferroptosis, we assessed the following key genes related to ferroptosis: \u003cem\u003eAIFM2\u003c/em\u003e (encoding FSP1), \u003cem\u003eGPX4, ACSL4\u003c/em\u003e, and \u003cem\u003eSLC7A11\u003c/em\u003e (cystine/glutamate transporter)\u003csup\u003e\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e,\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e,\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e,\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e\u003c/sup\u003e. Real-time RT-PCR showed that apomorphine slightly suppressed the upregulation of \u003cem\u003eSLC7A11\u003c/em\u003e; however, the differences were not statistically significant. Apomorphine also did not affect the mRNA expression of \u003cem\u003eAIFM2, GPX4\u003c/em\u003e, or \u003cem\u003eACSL4\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA-D).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eThe anti-ferrotosis effect of apomorphine is not related to the dopamine receptor agonist action of apomorphine.\u003c/b\u003e Our previous screening did not show the cell protective effects of other dopamine agonists (Ropinirole hydrochloride and Pramipexole dihydrochloride; both specific to D2 receptors) (Supplemental Fig.\u0026nbsp;2). Therefore, to evaluate whether or not the agonistic action of apomorphine was related to its cell-protective effect, we examined the effect of the addition of agonists and/or antagonists of each dopamine receptor on the cell-protective effect of apomorphine.\u003c/p\u003e \u003cp\u003ePrior to this, the expression of each dopamine receptor in the cells was examined by RT-PCR. The expression of four types of dopamine receptors\u0026mdash;D1R, D2R, D4R, and D5R\u0026mdash;was confirmed in LS and control fibroblasts, but the expression of D3R was not detected in our fibroblasts (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eA). Therefore, we examined the action of each dopamine receptor on the protection of apomorphine, except for D3R.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eFirst, we examined the contribution of D4R to the effects of apomorphine using agonists and antagonists of D4R, based on a previous report \u003csup\u003e\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u003c/sup\u003e. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eB, the D4R agonists did not enhance the effect of apomorphine, and the antagonists did not inhibit this effect. Similarly, antagonists of D1R and D2R did not affect the effect of apomorphine (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eC, D). Regarding D5R, as there was no commercially available antagonist for D5R, only the D5R agonist was used, but it did not provide any cell-protective effect (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eE).\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eIn the present study, we showed that fibroblasts from mitochondrial diseases were vulnerable to ferroptosis, and apomorphine markedly protected fibroblasts from ferroptosis induced by RSL3 in various types of mitochondrial diseases as well as in normal fibroblasts. The effect of ferroptosis on cell protection was comparable to that of the specific ferroptosis inhibitors Fer-1 and Lip-1. We observed that BSO- or RSL3-induced ferroptosis was not inhibited by inhibitors of apoptosis (Z-VAD-FMK) or necroptosis (GSK-872), and \u003cem\u003ePTGS2\u003c/em\u003e, which is known to be a marker of ferroptosis, was decreased by the addition of apomorphine. In addition, we confirmed that the effects of apomorphine were not related to its agonistic dopamine action.