Mito-apocynin protects against kainic acid-induced excitotoxicity by ameliorating mitochondrial impairment

Mito-apocynin protects against kainic acid-induced excitotoxicity by ameliorating mitochondrial impairment | 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 Research Article Mito-apocynin protects against kainic acid-induced excitotoxicity by ameliorating mitochondrial impairment Miaomiao Lin, Xiaorui Wan, Huanchen Wu, Na Liu, Yiyue Jiang, Yichao Sheng, and 5 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4537012/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 17 Mar, 2025 Read the published version in Molecular Neurobiology → Version 1 posted 11 You are reading this latest preprint version Abstract Neurodegenerative diseases are major global health problems with increasing incidence rates. A large amount of data suggests that excitotoxicity is a potential target of neurodegenerative diseases. However, effective pharmacological interventions against excitotoxicity are lacking. We aimed to elucidate the neuroprotective effect and mechanism of the mitochondrion-targeted NOX inhibitor mito-apocynin on kainic acid (KA)-induced excitotoxicity. We found that KA impaired mitochondrial morphology and led to impaired mitochondrial energy metabolism and dysfunction. In Western blotting experiments, KA disrupted mitochondrial quality control. In Nissl staining and CCK8 experiments, Mito-apocynin attenuated the death of neurons due to excitotoxic damage induced by KA both in vivo and in vitro . Mito-apocynin ameliorated neurobehavior induced by KA deficits in vivo and mitochondrial dysfunction in vitro . Mito-apocynin significantly reversed the increase in NOX4 levels caused by KA in the mitochondria of the striatum, decreased phosphorylated DRP1 (Ser616)/total DRP1 and increased PGC-1α, PINK1 and Parkin protein expression in the total striatum. In summary, Mito-apocynin alleviated oxidative stress, maintained normal mitochondrial function and energy metabolism levels, and promoted the balance of mitochondrial quality control by regulating the expression of NOX in mitochondria, thus reducing KA-induced excitatory toxic damage. Mito-apocynin KA Excitotoxicity Mitochondrial dysfunction NADPH oxidase Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Introduction Neurodegenerative diseases are major global health problems with increasing incidence rates [ 1 , 2 ]. Ischemic stroke is thought to be caused by excitotoxicity induced by glutamate receptor overactivation [ 3 , 4 ]. A large amount of data suggests that excitotoxicity is a potential target of neurodegenerative diseases [ 5 , 6 ]. Dysregulation of neuronal calcium homeostasis, stress caused by oxidants and dysfunction of the mitochondria result from glutamate release or decreased uptake [ 6 – 9 ]. Excitotoxicity is a hallmark of cellular responses to stress that occur in hypoxic/ischemic injury and diseases associated with neurodegeneration [ 10 , 11 ]. However, effective pharmacological interventions against excitotoxicity are lacking. Therefore, the molecular mechanism of neuronal damage caused by excitotoxicity urgently needs to be studied to find new ideas for preventing and treating neurodegenerative diseases [ 12 ]. Our previous studies have established that kainic acid (KA) receptors are predominantly distributed in the striatal region. We successfully modeled neuroexcitatory damage by KA injection in the right striatum of the mouse brain, with no effect on the left striatum or cerebral cortex. Supplementation with NADPH protected neurons from KA-induced excitotoxic injury [ 13 , 14 ]. However, NADPH has a dual role. In addition to acting as an antioxidant, it is a substrate for NADPH oxidase (NOX), a chemical that generates oxygen radicals (ROS) by oxidizing NADPH [ 15 , 16 ]. There are three primary NOX isoforms expressed in the central nervous system: NOX1, NOX2 and NOX4[ 17 ]. We found that after exposure to excitotoxins, NOX4 expression was significantly increased[ 14 ]. The production of NOX is closely related to ROS, making it a promising target as a new treatment for neurodegenerative diseases [ 1 , 17 , 18 ]. Various diseases associated with neurodegeneration lead to increased NOX activity and expression [ 19 ]. NOX-mediated superoxide generation is a necessary mechanism of excitotoxic death resulting from NMDA receptor activation [ 20 ]. ROS are mainly produced by NOX and mitochondria [ 21 ]. NOX-generated ROS can damage mitochondria [ 22 ]. In some cases, ROS produced by mitochondria may also stimulate NOX [ 23 ]. Therefore, mitochondrial-NOX crosstalk may result in an overproduction of ROS, which may be a new pharmacological target [ 24 , 25 ]. Recently, researchers have found that NOX4 is also localized in mitochondria and are increasingly interested in the interaction between NOX4 and mitochondria [ 26 ]. Previously, ROS were believed to come mainly from the mitochondrial respiratory chain in cell [ 27 ]. According to the literature, ROS may be produced in the mitochondrial respiratory chain rather than by the mitochondria [ 26 ]. Mitochondrial NOX4 is one of the main sources of mitochondrial superoxide [ 28 , 29 ]. NOX4 regulates mitochondrial energy metabolism, biogenesis and mitochondrial DNA repair in cardiovascular diseases [ 30 ]. Therefore, it is important to explore the changes in mitochondrial NOX levels during excitotoxicity and the role of mitochondrial NOX in excitotoxicity. The purpose of this study was to determine whether the mitochondrion-targeted NOX inhibitor mitoapocynin protects against KA-induced excitotoxicity and assess its effects on excitotoxicity-induced mitochondrial damage. Materials and Methods Animals and Drug Administration SPF-grade Institute of Cancer Research (ICR) male mice, 25–30 g, were purchased from ZhaoYan (Suzhou) Company. SPF animal centers maintained constant temperatures of 22°C and a humidity of 50%-60% for the rearing of mice. Institutional regulations on animal health apply to the use of these animals. In all cases, Soochow University's Institutional Animal Care and Use Committee approved the protocols. Stereotaxic apparatuses were used to fix the mice after chloral hydrate anesthesia (400 mg/kg). The right striatum was injected with 0.625 nmol of KA (Sigma Aldrich, K0250). Coordinates of the right striatum: 0.8 mm anterior to the bregma, 1.8 mm lateral to the sagittal suture, and 3.5 mm ventral to the pial surface. The injection volume was 1 µl, and the injection speed was 0.5 µl/min. Mito-apocynin (HY-135869, MCE, ig) was preadministered one day before KA injection [ 13 , 14 ]. Isolation of tissue mitochondrial and cytoplasmic fractions After the mice were sacrificed, the striatum was isolated. A 10-fold volume of Mitochondrial Isolation Reagent A solution (Beyotime Biotechnology, C3606) with 1% PMSF (Beyotime Biotechnology, ST505) was added and homogenized 30 times. Five minutes of centrifugation at 600 ×g was performed on the tissue homogenate. The supernatant was collected for the isolation of mitochondria. In the supernatant, 10 minutes of centrifugation were carried out at 11,000 ×g. This supernatant was aspirated as a cytoplasmic fraction. The precipitate was resuspended by adding 100 µl of Mitochondrial Isolation Reagent A solution. Afterward, it was centrifuged for ten minutes at 11,000 ×g. The resuspension was transferred to an EP tube containing 22%/55% Percoll and centrifuged at 20,000 ×g for 20 min. The white flocculent between the Percoll gradient was the fraction of mitochondria. Western blotting By our previous description, Western blotting was performed [ 31 ]. Antibody source: NOX1 antibody (Proteintech, 17772-1-AP); NOX2 antibody (Santa Cruz, sc-130543); NOX4 antibody (Abcam, ab109225); DRP1 antibody (Cell Signaling Technology, 5931S); DRP1 (phospho-ser616) antibody (SAB, 12749); PINK1 antibody (Absin, abs100425); Parkin antibody (Santa Cruz, sc-2282); PGC-1α antibody (Santa Cruz, sc-3067); GAPDH antibody (Abcam, ab8245); β-actin antibody (Sigma Aldrich, A5441); α-Tubulin antibody (Abcam, ab7291); VDAC