\u003c/p\u003e \u003cp\u003eFerroptosis is an iron-dependent form of cell death caused by the accumulation of lipid hydroperoxides and is distinguished from other forms of cell death, such as apoptosis and necroptosis\u003csup\u003e\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e,\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003e. Ferroptosis occurs when GPX4 is directly or indirectly inhibited by GSH depletion, which leads to the accumulation of membrane lipid peroxidation and results in cell death\u003csup\u003e\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e,\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e,\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003e. RSL3 is commonly used as a specific inducer of ferroptosis and inactivates GPX4, leading to excessive lipid peroxidation, which causes cell death\u003csup\u003e\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003e. In contrast, ferroptosis can be suppressed by lipophilic antioxidants, inhibitors of lipid peroxidation, iron chelators, and polyunsaturated fatty acids\u003csup\u003e\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003e. Currently, several biomarkers of ferroptosis exist, including protein markers such as PTGS2 (which encodes COX-2), lipid peroxidation, and ROS\u003csup\u003e\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e,\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e,\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e,\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eWe have already shown that apomorphine suppresses the production of ROS\u003csup\u003e\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u003c/sup\u003e. In the present study, we showed that apomorphine prevented lipid peroxidation and upregulation of the mRNA expression of \u003cem\u003ePTGS2\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e), which are key features of ferroptosis. Studies on cancer cells have indicated that ferroptosis can directly increase the expression of \u003cem\u003ePTGS2\u003c/em\u003e and cause inflammation by accelerating AA metabolism and promoting the secretion of pro-inflammatory molecules\u003csup\u003e\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e,\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e\u003c/sup\u003e. The downregulation of \u003cem\u003ePTGS2\u003c/em\u003e may be related to the inhibition of various cytokines and chemokines by apomorphine\u003csup\u003e\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u003c/sup\u003e. Recently, ferroptosis has been suggested to be involved in epilepsy with mitochondrial disease\u003csup\u003e\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/sup\u003e. Our study also supports the hypothesis that fibroblasts from various mitochondrial diseases are vulnerable to ferroptosis.\u003c/p\u003e \u003cp\u003eApomorphine reportedly protects against glutamate-induced oxidative cell death via dopamine receptors, especially D4, in the HT22 cell line\u003csup\u003e\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u003c/sup\u003e. In this assay system, the protective effect was reversed by D4 antagonists but not by D1, D2, or D3 antagonists. A selective D4 agonist also protects neurons from glutamate-induced cell death. However, in our assay system using fibroblasts, the D4 agonist did not exert cell protective effects, and the D4 antagonist did not prevent the cell-protective effect of apomorphine. Furthermore, our data do not support the involvement of subtypes D1, D2, and D5. Therefore, we conclude that the cell-protective effect of apomorphine is not related to dopamine receptors in our system. Dopamine agonistic action causes several common side effects, especially digestive symptoms, such as nausea, vomiting, loss of appetite, and constipation\u003csup\u003e\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e\u003c/sup\u003e. Our research presents a promising avenue for the potential development of apomorphine derivatives that offer cellular protection without the undesirable effects associated with dopamine agonists.\u003c/p\u003e \u003cp\u003eThe limitation of the present study was that we were unable to identify the mechanism underlying the protection from ferroptosis, as we only performed an RT-PCR assay of key inhibitory genes for ferroptosis. Elucidation of the binding proteins that explain anti-ferroptosis and their downstream signaling cascades is the next research question that should be explored.\u003c/p\u003e \u003cp\u003eIn conclusion, we found that fibroblasts from patients with mitochondrial diseases were vulnerable to ferroptosis, which is inhibited by apomorphine in this study. The cell-protective effect of apomorphine has long been believed to be a result of D4R agonistic action, but we first revealed that at least part of its anti-ferroptosis effect is not related to the dopamine receptor agonist action. Our study suggests that ferroptosis may be a potential therapeutic target for mitochondrial disease and also provides hope for the creation of new drugs that maintain their cell-protective effect without the dopamine agonist effect, to avoid the adverse effects of dopamine.\u003c/p\u003e"},{"header":"Materials and methods","content":"\u003cp\u003e \u003cb\u003eSubjects.\u003c/b\u003e This study was approved by the Ethics Committee of Jichi Medical University (Approval Number: J21-014) and all methods of this study were performed in accordance with the relevant guidelines and regulations. Fibroblasts were obtained from six patients at Jichi Medical University, Kanagawa Children\u0026rsquo;s Medical Center, and Saitama Medical University. The collection was conducted under the approval of the Ethics Committee of each institution, with approval numbers J21-014, H2021-094, and 11303-06, respectively. Written informed consent was obtained from the parents of each patient. And we used fibroblasts from healthy individuals as controls (normal human dermal fibroblasts purchased from PromoCell Company [#C-12300; Heidelberg, Germany]). Our four patients were diagnosed with LS and MELAS, as previously described \u003csup\u003e\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u003c/sup\u003e. Two of the patients had genetically identified cases of LS, including one with an m.10158 T\u0026thinsp;\u0026gt;\u0026thinsp;C, p(S34P) mutation in \u003cem\u003eMT-ND3\u003c/em\u003e (Case 1; LS\u003csup\u003eND\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/sup\u003e) and one with a c.55 C\u0026thinsp;\u0026gt;\u0026thinsp;T, p(P19S) mutation in \u003cem\u003eNDUFA1\u003c/em\u003e (Case 2; LS\u003csup\u003eNDUFA\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003e) \u003csup\u003e\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e,\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e\u003c/sup\u003e. Both ND3 and NDUFA1 are subunits of Complex I in the mitochondrial respiratory chain. Fibroblasts were also obtained from two patients with MELAS, including one with an m.3243 A\u0026thinsp;\u0026gt;\u0026thinsp;G mutation in tRNA-Leu (Case 3; MELAS\u003csup\u003etRNA\u0026minus;Leu\u003c/sup\u003e) and one with an m.5541 C\u0026thinsp;\u0026gt;\u0026thinsp;T mutation in tRNA-Trp (Case 4; MELAS\u003csup\u003etRNA\u0026minus;Trp\u003c/sup\u003e) \u003csup\u003e\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e\u003c/sup\u003e. We additionally obtained fibroblasts from patients with two other types of mitochondrial diseases: mitochondrial cardiomyopathy (Case 5; MC\u003csup\u003eND\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u003c/sup\u003e) and Kearns-Sayer syndrome (KSS) (Case 6; KSS\u003csup\u003elarge deletion\u003c/sup\u003e). One patient with mitochondrial cardiomyopathy who presented with HCM and died at 4 months old had an m.13513G\u0026thinsp;\u0026gt;\u0026thinsp;A mutation in the ND5 subunit of complex I (\u003cem\u003eMT-ND5\u003c/em\u003e m.13513G\u0026thinsp;\u0026gt;\u0026thinsp;A), with 78.87% heteroplasmy. The patient with KSS was genetically identified as having a large deletion (m.8290\u0026ndash;13802) of mitochondrial DNA (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). Fibroblasts with fewer than 20 passages from patients and controls were used in the experiments. The heteroplasmic rate was analyzed by deep sequencing of mutated regions.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eFibroblast cell lines from patients with mitochondrial disease.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"7\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCase\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eCell ID\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eDisease\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eAge\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eGene mutation\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003eProtein\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c7\"\u003e \u003cp\u003eMutation\u003c/p\u003e \u003cp\u003erate (%)\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eLS \u003csup\u003eND\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eLeigh\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003em.10158 T\u0026thinsp;\u0026gt;\u0026thinsp;C,\u003c/p\u003e \u003cp\u003ep.