antibody (Cell Signaling Technology, 12454). Transmission electron microscopy Isolation of the striatum after heart perfusion. Preparation of 2 mm × 2 mm ultrathin tissue sections. Store at 4°C in electron microscope fixative (Servicebio, G1102). Samples were double-stained using lead-uranium. The copper mesh containing the samples was placed in a transmission electron microscope for observation. Nissl staining Whole brains were isolated after heart perfusion and fixed in 4% paraformaldehyde. Sucrose was dehydrated, and coronal sections were prepared using a vibrating slicer. Nissl staining (Beyotime Biotechnology, C0117) was performed for 30 min. For 2 min, the sections were dehydrated and decolorized in 75%, 95%, and 100% ethanol. Xylene was used to permeabilize the slices for 10 minutes, followed by sealing with neutral resin. Neuronal morphology was observed under a microscope, and the number of normal neurons in the striatal center was counted at 20× magnification. Behavioral test Cylinder test: Mice were placed in a 10 cm diameter glass round cylinder. The number of unilateral and bilateral forelimb contacts with the cylinder wall was recorded within 3 min. The proportion of unilateral contact was counted. Adhesive removal test: The mice were acclimated to their new environment before starting the experiment. A 0.2-inch piece of tape was applied to the nose of the mice. The time needed to remove the tape from the forelimbs of the mice was recorded for no more than 1 min. Inverted grid test: A pretest was performed on the day of the experiment to acclimatize the mice to the test. The mice were placed in the center of a 15 cm 2 horizontal square grid. The grid was raised 20 cm from the table, and the grid was flipped up and down. The time that the mice remained on the grid was recorded for no more than 1 min. Primary neuron culture Separate the cerebral cortex of 18-day-old ICR mouse embryos. Add 2.5% trypsin and digest at 37°C for 15 min. After termination of digestion, add DNAase and gently blow 100 times. Centrifuge at 200 ×g for 5 min. Resuspend the cells by adding neuronal medium and filter the cells through a 40 µm cell sieve. Cell suspensions were diluted to 10 6 cells/ml and incubated at 37°C with 5% CO 2 . Measurement of ATP levels The experiment was performed according to the instructions provided with the ATP assay kit (Beyotime Biotechnology, S0026). The cells were collected, and 50 µl of ATP assay lysate was added. Five minutes at 12,000 ×g in a centrifuge. Collection of supernatants for ATP level determination. Place 100 µl of ATP assay working solution into each well in a black 96-well plate. This was allowed to sit at room temperature and was protected from light for 3–5 minutes. Add 20 µl of sample to the assay wells. RLU values were determined. The protein concentration was also measured by a BCA kit (Takara Bio, T9300A). The ATP level was expressed as nmol/mg. Cell viability Ninety-six-well plates were used to inoculate primary neurons. Pretreatment with Mito-apocynin for 4 h was followed by treatment with 100 µM KA for 8 h. To measure cell viability, we used a kit called CCK-8 (DOJINDO, CK04). The reaction was carried out at 37°C for 3 h. Microplate readers were used to measure absorbance at 450 nm. Measurement of mitochondrial membrane potential The membrane potential of the mitochondria was detected using JC-1 (Beyotime Biotechnology, C2006). Primary cortical neurons were inoculated in a 24-well plate. We pretreated neurons with Mito-apocynin for 4 h and then treated them with 100 µM KA for 8 h. The medium was aspirated, and the cells were washed twice in HBSS. Working solution for JC-1 staining should be added in 0.3 ml. The mixture was incubated for 20 min at 37°C in the dark. The cells were washed with JC-1 buffer 3 times. The cells were observed under a fluorescence microscope. Measurement of mitochondrial superoxide levels Mitochondrial superoxide levels were detected using MitoSOX Red mitochondrial superoxide indicator (Yeasen, 40778ES50). Primary cortical neurons were inoculated in 24-well plates. Neurons were pretreated with Mito-apocynin for 4 h, followed by treatment with 100 µM KA for 8 h. The medium was aspirated, and the cells were washed twice with HBSS. Add 0.3 ml MitoSOX Red working solution and incubate for 10 min at 37°C protected from light. The HBSS disinfection was repeated three times, and the cells were stained again with Hoechst's reagent in 0.3 ml. wash twice with HBSS. The cells were observed under a fluorescence microscope. Statistical analysis Statistical analysis and graphing of experimental data were performed using GraphPad Prism 8. The mean and standard error of the experimental data are expressed as the mean ± SEM. Analysis of the differences between the two data groups was performed using an unpaired t-test, one-way analysis of variance for multigroup data was performed using one-way ANOVA, two-way ANOVA was used for multivariate analysis of differences in multiple groups of data, statistical significance was determined by *P < 0.05, and no */# in the statistical graph indicates no significant difference. Results KA-induced excitotoxicity upregulated the expression level of NOX4 in mitochondria. Our previous studies showed that NADPH protects against KA-induced excitotoxic injury [ 13 ]. NADPH exerts neuroprotective effects as an antioxidant, but it is also utilized by NOX to generate ROS [ 31 ]. Therefore, the therapeutic window of NADPH is narrow. This dual effect of NADPH suggests that the role of NOX should be considered when studying the mechanism of excitotoxicity [ 32 ]. We previously found increased levels of NOX4 expression in KA-induced excitotoxicity [ 14 ]. Changes in the protein levels of NOX1 and NOX2 are not yet known. To investigate the involvement of the NOX family in excitotoxicity, KA, a glutamate analog, was stereotaxically injected into the right striatum of mice to create an excitotoxicity animal model. The control group was injected with an equal amount of normal saline in the same way. We examined indicators of NOX expression at high levels or low levels at different times after KA treatment. KA upregulated NOX4 and NOX2 expression but not NOX1 (Fig. 1 a-f). NOX4 has constitutive activity [ 33 ]. In cardiovascular disease, NOX4 was shown to be localized in cardiac mitochondria under pathological conditions and may be associated with ROS produced by mitochondrial respiration and NOX[ 28 ]. Furthermore, NOX4 has been reported to bind to mitochondrial respiratory chain complex I. As a result, NOX4 may play a role in the generation of ROS mediated by both NOX and the mitochondrial respiratory chain. When KA injections were administered, mitochondrial NOX4 expression was significantly increased, but cytoplasmic NOX4 was not affected (Fig. 2 a-c). NOX2 and NOX1 in mitochondria did not appear to be affected by KA (Fig. 2 d-h). However, KA upregulated the expression of NOX2 in the cytoplasm (Fig. 2 d-f). Given the critical role of mitochondrial NOX4 in KA-induced excitotoxic injury and its potential importance in regulating mitochondrial function, we subsequently paid special attention to mitochondrial NOX4 when investigating the protective role of Mito-apocynin. NOX4 and NOX2 are the major contributors to ROS in KA-induced excitotoxic injury. NOX4 may have functional regulatory implications for mitochondrial ROS formation. NOX4 has previously been identified as containing a potential mitochondrial localization signal. Localized mitochondrial NOX4 can bind to mitochondrial complex I, and this interaction may have relevance to the regulation of mitochondrial function. In the presence of a KA-induced increase in NOX4 expression, the level of NOX4 in mitochondria increases, which may contribute to the development of mitochondrial ROS or regulate the activity of complex I on the mitochondrial respiratory chain, implying that both energy metabolism and quality control in mitochondria are affected. KA-induced excitotoxicity impairs mitochondrial morphology and disturbs quality control