(S34P)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eND3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e90\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eLS \u003csup\u003eNDUFA\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eLeigh\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003ec.55 C\u0026thinsp;\u0026gt;\u0026thinsp;T,\u003c/p\u003e \u003cp\u003ep.(P19S)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eNDUFA1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eNuclear\u003c/p\u003e \u003cp\u003egene\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eMELAS \u003csup\u003etRNA\u0026minus;Leu\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eMELAS\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e14\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003em.3243 A\u0026thinsp;\u0026gt;\u0026thinsp;G\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e(tRNA-Leu)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e21\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eMELAS \u003csup\u003etRNA\u0026minus;Trp\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eMELAS\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e23\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003em.5541 C\u0026thinsp;\u0026gt;\u0026thinsp;T\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e(tRNA-Trp)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e49\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eMC \u003csup\u003eND\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003emitochondrial cardiomyopathy\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1M\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003em.13513 G\u0026thinsp;\u0026gt;\u0026thinsp;A\u003c/p\u003e \u003cp\u003ep.(D393N)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eND5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e79\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eKSS \u003csup\u003elarge deletion\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eKSS\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003em.8290-13802del\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e\u0026minus;\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e\u0026minus;\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003ePromo1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eControl\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u0026minus;\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e\u0026minus;\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e\u0026minus;\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003ctfoot\u003e \u003ctr\u003e\u003ctd colspan=\"7\"\u003eLS: Leigh syndrome, MELAS: myopathy encephalopathy, lactic acidosis, and stroke-like episodes. Respiratory chain activities from fibroblasts: LS\u003csup\u003eND\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/sup\u003e - Complex Ⅰ 9.8%, II 93.9%, III 94.5%, IV 47.6%, and CS 100.9%. LS\u003csup\u003eNDUFA\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003e - Complex Ⅰ 27.6%, Ⅱ 104.3%, Ⅲ 70.1%, Ⅳ 77.7%, CS 77.4%. MELAS\u003csup\u003etRNA\u0026minus;Leu\u003c/sup\u003e - Not available. MELAS\u003csup\u003etRNA\u0026minus;Trp\u003c/sup\u003e - Complex I 48%, II 103%, III 65%, IV 18%, MC \u003csup\u003eND\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u003c/sup\u003e \u0026ndash; not available. KSS \u003csup\u003elarge deletion\u003c/sup\u003e \u0026ndash; not available.\u003c/td\u003e\u003c/tr\u003e \u003c/tfoot\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eCell culture and growth conditions\u003c/b\u003e. Fibroblasts from patients were cultured in 1.0 g/L low-glucose Dulbecco\u0026rsquo;s Modified Eagle\u0026rsquo;s medium (DMEM) supplemented with 10% fetal bovine serum (FBS), 100 units/mL penicillin, and 100 \u0026micro;g/mL streptomycin. Cells were incubated at 37\u0026deg;C in 5% CO\u003csub\u003e2\u003c/sub\u003e.\u003c/p\u003e \u003cp\u003e \u003cb\u003eReagents.\u003c/b\u003e l-Butionine (S, R)-sulfoximine (BSO, No. B690270), a glutathione synthesis inhibitor, was purchased from Wako Pure Chemical Industries (Tokyo, Japan). RSL3 (No. S8155) was purchased from Selleck Chemicals (Houston, TX, USA). Fer-1 (No. SML0583) and Lip-1 (No. SML1414) were obtained from Sigma-Aldrich (St. Louis, MO, USA). Z-VAD-FMK (No. 3188-v) and GSK-872 (No. HY-101872) were purchased from Peptide Institute Inc. (Osaka, Japan) and MedChemExpress (Shanghai, China), respectively. All reagents were dissolved in dimethyl sulfoxide (DMSO).\u003c/p\u003e \u003cp\u003e \u003cb\u003eCell viability assays.\u003c/b\u003e The BSO assay was performed as previously described \u003csup\u003e\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u003c/sup\u003e. We performed several cell viability assays to investigate ferroptosis. Ferroptotic cell death was induced in fibroblasts by ferroptosis inducers (RSL3 at 50\u0026ndash;100 nM). In these cell viability experiments, fibroblasts were cultured to semi-confluence and plated at 5,000 cells per well in a 96-well culture plate in normal medium. After incubation for 24 h, apomorphine and several compounds were added as positive controls (Fer-1, or Lip-1) and cultured in the assay medium. All compounds were applied at a concentration of 1 \u0026micro;M unless otherwise indicated. After incubating the cell plates for 24 h at 37\u0026deg;C (95% humidity and 5% CO\u003csub\u003e2\u003c/sub\u003e), a cell viability assay was performed using Cell Count Reagent SF (Nacalai Tesque, Kyoto, Japan). Fluorescence intensity was measured using a Benchmark Plus microplate reader (Bio-Rad, Hercules, CA, USA) according to the manufacturer\u0026rsquo;s instructions.\u003c/p\u003e \u003cp\u003e \u003cb\u003eThe assessment of lipid peroxidation.\u003c/b\u003e Lipid peroxidation was examined using the fluorescent dye C11-BODIPY581/591 (No. D3861; Thermo Fisher Scientific, Waltham, MA, USA)\u003csup\u003e\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u003c/sup\u003e. Fibroblasts were cultured to semi-confluence and then plated at 7,000 cells per well in a 96-well culture plate in normal medium. After incubation for 24 h, BSO and several other compounds (e.g. apomorphine and Fer-1) were added to each well. After incubation for 24 h, the cells were labeled with 5 \u0026micro;M C11-BODIPY581/591 for 30 min in the assay medium. The nuclei were stained with Hoechst 33342. Representative images were obtained using a Keyence All-in-One Fluorescence BZ-X810 microscope (Keyence Co., Itasca, IL, USA).\u003c/p\u003e \u003cp\u003e \u003cb\u003eRNA extraction and real-time RT-PCR.\u003c/b\u003e Total RNA was extracted from fibroblasts using an RNeasy\u0026reg; Mini Kit (QIAGEN, Valencia, CA, USA) according to the manufacturer\u0026rsquo;s instructions \u003csup\u003e\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e\u003c/sup\u003e. Total RNA was reverse transcribed to cDNA, followed by amplification by PCR with a Superscript VILO cDNA synthesis kit at 60\u0026deg;C (Invitrogen; Thermo Fisher Scientific). Real-time RT-PCR was performed using the SYBR Green system with a primer set that amplified a fragment of the target genes to measure the mRNA expression. The following primers were used: \u003cem\u003ePTGS2\u003c/em\u003e (Forward primer 5\u0026rsquo;-GCCTGAATGTGCCATAA-GACTGAC-3\u0026rsquo;, Reverse primer 5\u0026rsquo;-AAACCCACAGTG-CTTGACACACA \u0026minus;\u0026thinsp;3\u0026rsquo;), \u003cem\u003eAIFM2\u003c/em\u003e (Forward primer 5\u0026rsquo;-ATGGTTCGGCTGACCAAGAG-3\u0026rsquo;, Reverse primer 5\u0026rsquo;-GCCACCACATCATTGGCATC-3\u0026rsquo;), \u003cem\u003eGPX4\u003c/em\u003e (Forward primer 5\u0026rsquo;-GCCTTCCCGTGTAACCAGT-3\u0026rsquo;, Reverse primer 5\u0026rsquo;-GCGAACTCTTTGATCT-CTTCG-3\u0026rsquo;), \u003cem\u003eACSL4\u003c/em\u003e (Forward primer 5\u0026rsquo;-CCCTGAAGGATTTGAGATTCACA-3\u0026rsquo;, Reverse primer 5\u0026rsquo;-CCTTAGGT-CGGCCAGTAGAAC-3\u0026rsquo;), \u003cem\u003eSLC7A11\u003c/em\u003e (Forward primer 5\u0026rsquo;-ATGCAGTGGCAGTGA-CCTTT-3\u0026rsquo;, Reverse primer 5\u0026rsquo;-GGCAACAAAGATCG-GAACTG-3\u0026rsquo;), and \u003cem\u003eGAPDH\u003c/em\u003e (Forward primer 5\u0026rsquo;-CTTTGTCAAGCTCATTTCCTGG \u0026minus;\u0026thinsp;3\u0026rsquo;, Reverse primer 5\u0026rsquo;-TCTTC-CTCTTGTGCTCTTGC \u0026minus;\u0026thinsp;3\u0026rsquo;). The reactions were performed in triplicate. Gene expression was normalized to that of \u003cem\u003eGAPDH\u003c/em\u003e, and the data were analyzed in the Excel software program (Microsoft, Redmond, WA, USA) using the ΔΔCt method.