systems. Overactivation of glutamate receptors damages mitochondria [ 34 ]. The cytoplasmic matrix of neurons in the KA-treated group became lighter, the mitochondria swelled, the mitochondrial matrix also became lighter, and the mitochondrial cristae appeared ruptured (Fig. 3 a). The mitochondrial quality control system is critical for maintaining functional mitochondria [ 35 ]. A rapid increase in neuronal mitochondrial biogenesis after hypoxic-ischemic brain injury is an endogenous neuroprotective response to hypoxic-ischemic injury [ 36 ]. PGC-1α is a key protein that regulates mitochondrial biogenesis [ 37 ]. The expression level of PGC1-1α was significantly increased after KA injection (Fig. 3 b-c). An organelle with a high degree of dynamic activity, mitochondria undergo continuous fission and fusion [ 38 ]. Healthy cells maintain equilibrium between fissions and fusions, thus maintaining mitochondrial homeostasis [ 39 ]. The decrease in the phosphorylated DRP1 (Ser616)/total DRP1 ratio indicated impaired mitochondrial dynamics (Fig. 3 d-e). When mild and transient stress induces mitochondrial autophagy, we call it ‘stress-induced mitophagy’ [ 40 ]. Autophagy-related stress is primarily induced by ROS [ 41 ]. PINK1 and Parkin synergistically promote autophagic clearance of damaged mitochondria. activation of mitochondrial autophagy [ 42 ]. Upregulation of PINK1 and Parkin indicated activation of mitochondrial autophagy (Fig. 3 f-i). Crosstalk between NOX and mitochondria-derived ROS with mutual feedback leads to an amplification of ROS in a vicious cycle, ultimately leading to mitochondrial dysfunction and neuronal damage [ 44 ]. However, current therapeutic approaches using ROS blockers are not always effective. Consequently, we need new therapeutic strategies for treating diseases resulting from oxidative stress, such as neurodegenerative diseases. Therefore, we focused on studying mito-apocynin, a mitochondrion-targeted NOX inhibitor, which may be an effective therapy against NOX and mitochondrial ROS crosstalk and help to investigate the role and mechanism of mitochondrial NOX in KA-mediated mitochondrial damage. Mito-apocynin protects striatal neurons from KA-induced excitotoxicity After KA injection, the opening of glutamate receptors leads to massive calcium inward flow, causing seizure-like symptoms and motor dysfunction in the affected trunk during the acute phase. Importantly, neuronal death does not occur immediately but is secondary to a series of cascading responses triggered by calcium signaling, a progressive neurodegenerative process. Therefore, we chose to observe neuronal death on day 14 after KA modeling to fully assess the long-term effects of the injury. To explore the potential role of NOX inhibitors in neuroprotection, we used Mito-apocynin, a mitochondria-targeted NOX inhibitor. The NOX inhibitor Apocynin binds to the cationic portion of the mitochondria-targeting moiety triphenylphosphonic acid to generate the mitochondrion-targeted NOX inhibitor Mito-apocynin [ 43 , 44 ]. The blood-brain barrier is highly lipophilic, so mito-apocynin can cross through to reach the striatum [ 45 ]. Mito-apocynin was preadministered to mice one day before KA injection. Mito-apocynin was then administered once a day, and brain tissue was isolated for Nissl staining after 14 days (Fig. 4 a). Exogenous supplementation with Mito-apocynin significantly ameliorated KA-induced neuronal crinkling and loss of Nisin vesicles (Fig. 4 b). Additionally, Mito-apocynin reduced the number of neuronal deaths in a dose-dependent manner (Fig. 4 c). Therefore, we chose the best protective effect of 75 µg/kg of Mito-apocynin for the follow-up study in vivo . Mito-apocynin ameliorates KA-induced motor behavioral deficits in mice Striatal injury leads to altered motor and muscle control in mice, mainly in the forelimbs [ 46 ]. In a previous study, we found that KA-induced excitotoxicity leads to motor dysfunction in mice [ 47 ]. Therefore, we performed the cylinder test, the adhesive removal test and the inverted grid test as indicators to evaluate striatal damage [ 48 ]. Healthy mice tend to use both limbs to touch the container wall, can remove adhesive labels in a short time and have high muscle tone in the limbs. KA treatment resulted in an increased proportion of mice touching the wall unilaterally, a longer time to remove adhesive labels and a shorter time to maintain them on the grid. Treatment with Mito-apocynin promoted recovery of motor function in mice (Fig. 5 a-d). Mito-apocynin ameliorates KA-induced cytotoxicity and mitochondrial dysfunction To explore whether Mito-apocynin also has a restoring effect on KA-induced mitochondrial damage in vitro , we examined the experiments shown in Fig. 6 a. In primary neurons, Mito-apocynin treatment reduced KA-induced neurotoxicity (Fig. 6 b). In excitotoxicity, mitochondrial membrane potential run-down is a key step in cell death [ 9 , 49 ]. The membrane potential of mitochondria was examined by staining with JC-1, and a decrease in mitochondrial membrane potential caused a decrease in JC-1 polymers (red fluorescence) and an increase in JC-1 monomers (green fluorescence) in the mitochondrial matrix, and fluorescence observations showed a significant decrease in the relative proportions of red and green fluorescence of KA-induced JC-1 (Fig. 6 d-e). Mitochondrial defects lead to insufficient ATP production (Fig. 6 c). Also produces too much superoxide [ 50 , 51 ]. Mitochondrial superoxide levels are detected using the MitoSOX Red Mitochondrial Superoxide Indicator, with higher fluorescence intensity indicating more superoxide production (Fig. 6 f-g). The above results suggest that Mito-apocynin treatment ameliorates KA-induced mitochondrial dysfunction in vitro. Mito-apocynin inhibits KA-induced upregulation of mitochondrial NOX4 and promotes restoration of the mitochondrial quality control system We speculate that mitochondrial NOX4 may be responsible for mitochondrial damage in excitotoxicity. Mito-apocynin is mitochondria-selective and inhibits KA-induced upregulation of mitochondrial NOX4 (Fig. 7 a-c). The protective effect of Mito-apocynin may be dependent on its inhibition of mitochondrial NOX4 expression, thereby reducing mitochondrial ROS production. Immunoblotting data showed that Mito-apocynin treatment reversed the KA-induced decrease in the phosphorylated DRP1 (Ser616)/total DRP1 ratio and significantly reversed KA-induced upregulation of PGC-1α, PINK1 and Parkin (Fig. 7 d-k). We conclude that the mitochondrion-targeted NOX inhibitor mito-apocynin may attenuate KA-mediated mitochondrial damage by inhibiting changes in mitochondrial NOX. Discussion Glutamate is one of the most common neurotransmitters in the mammalian central nervous system, and it mediates excitatory neurotransmission [ 52 ]. However, high levels of glutamatergic input can cause excitotoxicity, leading to neuronal cell death after acute brain injury, such as stroke or trauma [ 53 ]. An increasing amount of research has focused on understanding the mechanisms underlying excitotoxic injury and revealing potential therapeutic strategies targeting these mechanisms [ 54 , 55 ]. In the current study, we constructed an animal model of excitotoxicity by injecting the glutamate analog KA into the striatum. In the KA-mediated excitotoxicity animal models, upregulation of NOX4 and NOX2 was observed. Unlike NOX1 and NOX2, NOX4 activity is controlled by its expression level rather than cytosolic regulatory proteins [ 33 ]. NOX is also directly associated with mitochondria, and when NOX in mitochondria is activated, its interactions with electron transport chain elements may cause mitochondria to produce superoxide indirectly[ 56 ]. The expression of NOX4 is upregulated in mitochondrial fractions. NOX4 in mitochondria is thought to be a central mediator of reduced oxidative stress and may mediate an imbalance in mitochondrial function and cellular injury in a variety of diseases. Therefore, mitochondrial NOX4 is considered a major target for designing new therapeutic strategies to uncover effective therapies for various diseases characterized by oxidative stress, including neurodegenerative diseases[ 56 , 57 ]. Calcium overload is caused by excessive glutamate receptor activation, which leads to disruption of mitochondrial morphology and mitochondrial dysfunction. Exposure of mitochondria to high levels of KA leads to disturbances in mitochondrial dynamics and metabolism and mitochondrial autophagy. An important factor that determines how neurons respond to glutamate is their bioenergetic state. This suggests that to counter excitotoxicity, mitochondrial function must be targeted[ 34 ]. Although new therapies may target glutamate receptors or glutamate uptake, pharmacological strategies that promote mitochondrial function may also prove beneficial. The NOX family is a new class of pharmacological targets for the treatment of neurodegenerative diseases because of the roles of NOXs in oxidant production and their possible role in the regulation of neurons. Mito-apocynin has higher mitochondrial selectivity than other NOX inhibitors. Mito-apocynin, which can reach the striatum, was administered orally. Supplementation with Mito-apocynin attenuated excitotoxic damage. Here, we confirmed the protective effect of mito-apocynin against KA-induced excitotoxicity. However, apocynin is a nonspecific NOX inhibitor, and even though mito-apocynin was able to inhibit KA-induced upregulation of mitochondrial NOX4, it is still difficult to exclude its effect on other NOX isoforms in mitochondria. However, due to the lack of animal models that can be used to elucidate the subtype- and subcellular-specific function of NOX4 in vivo , the role of NOX4 in excitotoxicity-mediated mitochondrial injury and neurodegenerative diseases requires further in-depth study. In summary, in KA-induced excitotoxicity, mitochondrial NOX expression is upregulated, and this change is accompanied by mitochondrial dysfunction and impairment of the mitochondrial quality control system. Mito-apocynin, an exogenous mitochondrion-targeted NOX inhibitor, can exert neuroprotective effects by inhibiting mitochondrial NOX expression and ameliorating mitochondrial dysfunction and mitochondrial quality control system impairment, thereby reversing KA-induced mitochondrial damage (Fig. 8 ). Declarations Ethics Approval The study has been examined and certified by the Ethics Committee of Soochow University, and informed consent was obtained from all participants included in the study, in agreement with institutional guidelines. Consent for Publication All authors have approved for publication. Competing interests The authors declare no competing interests. Author Contribution MML and YW designed the study. XRW, HCW,NL, YYJ, YCS and JW assisted with the experiments. MML, YW, HDX, JX and ZHQ contributed to the drafting of the manuscript and figures. All authors have approved the final article. Acknowledgments This work was supported by the Natural Science Foundation of Jiangsu Province (No. BK20221360), the National Natural Science Foundation of China (No. 81671252, 81730092, 81773768) and the Priority Academic Program Development of the Jiangsu Higher Education Institutes (PAPD). Data Availability The authors declare the availability of data and material. References Hou L, Zhang L, Hong JS, Zhang D, Zhao J, Wang Q (2020) Nicotinamide Adenine Dinucleotide Phosphate Oxidase and Neurodegenerative Diseases: Mechanisms and Therapy. Antioxid Redox Signal 33(5):374–393. http://doi.rog/10.1089/ars.2019.8014 Hou Y, Dan X, Babbar M, Wei Y, Hasselbalch SG, Croteau DL, Bohr VA (2019) Ageing as a risk factor for neurodegenerative disease. 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Cite Share Download PDF Status: Published Journal Publication published 17 Mar, 2025 Read the published version in Molecular Neurobiology → Version 1 posted Editorial decision: Revision requested 01 Jan, 2025 Reviews received at journal 01 Jan, 2025 Reviewers agreed at journal 26 Dec, 2024 Reviewers agreed at journal 24 Dec, 2024 Reviews received at journal 01 Oct, 2024 Reviewers agreed at journal 09 Sep, 2024 Reviewers agreed at journal 08 Sep, 2024 Reviewers invited by journal 16 Jun, 2024 Editor assigned by journal 09 Jun, 2024 Submission checks completed at journal 09 Jun, 2024 First submitted to journal 05 Jun, 2024 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. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. 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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-4537012","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":317348338,"identity":"e1f34dd3-8d4f-4ba4-9cc6-07f95d3b799c","order_by":0,"name":"Miaomiao Lin","email":"","orcid":"","institution":"Soochow University","correspondingAuthor":false,"prefix":"","firstName":"Miaomiao","middleName":"","lastName":"Lin","suffix":""},{"id":317348339,"identity":"37ace33c-4dbc-4143-ac2c-e8c5ed9e5ca2","order_by":1,"name":"Xiaorui Wan","email":"","orcid":"","institution":"Soochow University","correspondingAuthor":false,"prefix":"","firstName":"Xiaorui","middleName":"","lastName":"Wan","suffix":""},{"id":317348340,"identity":"796af0fa-aa71-4f98-b3d8-92f14ce3ff2c","order_by":2,"name":"Huanchen Wu","email":"","orcid":"","institution":"Soochow University","correspondingAuthor":false,"prefix":"","firstName":"Huanchen","middleName":"","lastName":"Wu","suffix":""},{"id":317348341,"identity":"254a98d5-c178-43db-9a67-b0d142535e60","order_by":3,"name":"Na Liu","email":"","orcid":"","institution":"Soochow University","correspondingAuthor":false,"prefix":"","firstName":"Na","middleName":"","lastName":"Liu","suffix":""},{"id":317348342,"identity":"3037a3a1-028d-4c4f-91c8-fe62b94abf35","order_by":4,"name":"Yiyue Jiang","email":"","orcid":"","institution":"Soochow University","correspondingAuthor":false,"prefix":"","firstName":"Yiyue","middleName":"","lastName":"Jiang","suffix":""},{"id":317348343,"identity":"b6b14aa7-d825-4f56-a88e-189a1b18eddf","order_by":5,"name":"Yichao Sheng","email":"","orcid":"","institution":"Soochow University","correspondingAuthor":false,"prefix":"","firstName":"Yichao","middleName":"","lastName":"Sheng","suffix":""},{"id":317348344,"identity":"13becc7d-0e41-4982-bd28-84571ebe1679","order_by":6,"name":"Jing Wang","email":"","orcid":"","institution":"Soochow University","correspondingAuthor":false,"prefix":"","firstName":"Jing","middleName":"","lastName":"Wang","suffix":""},{"id":317348345,"identity":"b1c83c07-a1e7-4a69-9cc6-2bf21b005bf4","order_by":7,"name":"Haidong Xu","email":"","orcid":"","institution":"Soochow University","correspondingAuthor":false,"prefix":"","firstName":"Haidong","middleName":"","lastName":"Xu","suffix":""},{"id":317348346,"identity":"558e28ed-5c68-49f8-bd46-db49a4e34c21","order_by":8,"name":"Jie Xue","email":"","orcid":"","institution":"Soochow University","correspondingAuthor":false,"prefix":"","firstName":"Jie","middleName":"","lastName":"Xue","suffix":""},{"id":317348347,"identity":"daf00a9b-9cd9-4c9f-aab4-5f3fa3bf1870","order_by":9,"name":"Zhenghong Qin","email":"","orcid":"","institution":"Soochow University","correspondingAuthor":false,"prefix":"","firstName":"Zhenghong","middleName":"","lastName":"Qin","suffix":""},{"id":317348353,"identity":"6d1f621d-caf9-4eb4-a553-d5f86eeac1fb","order_by":10,"name":"Yan Wang","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA2UlEQVRIiWNgGAWjYBADOSA2AGJm4rUYk64lsYFoLQbHzx6T+LmjNn3+jOSNHxgqrBMb2M8ewK/lTF6aZO+Z47kbbqQVSzCcSU9s4MlLwK/lQI7ZDd62Y7kbJHIMJBjbDic2SPAY4Ndy/o3Zzb9tx9LlZ+QY/2D8R4yWGzlmt3nbahIYgAwJxgYitEjeeGP+W7btgOGGM8/KLBKOpRu38eTg18J3PsfY8G1bnbx8e/LmGx9qrGX72c/g16JwAEwdhvASgJgNr3ogkG8AU3WE1I2CUTAKRsFIBgBUdEnKujVoHQAAAABJRU5ErkJggg==","orcid":"","institution":"Soochow University","correspondingAuthor":true,"prefix":"","firstName":"Yan","middleName":"","lastName":"Wang","suffix":""}],"badges":[],"createdAt":"2024-06-06 03:24:31","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4537012/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4537012/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1007/s12035-025-04827-3","type":"published","date":"2025-03-17T15:57:36+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":58907930,"identity":"20ab6594-1c20-4fca-bf8a-c68180ca2db9","added_by":"auto","created_at":"2024-06-24 03:27:23","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":572865,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eNADPH oxidase subtype NOX4, NOX2 and NOX1 protein expression in KA-induced excitotoxicity. \u003c/strong\u003eMice were killed 6, 12, 24, 48 and 96 h after KA injection for western blotting. (\u003cstrong\u003ea\u003c/strong\u003e, \u003cstrong\u003eb\u003c/strong\u003e) Time course of KA-induced changes in NOX4 protein expression in the striatum. n=4. (\u003cstrong\u003ec\u003c/strong\u003e, \u003cstrong\u003ed\u003c/strong\u003e) Time course of KA-induced changes in NOX2 protein expression in the striatum. n=6. (\u003cstrong\u003ee\u003c/strong\u003e, \u003cstrong\u003ef\u003c/strong\u003e) Time course of KA-induced changes in NOX1 protein expression in the striatum. n=6. Data are expressed as the mean ± SEM. *** \u003cem\u003eP \u003c/em\u003e\u0026lt; 0.001 versus control, one-way ANOVA, Dunnett test.