\u003c/p\u003e \u003cp\u003e \u003cb\u003eWestern blotting.\u003c/b\u003e For whole-cell extracts, cells were lysed in TNE buffer (20 mM Tris-HCL at pH 7.4, 150 mM NaCl, 1 mM EDTA at pH7.4). Protein concentrations were determined using the Qubit\u0026reg; Protein Assay Kit (Invitrogen; Thermo Fisher Scientific). Whole-cell lysates were mixed with an equal volume of 2\u0026times; sodium dodecyl sulfate (SDS) sample buffer and boiled. Western blotting was performed using 20 \u0026micro;g of total protein, and immunoprecipitation was performed using XV PANTERA GEL (NXV-361HP; DRC Co., Ltd, Tokyo, Japan). Total cell proteins were separated by 10% SDS-polyacrylamide gel electrophoresis (PAGE) and transferred to a PVDF membrane by electrotransfer. The membrane was blocked for 1 h at room temperature (RT) using 5% skimmed milk/phosphate-buffered saline with Tween (PBST) and then incubated with the following primary antibodies: rabbit monoclonal anti-PTGS2 (Cox2(D5H5)XP #12282; Cell Signaling, Danvers, MA, USA) at 1:1000 and mouse monoclonal anti-beta-actin (A1978; Sigma-Aldrich) at 1:5000 in PBST overnight at 4\u0026deg;C. After washing with PBST 3 times for 10 min each, the membrane was incubated with the following secondary antibodies: anti-rabbit IgG horseradish peroxidase (HRP; Cell Signaling) at 1:3000 and anti-mouse IgG HRP (Santa Cruz Biotechnology, Santa Cruz, CA, USA) at 1:2000 for 1 h at RT. After washing with PBST 3 times for 15 min each, the membrane was incubated with Hyper HRP Substrate (TAKARA BIO INC., Ohtsu, Japan) for 2 min. Finally, chemiluminescence from the membrane was imaged using Amersham Imager 680 (GE Healthcare UK Ltd., Little Chalfont, UK). Protein intensities were measured using the ImageJ software program. Relative protein levels or protein abundances were normalized to those in the control group.\u003c/p\u003e \u003cp\u003e \u003cb\u003eDetection of dopamine gene receptor (DRD1-DRD5) expression on control and patient-derived fibroblasts by RT-PCR.\u003c/b\u003e RNA was extracted from isolated control and LS patient fibroblasts (LS\u003csup\u003eND\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/sup\u003e) using an RNeasy Mini Kit (QIAGEN) according to the manufacturer\u0026rsquo;s protocol. Commercial human Adult Normal Tissue: Brain: Frontal Lobe (BioChain, Newark, CA, USA) was used as the Positive Control. Total RNA (1500\u0026ndash;2000 ng) was reverse-transcribed into first-strand cDNA using the Superscript VILO cDNA Synthesis kit (Invitrogen; Thermo Fisher Scientific). RT-PCR was performed using intron-spanning primers (Supplemental Table\u0026nbsp;1). The D1R/D2R/D5R cycle conditions were as follows: 94\u0026deg;C for 2 min for polymerase activation, followed by 45 cycles at 98\u0026deg;C for 10 s, 62\u0026deg;C for 30 s, 68\u0026deg;C for 1 min. The D3R cycle conditions were as follows: 94\u0026deg;C for 2 min for polymerase activation, followed by 45 cycles of 98\u0026deg;C for 10 s, 62\u0026deg;C for 15 s, 72\u0026deg;C for 1 min. The D4R cycle conditions were as follows: 94\u0026deg;C for 2 min for polymerase activation, followed by 5 cycles of 98\u0026deg;C for 10 s, 68\u0026deg;C for 1 min, followed by 5 cycles of 98\u0026deg;C for 10 s, 66\u0026deg;C for 1 min, followed by 5 cycles of 98\u0026deg;C for 10 s, 64\u0026deg;C for 1 min, followed by 25 cycles of 98\u0026deg;C for 10 s, 62\u0026deg;C for 1 min, 68\u0026deg;C for 7 min. The products were resolved on a 2% agarose gel containing ethidium bromide and photographed.\u003c/p\u003e \u003cp\u003e \u003cb\u003eInteractions between apomorphine and dopamine receptor agonists or antagonists: Effects on cell viability.