\u003c/p\u003e","description":"","filename":"Figure1.png","url":"https://assets-eu.researchsquare.com/files/rs-4537012/v1/4272eecce4b24215d4d89b90.png"},{"id":58908129,"identity":"7daa49dd-a48a-4f30-98d1-e46fd440751c","added_by":"auto","created_at":"2024-06-24 03:35:23","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":621539,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eMitochondrial and cytoplasmic fraction NOX4, NOX2 and NOX1 protein expression in KA-induced excitotoxicity. \u003c/strong\u003eMice were killed 6, 12, and 24 h after KA injection, and then the mitochondrial and cytoplasmic fractions were isolated for western blotting. (\u003cstrong\u003ea\u003c/strong\u003e-\u003cstrong\u003ec\u003c/strong\u003e) Time course of KA-induced changes in NOX4 protein expression in the mitochondrial and cytoplasmic fractions of the striatum. n=5. (\u003cstrong\u003ed\u003c/strong\u003e) Time course of KA-induced changes in NOX2 and NOX1 protein expression in the mitochondrial and cytoplasmic fractions of the striatum. (\u003cstrong\u003ee\u003c/strong\u003e-\u003cstrong\u003ef\u003c/strong\u003e) Quantification of western blotting analysis of NOX2. n=6. (\u003cstrong\u003eg\u003c/strong\u003e-\u003cstrong\u003eh\u003c/strong\u003e) Quantification of western blotting analysis of NOX1. n=3. Data are expressed as the mean ± SEM. * \u003cem\u003eP \u003c/em\u003e\u0026lt; 0.05 versus control, ** \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01 versus control, one-way ANOVA, Dunnett test.\u003c/p\u003e","description":"","filename":"Figure2.png","url":"https://assets-eu.researchsquare.com/files/rs-4537012/v1/d90cceba0a245938998c2b73.png"},{"id":58907932,"identity":"9b2dcec3-fa65-4545-befa-37592897285a","added_by":"auto","created_at":"2024-06-24 03:27:23","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":3058659,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eKA-induced excitotoxicity impairs mitochondrial morphology and disturbs quality control systems. \u003c/strong\u003e(\u003cstrong\u003ea\u003c/strong\u003e) Mice were killed 48 h after KA injection.\u003cstrong\u003e \u003c/strong\u003eStriatum tissue was collected for transmission electron microscopy\u003cstrong\u003e. \u003c/strong\u003eRepresentative electron microscopy micrographs of mitochondria in striatal neurons. Images C-D are representative areas indicated by the red wireframes in A-B. Images E-F show representative mitochondria indicated by red arrows in C-D. Scale bar = 5 μm in A-B; Scale bar = 2 μm in C-D; Scale bar = 100 nm in E-F. (\u003cstrong\u003eb\u003c/strong\u003e, \u003cstrong\u003ec\u003c/strong\u003e) Mice were killed 6, 12, 24, 48 and 96 h after KA injection for western blotting. Time course of KA-induced changes in PGC-1α in the striatum. n=6. (\u003cstrong\u003ed\u003c/strong\u003e, \u003cstrong\u003ee\u003c/strong\u003e) Time course of KA-induced changes in phospho-DRP1 (Ser616)/total DRP1 in the striatum. n=4. (\u003cstrong\u003ef\u003c/strong\u003e, \u003cstrong\u003eg\u003c/strong\u003e) Time course of KA-induced changes in PINK1 protein expression in the striatum. n=5. (\u003cstrong\u003eh\u003c/strong\u003e, \u003cstrong\u003ei\u003c/strong\u003e) Time course of KA-induced changes in Parkin protein expression in the striatum. n=4. Data are expressed as the mean ± SEM. * \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05 versus control,** \u003cem\u003eP\u003c/em\u003e\u0026lt; 0.01 versus control, *** \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.001 versus control, one-way ANOVA, Dunnett test.\u003c/p\u003e","description":"","filename":"Figure3.png","url":"https://assets-eu.researchsquare.com/files/rs-4537012/v1/0f37f5305cb04b14028b0ff7.png"},{"id":58907936,"identity":"41c76f9a-6a2c-4530-89fb-142fd7b1700b","added_by":"auto","created_at":"2024-06-24 03:27:23","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":3196078,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eEffects of Mito-apocynin on KA-induced excitotoxic injury\u003c/strong\u003e\u003cem\u003e\u003cstrong\u003e in vivo. \u003c/strong\u003e\u003c/em\u003e(\u003cstrong\u003ea\u003c/strong\u003e) Study road map of the animal experiment. Mice were administered 18.75 μg/kg, 37.5 μg/kg, and 75 μg/kg Mito-apocynin (i.g.) 1 day in advance. Unilateral injection of KA (0.625 nmol) was used to construct an animal model of excitotoxicity. Mito-apocynin was administered continuously for 14 days. Brain sections were stained with Nissl’s stain. (\u003cstrong\u003eb\u003c/strong\u003e) Representative images were taken in the center of the drug injection. (Scale bar = 500 μm in A-F; Scale bar = 100 μm in G-L; Scale bar = 50 μm in M-R; and 20 µm in S-X) (\u003cstrong\u003ec\u003c/strong\u003e) Quantitative results of normal morphological neurons. n=3. Data are expressed as the mean ± SEM.\u003cstrong\u003e \u003c/strong\u003e*** \u003cem\u003eP \u003c/em\u003e\u0026lt; 0.001 versus control, ### \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.001 versus KA-treated group, one-way ANOVA, Tukey test.\u003c/p\u003e","description":"","filename":"Figure4.png","url":"https://assets-eu.researchsquare.com/files/rs-4537012/v1/f27542acd0bcad9dca690d27.png"},{"id":58907933,"identity":"fa8ee6b7-e833-481d-a531-fe1253e42a66","added_by":"auto","created_at":"2024-06-24 03:27:23","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":296231,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eMito-apocynin reverses KA-induced motor behavioral impairment. \u003c/strong\u003eMice were administered 75 μg/kg Mito-apocynin 1 day in advance. Behavioral tests were performed at 0 h, 1.5 h, 3 h, 6 h, 12 h and 24 h after KA injection. (\u003cstrong\u003ea\u003c/strong\u003e) Cylinder test. (\u003cstrong\u003eb\u003c/strong\u003e) Adhesive removal test. (\u003cstrong\u003ec\u003c/strong\u003e) Inverted grid test. (\u003cstrong\u003ed\u003c/strong\u003e) Neurobehavioral dysfunction scores. n=5. Data are expressed as the mean ± SEM. *** \u003cem\u003eP \u003c/em\u003e\u0026lt; 0.001 versus control, # P \u0026lt; 0.05 versus KA-treated group, ###\u003cem\u003e P\u003c/em\u003e \u0026lt; 0.001 versus KA-treated group, two-way ANOVA, Tukey test.\u003c/p\u003e","description":"","filename":"Figure5.png","url":"https://assets-eu.researchsquare.com/files/rs-4537012/v1/251c9602589df22a310b9dd3.png"},{"id":58907935,"identity":"50e19577-a2f0-4d69-bcf7-0e2eb00b5ddf","added_by":"auto","created_at":"2024-06-24 03:27:23","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":635001,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eMito-apocynin reverses KA-induced primary neuronal damage and mitochondrial dysfunction\u003c/strong\u003e\u003cem\u003e\u003cstrong\u003e in vitro.\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e \u003c/strong\u003e(\u003cstrong\u003ea\u003c/strong\u003e) Study road map of the experiment. Cells were pretreated with Mito-apocynin for 4 h and then treated with KA for 8 h. (\u003cstrong\u003eb\u003c/strong\u003e) Cell viability of primary neurons. Cells were pretreated with Mito-apocynin (0.25, 0.5, 1 and 2 μM) for 4 h and then treated with KA for 8 h. Cell viability was determined by a CCK8 kit. n=6. (\u003cstrong\u003ec\u003c/strong\u003e) ATP levels were measured by an ATP detection kit. n=6. (\u003cstrong\u003ed\u003c/strong\u003e) Mitochondrial membrane potential was measured by a JC-1 detection kit. Scale bar = 100 μm. (\u003cstrong\u003ee\u003c/strong\u003e) Quantification of the fluorescence ratio (red fluorescence/green fluorescence). n=6. (\u003cstrong\u003ef\u003c/strong\u003e) Mitochondrial superoxide was measured by the MitoSOX Red probe. Scale bar = 50 μm. (\u003cstrong\u003eg\u003c/strong\u003e) Quantification of the MitoSOX fluorescence intensity. n=6. Data are expressed as the mean ± SEM.\u003cstrong\u003e \u003c/strong\u003e** \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01 versus control, *** \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.001 versus control, # P \u0026lt; 0.05 versus KA-treated group, ## \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01 versus KA-treated group, ### \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.001 versus KA-treated group, one-way ANOVA, Tukey test.\u003c/p\u003e","description":"","filename":"Figure6.png","url":"https://assets-eu.researchsquare.com/files/rs-4537012/v1/28ef242e281e656a4e2cd34b.png"},{"id":58908130,"identity":"60abcb7c-0cd4-449c-9138-cda5e08e96cd","added_by":"auto","created_at":"2024-06-24 03:35:23","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":334307,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eMito-apocynin reverses KA-induced upregulation of the expression levels of mitochondrial fraction NOX4 and ameliorates the disturbance of the mitochondrial quality control system.\u003c/strong\u003e (\u003cstrong\u003ea\u003c/strong\u003e) Mice were pretreated with mito-apocynin (75 μg/kg) 1 day before KA (0.625 nmol) injection and then killed 6 h later, and striatal mitochondrial and cytoplasmic fractions were isolated. Representative image of a western blotting for NOX4. (\u003cstrong\u003eb\u003c/strong\u003e) Quantification of western blotting analysis of mitochondrial NOX4. n=3. (\u003cstrong\u003ec\u003c/strong\u003e) Quantification of western blotting for cytoplasmic NOX4. n=3. (\u003cstrong\u003ed\u003c/strong\u003e-\u003cstrong\u003ee\u003c/strong\u003e) Mice were pretreated with mito-apocynin (75 μg/kg) 1 day before KA (0.625 nmol) injection and then killed 48 h later to isolate proteins. Representative bands and semi-quantitation of western blotting for detecting PGC-1α. n=4. (\u003cstrong\u003ef\u003c/strong\u003e-\u003cstrong\u003eg\u003c/strong\u003e) Mice were pretreated with Mito-apocynin (75 μg/kg) 1 day before KA (0.625 nmol) injection and then killed 48 h later to isolate proteins. Representative bands and semi-quantitation of western blotting for detecting DRP1 (phospho-Ser616) and total DRP1. n=5. (\u003cstrong\u003eh\u003c/strong\u003e-\u003cstrong\u003ei\u003c/strong\u003e) Mice were pretreated with Mito-apocynin (75 μg/kg) 1 day before KA (0.625 nmol) injection and then killed 24 h later to isolate proteins for western blotting. Representative bands and semi-quantitation of western blotting for detecting PINK1. n=4. (\u003cstrong\u003ej\u003c/strong\u003e-\u003cstrong\u003ek\u003c/strong\u003e) Representative bands and semi-quantitation of western blotting for detecting Parkin. n=6. Data are expressed as the mean ± SEM.\u003cstrong\u003e *\u003c/strong\u003e P \u0026lt; 0.05 versus control,\u003cstrong\u003e **\u003c/strong\u003e P \u0026lt; 0.01 versus control,\u003cstrong\u003e ***\u003c/strong\u003e P \u0026lt; 0.001 versus control, # P \u0026lt; 0.05 versus KA-treated group, ## P \u0026lt; 0.01 versus KA-treated group, ### P \u0026lt; 0.001 versus KA-treated group, one-way ANOVA, Tukey test.\u003c/p\u003e","description":"","filename":"Figure7.png","url":"https://assets-eu.researchsquare.com/files/rs-4537012/v1/58175ccb062ff894c2b765d5.png"},{"id":58907934,"identity":"924cd8aa-16cd-4067-9b9d-bc0a8b846d31","added_by":"auto","created_at":"2024-06-24 03:27:23","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":325184,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eMito-apocynin protects against KA-induced excitotoxicity by attenuating mitochondrial impairment. \u003c/strong\u003eKA upregulated the expression of the mitochondrial fraction NOX protein. KA led to mitochondrial dysfunction and disrupted the mitochondrial quality control system. Exogenous supplemental mitochondrion-targeted NOX inhibitor Mito-apocynin ameliorated KA-induced mitochondrial impairment by inhibiting mitochondrial NOX upregulation.\u003c/p\u003e","description":"","filename":"Figure8.png","url":"https://assets-eu.researchsquare.com/files/rs-4537012/v1/5de63d3a0e14b206ccbfefd6.png"},{"id":79120952,"identity":"29a64b4c-01a4-42df-8c7d-d6a4545d5af6","added_by":"auto","created_at":"2025-03-24 16:11:29","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":13368947,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4537012/v1/7ad87018-77c5-4267-b0e8-6a2a852a3bca.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Mito-apocynin protects against kainic acid-induced excitotoxicity by ameliorating mitochondrial impairment","fulltext":[{"header":"Introduction","content":"\u003cp\u003eNeurodegenerative diseases are major global health problems with increasing incidence rates [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. Ischemic stroke is thought to be caused by excitotoxicity induced by glutamate receptor overactivation [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. A large amount of data suggests that excitotoxicity is a potential target of neurodegenerative diseases [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. Dysregulation of neuronal calcium homeostasis, stress caused by oxidants and dysfunction of the mitochondria result from glutamate release or decreased uptake [\u003cspan additionalcitationids=\"CR7 CR8\" citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. Excitotoxicity is a hallmark of cellular responses to stress that occur in hypoxic/ischemic injury and diseases associated with neurodegeneration [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e, \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. However, effective pharmacological interventions against excitotoxicity are lacking. Therefore, the molecular mechanism of neuronal damage caused by excitotoxicity urgently needs to be studied to find new ideas for preventing and treating neurodegenerative diseases [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eOur previous studies have established that kainic acid (KA) receptors are predominantly distributed in the striatal region. We successfully modeled neuroexcitatory damage by KA injection in the right striatum of the mouse brain, with no effect on the left striatum or cerebral cortex. Supplementation with NADPH protected neurons from KA-induced excitotoxic injury [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e, \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. However, NADPH has a dual role. In addition to acting as an antioxidant, it is a substrate for NADPH oxidase (NOX), a chemical that generates oxygen radicals (ROS) by oxidizing NADPH [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e, \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. There are three primary NOX isoforms expressed in the central nervous system: NOX1, NOX2 and NOX4[\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. We found that after exposure to excitotoxins, NOX4 expression was significantly increased[\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. The production of NOX is closely related to ROS, making it a promising target as a new treatment for neurodegenerative diseases [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e, \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. Various