\u003c/b\u003e Cell viability assays to examine the influence of dopamine receptor agonists or antagonists on the effects of apomorphine were performed under RSL-induced oxidative stress. SKF 83566 cells (no. HY-103430A; MedChemExpress) was used as the D1R antagonist \u003csup\u003e\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e\u003c/sup\u003e, sulpiride (No. S4655; Selleck Chemicals) as the D2R antagonist \u003csup\u003e\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e\u003c/sup\u003e, PD168077 (No. HY-21098A; MedChemExpress) as the D4R agonist \u003csup\u003e\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e\u003c/sup\u003e; L745870 (No. HY-14325; MedChemExpress) as the D4R antagonist \u003csup\u003e\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e\u003c/sup\u003e, and SKF38393 (No. S7993; Selleck Chemicals) as the D5R agonist \u003csup\u003e\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e\u003c/sup\u003e. The concentrations used were determined based on the EC50 and Ki values \u003csup\u003e35 36 37 38 39\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eA cell viability assay was performed as previously described \u003csup\u003e\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u003c/sup\u003e. In brief, fibroblasts were cultured in 1.0 g/L low-glucose DMEM with 10% FBS at 37\u0026deg;C in 5% CO\u003csub\u003e2\u003c/sub\u003e until they reached semi-confluence. Fibroblasts were seeded at 5000 cells per well in a 96-well plate. After 24 h of incubation, we divided the cells into 3 groups: a DR agonist- or antagonist-treated group, a DR agonist- or antagonist-treated group at a concentration 10 times the EC50 or Ki values, and a DR agonist- or antagonist-treated group at a concentration 100 times the EC50 or Ki values. Apomorphine was added at a final concentration of 1 \u0026micro;M. After 24 h of incubation, the cell survival rate was checked using Cell Count Reagent SF (Nacalai Tesque). The cell viability of Apomorphin-treated group without RSL3 was used as 100% to compare other group\u0026rsquo;s cell viabilities.\u003c/p\u003e \u003cp\u003e \u003cb\u003eStatistical analyses\u003c/b\u003e. The results are expressed as the mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation. Comparisons between multiple-group means were performed using a one-way analysis of variance with Bonferroni\u0026rsquo;s post-hoc test. Statistical significance was set at P\u0026thinsp;\u0026lt;\u0026thinsp;0.05. Statistical analyses were performed using the GraphPad Prism software program (GraphPad Software Inc., La Jolla, CA, USA).\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eData availability.\u003c/strong\u003e The datasets and raw data used and/or analyzed during the current study are available from the corresponding author on reasonable request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgements\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe thank the patients and their families as well as all of the staff working, especially Miyuki Watanabe, Tomomi Oyama, and Narumi Omika at Jichi Children Medical Center Tochigi and Jichi Medical University Hospital.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor contributions\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eA.M., T.Y., and H.O. developed the strategy for the research; methodology, C.W., E.F.J., and N.Y.; performed the research, A.M., A.N., M.K., and N.O.; analyzed the data, A.M. and S.A.; resources, N.Y., Y.K., A.O., M.T.; writing\u0026mdash;original draft preparation, A.M.; writing\u0026mdash;review and editing, N.O. and H.O..\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis research was supported by a grant from the Project for Health Research on Infants, Children, Adolescents, and Young Adults from the Agency of Medical Research and Development, and a grant from the Japan Agency for Medical Research and Development (grant number im0210625h0001, 17ek0109270s0301, 22ek0109511h0002) to H.O. and JSPS KAKENHI to H.O. (JP21H03648).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no competing interests regarding the content of this article.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAdditional information\u003c/strong\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eSupplementary informations of this article can be found online.