diseases associated with neurodegeneration lead to increased NOX activity and expression [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. NOX-mediated superoxide generation is a necessary mechanism of excitotoxic death resulting from NMDA receptor activation [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eROS are mainly produced by NOX and mitochondria [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. NOX-generated ROS can damage mitochondria [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. In some cases, ROS produced by mitochondria may also stimulate NOX [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. Therefore, mitochondrial-NOX crosstalk may result in an overproduction of ROS, which may be a new pharmacological target [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e, \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. Recently, researchers have found that NOX4 is also localized in mitochondria and are increasingly interested in the interaction between NOX4 and mitochondria [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. Previously, ROS were believed to come mainly from the mitochondrial respiratory chain in cell [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. According to the literature, ROS may be produced in the mitochondrial respiratory chain rather than by the mitochondria [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. Mitochondrial NOX4 is one of the main sources of mitochondrial superoxide [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e, \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. NOX4 regulates mitochondrial energy metabolism, biogenesis and mitochondrial DNA repair in cardiovascular diseases [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. Therefore, it is important to explore the changes in mitochondrial NOX levels during excitotoxicity and the role of mitochondrial NOX in excitotoxicity.\u003c/p\u003e \u003cp\u003eThe purpose of this study was to determine whether the mitochondrion-targeted NOX inhibitor mitoapocynin protects against KA-induced excitotoxicity and assess its effects on excitotoxicity-induced mitochondrial damage.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eAnimals and Drug Administration\u003c/h2\u003e \u003cp\u003eSPF-grade Institute of Cancer Research (ICR) male mice, 25\u0026ndash;30 g, were purchased from ZhaoYan (Suzhou) Company. SPF animal centers maintained constant temperatures of 22\u0026deg;C and a humidity of 50%-60% for the rearing of mice. Institutional regulations on animal health apply to the use of these animals. In all cases, Soochow University's Institutional Animal Care and Use Committee approved the protocols.\u003c/p\u003e \u003cp\u003eStereotaxic apparatuses were used to fix the mice after chloral hydrate anesthesia (400 mg/kg). The right striatum was injected with 0.625 nmol of KA (Sigma Aldrich, K0250). Coordinates of the right striatum: 0.8 mm anterior to the bregma, 1.8 mm lateral to the sagittal suture, and 3.5 mm ventral to the pial surface. The injection volume was 1 \u0026micro;l, and the injection speed was 0.5 \u0026micro;l/min. Mito-apocynin (HY-135869, MCE, ig) was preadministered one day before KA injection [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e, \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003eIsolation of tissue mitochondrial and cytoplasmic fractions\u003c/h2\u003e \u003cp\u003eAfter the mice were sacrificed, the striatum was isolated. A 10-fold volume of Mitochondrial Isolation Reagent A solution (Beyotime Biotechnology, C3606) with 1% PMSF (Beyotime Biotechnology, ST505) was added and homogenized 30 times. Five minutes of centrifugation at 600 \u0026times;g was performed on the tissue homogenate. The supernatant was collected for the isolation of mitochondria. In the supernatant, 10 minutes of centrifugation were carried out at 11,000 \u0026times;g. This supernatant was aspirated as a cytoplasmic fraction. The precipitate was resuspended by adding 100 \u0026micro;l of Mitochondrial Isolation Reagent A solution. Afterward, it was centrifuged for ten minutes at 11,000 \u0026times;g. The resuspension was transferred to an EP tube containing 22%/55% Percoll and centrifuged at 20,000 \u0026times;g for 20 min. The white flocculent between the Percoll gradient was the fraction of mitochondria.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003eWestern blotting\u003c/h2\u003e \u003cp\u003eBy our previous description, Western blotting was performed [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]. Antibody source: NOX1 antibody (Proteintech, 17772-1-AP); NOX2 antibody (Santa Cruz, sc-130543); NOX4 antibody (Abcam, ab109225); DRP1 antibody (Cell Signaling Technology, 5931S); DRP1 (phospho-ser616) antibody (SAB, 12749); PINK1 antibody (Absin, abs100425); Parkin antibody (Santa Cruz, sc-2282); PGC-1α antibody (Santa Cruz, sc-3067); GAPDH antibody (Abcam, ab8245); β-actin antibody (Sigma Aldrich, A5441); α-Tubulin antibody (Abcam, ab7291); VDAC antibody (Cell Signaling Technology, 12454).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003eTransmission electron microscopy\u003c/h2\u003e \u003cp\u003eIsolation of the striatum after heart perfusion. Preparation of 2 mm \u0026times; 2 mm ultrathin tissue sections. Store at 4\u0026deg;C in electron microscope fixative (Servicebio, G1102). Samples were double-stained using lead-uranium. The copper mesh containing the samples was placed in a transmission electron microscope for observation.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003eNissl staining\u003c/h2\u003e \u003cp\u003eWhole brains were isolated after heart perfusion and fixed in 4% paraformaldehyde. Sucrose was dehydrated, and coronal sections were prepared using a vibrating slicer. Nissl staining (Beyotime Biotechnology, C0117) was performed for 30 min. For 2 min, the sections were dehydrated and decolorized in 75%, 95%, and 100% ethanol. Xylene was used to permeabilize the slices for 10 minutes, followed by sealing with neutral resin. Neuronal morphology was observed under a microscope, and the number of normal neurons in the striatal center was counted at 20\u0026times; magnification.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eBehavioral test\u003c/h2\u003e \u003cp\u003eCylinder test: Mice were placed in a 10 cm diameter glass round cylinder. The number of unilateral and bilateral forelimb contacts with the cylinder wall was recorded within 3 min. The proportion of unilateral contact was counted.\u003c/p\u003e \u003cp\u003eAdhesive removal test: The mice were acclimated to their new environment before starting the experiment. A 0.2-inch piece of tape was applied to the nose of the mice. The time needed to remove the tape from the forelimbs of the mice was recorded for no more than 1 min.\u003c/p\u003e \u003cp\u003eInverted grid test: A pretest was performed on the day of the experiment to acclimatize the mice to the test. The mice were placed in the center of a 15 cm\u003csup\u003e2\u003c/sup\u003e horizontal square grid. The grid was raised 20 cm from the table, and the grid was flipped up and down. The time that the mice remained on the grid was recorded for no more than 1 min.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003ePrimary neuron culture\u003c/h2\u003e \u003cp\u003eSeparate the cerebral cortex of 18-day-old ICR mouse embryos. Add 2.5% trypsin and digest at 37\u0026deg;C for 15 min. After termination of digestion, add DNAase and gently blow 100 times. Centrifuge at 200 \u0026times;g for 5 min. Resuspend the cells by adding neuronal medium and filter the cells through a 40 \u0026micro;m cell sieve. Cell suspensions were diluted to 10\u003csup\u003e6\u003c/sup\u003e cells/ml and incubated at 37\u0026deg;C with 5% CO\u003csub\u003e2\u003c/sub\u003e.