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCorrespondence\u003c/strong\u003e and requests for materials should be addressed to A.M. or H.O..\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eHsieh, G. 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Dopamine receptor pharmacology. \u003cem\u003eTrends Pharmacol Sci\u003c/em\u003e \u003cstrong\u003e15\u003c/strong\u003e, 264-270, doi:10.1016/0165-6147(94)90323-9 (1994).\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"","lastPublishedDoi":"10.21203/rs.3.rs-3361719/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-3361719/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eOriginally, apomorphine was a broad-spectrum dopamine agonist with an affinity for all subtypes of the Dopamine D1 receptor to the D5 receptor. We previously identified apomorphine as a potential therapeutic agent for mitochondrial diseases by screening a chemical library of fibroblasts from patients with mitochondrial diseases. In this study, we showed that apomorphine prevented ferroptosis in fibroblasts from various types of mitochondrial diseases as well as in normal controls. Well-known biomarkers of ferroptosis include protein markers such as prostaglandin endoperoxide synthase 2 (PTGS2), a key gene for ferroptosis-related inflammation PTGS2, lipid peroxidation, and reactive oxygen species. Our findings that apomorphine induced significant downregulation of \u003cem\u003ePtgs2\u003c/em\u003e, and suppressed lipid peroxide to the same extent as other inhibitors of ferroptosis also indicate that apomorphine suppresses ferroptosis. To our knowledge, this is the first study to report that the anti-ferroptosis effect of apomorphine is not related to dopamine receptor agonist action and that apomorphine is a potent inhibitor of ferroptotic cell death independent of dopaminergic receptors.\u003c/p\u003e","manuscriptTitle":"Apomorphine is a potent inhibitor of ferroptosis independent of dopaminergic receptors","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2023-10-03 18:08:17","doi":"10.21203/rs.3.rs-3361719/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2024-01-03T08:43:50+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2023-10-31T16:37:25+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2023-10-14T22:00:40+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"01906579-c15e-44a8-a157-7660801621f8","date":"2023-10-14T02:12:20+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"70607a60-fd4a-4bd5-aa37-6fdb890891a1","date":"2023-10-13T15:09:41+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2023-09-26T21:07:06+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2023-09-26T20:49:51+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"","date":"2023-09-26T05:18:23+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2023-09-26T05:13:14+00:00","index":"","fulltext":""},{"type":"submitted","content":"Scientific Reports","date":"2023-09-16T16:29:22+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"b382056b-c851-46ed-970e-c26ca87e6c25","owner":[],"postedDate":"October 3rd, 2023","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[{"id":25137493,"name":"Health sciences/Medical research/Drug development"},{"id":25137494,"name":"Biological sciences/Drug discovery/Pharmacology/Clinical pharmacology"},{"id":25137495,"name":"Biological sciences/Drug discovery/Drug delivery"},{"id":25137496,"name":"Biological sciences/Drug discovery/Pharmaceutics"}],"tags":[],"updatedAt":"2024-03-04T15:06:11+00:00","versionOfRecord":{"articleIdentity":"rs-3361719","link":"https://doi.org/10.1038/s41598-024-55293-1","journal":{"identity":"scientific-reports","isVorOnly":false,"title":"Scientific Reports"},"publishedOn":"2024-02-27 15:01:19","publishedOnDateReadable":"February 27th, 2024"},"versionCreatedAt":"2023-10-03 18:08:17","video":"","vorDoi":"10.1038/s41598-024-55293-1","vorDoiUrl":"https://doi.org/10.1038/s41598-024-55293-1","workflowStages":[]},"version":"v1","identity":"rs-3361719","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-3361719","identity":"rs-3361719","version":["v1"]},"buildId":"rHA-KDH7Qsr4HCuvH75dn","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}