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003eMeasurement of ATP levels\u003c/h2\u003e \u003cp\u003eThe experiment was performed according to the instructions provided with the ATP assay kit (Beyotime Biotechnology, S0026). The cells were collected, and 50 \u0026micro;l of ATP assay lysate was added. Five minutes at 12,000 \u0026times;g in a centrifuge. Collection of supernatants for ATP level determination. Place 100 \u0026micro;l of ATP assay working solution into each well in a black 96-well plate. This was allowed to sit at room temperature and was protected from light for 3\u0026ndash;5 minutes. Add 20 \u0026micro;l of sample to the assay wells. RLU values were determined. The protein concentration was also measured by a BCA kit (Takara Bio, T9300A). The ATP level was expressed as nmol/mg.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eCell viability\u003c/h2\u003e \u003cp\u003eNinety-six-well plates were used to inoculate primary neurons. Pretreatment with Mito-apocynin for 4 h was followed by treatment with 100 \u0026micro;M KA for 8 h. To measure cell viability, we used a kit called CCK-8 (DOJINDO, CK04). The reaction was carried out at 37\u0026deg;C for 3 h. Microplate readers were used to measure absorbance at 450 nm.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eMeasurement of mitochondrial membrane potential\u003c/h2\u003e \u003cp\u003eThe membrane potential of the mitochondria was detected using JC-1 (Beyotime Biotechnology, C2006). Primary cortical neurons were inoculated in a 24-well plate. We pretreated neurons with Mito-apocynin for 4 h and then treated them with 100 \u0026micro;M KA for 8 h. The medium was aspirated, and the cells were washed twice in HBSS. Working solution for JC-1 staining should be added in 0.3 ml. The mixture was incubated for 20 min at 37\u0026deg;C in the dark. The cells were washed with JC-1 buffer 3 times. The cells were observed under a fluorescence microscope.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eMeasurement of mitochondrial superoxide levels\u003c/h2\u003e \u003cp\u003eMitochondrial superoxide levels were detected using MitoSOX Red mitochondrial superoxide indicator (Yeasen, 40778ES50). Primary cortical neurons were inoculated in 24-well plates. Neurons were pretreated with Mito-apocynin for 4 h, followed by treatment with 100 \u0026micro;M KA for 8 h. The medium was aspirated, and the cells were washed twice with HBSS. Add 0.3 ml MitoSOX Red working solution and incubate for 10 min at 37\u0026deg;C protected from light. The HBSS disinfection was repeated three times, and the cells were stained again with Hoechst's reagent in 0.3 ml. wash twice with HBSS. The cells were observed under a fluorescence microscope.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eStatistical analysis\u003c/h2\u003e \u003cp\u003eStatistical analysis and graphing of experimental data were performed using GraphPad Prism 8. The mean and standard error of the experimental data are expressed as the mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SEM. Analysis of the differences between the two data groups was performed using an unpaired t-test, one-way analysis of variance for multigroup data was performed using one-way ANOVA, two-way ANOVA was used for multivariate analysis of differences in multiple groups of data, statistical significance was determined by *P\u0026thinsp;\u0026lt;\u0026thinsp;0.05, and no */# in the statistical graph indicates no significant difference.\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cp\u003e \u003cb\u003eKA-induced excitotoxicity upregulated the expression level of NOX4 in mitochondria.\u003c/b\u003e \u003c/p\u003e \u003cp\u003eOur previous studies showed that NADPH protects against KA-induced excitotoxic injury [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. NADPH exerts neuroprotective effects as an antioxidant, but it is also utilized by NOX to generate ROS [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]. Therefore, the therapeutic window of NADPH is narrow. This dual effect of NADPH suggests that the role of NOX should be considered when studying the mechanism of excitotoxicity [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]. We previously found increased levels of NOX4 expression in KA-induced excitotoxicity [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. Changes in the protein levels of NOX1 and NOX2 are not yet known. To investigate the involvement of the NOX family in excitotoxicity, KA, a glutamate analog, was stereotaxically injected into the right striatum of mice to create an excitotoxicity animal model. The control group was injected with an equal amount of normal saline in the same way. We examined indicators of NOX expression at high levels or low levels at different times after KA treatment. KA upregulated NOX4 and NOX2 expression but not NOX1 (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ea-f).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eNOX4 has constitutive activity [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]. In cardiovascular disease, NOX4 was shown to be localized in cardiac mitochondria under pathological conditions and may be associated with ROS produced by mitochondrial respiration and NOX[\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. Furthermore, NOX4 has been reported to bind to mitochondrial respiratory chain complex I. As a result, NOX4 may play a role in the generation of ROS mediated by both NOX and the mitochondrial respiratory chain. When KA injections were administered, mitochondrial NOX4 expression was significantly increased, but cytoplasmic NOX4 was not affected (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ea-c). NOX2 and NOX1 in mitochondria did not appear to be affected by KA (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ed-h). However, KA upregulated the expression of NOX2 in the cytoplasm (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ed-f). Given the critical role of mitochondrial NOX4 in KA-induced excitotoxic injury and its potential importance in regulating mitochondrial function, we subsequently paid special attention to mitochondrial NOX4 when investigating the protective role of Mito-apocynin.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eNOX4 and NOX2 are the major contributors to ROS in KA-induced excitotoxic injury. NOX4 may have functional regulatory implications for mitochondrial ROS formation. NOX4 has previously been identified as containing a potential mitochondrial localization signal. Localized mitochondrial NOX4 can bind to mitochondrial complex I, and this interaction may have relevance to the regulation of mitochondrial function. In the presence of a KA-induced increase in NOX4 expression, the level of NOX4 in mitochondria increases, which may contribute to the development of mitochondrial ROS or regulate the activity of complex I on the mitochondrial respiratory chain, implying that both energy metabolism and quality control in mitochondria are affected.\u003c/p\u003e \u003cp\u003e \u003cb\u003eKA-induced excitotoxicity impairs mitochondrial morphology and disturbs quality control systems.\u003c/b\u003e \u003c/p\u003e \u003cp\u003eOveractivation of glutamate receptors damages mitochondria [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]. The cytoplasmic matrix of neurons in the KA-treated group became lighter, the mitochondria swelled, the mitochondrial matrix also became lighter, and the mitochondrial cristae appeared ruptured (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ea). The mitochondrial quality control system is critical for maintaining functional mitochondria [\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e]. A rapid increase in neuronal mitochondrial biogenesis after hypoxic-ischemic brain injury is an endogenous neuroprotective response to hypoxic-ischemic injury [\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e]. PGC-1α is a key protein that regulates mitochondrial biogenesis [\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e]. The expression level of PGC1-1α was significantly increased after KA injection (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eb-c). An organelle with a high degree of dynamic activity, mitochondria undergo continuous fission and fusion [\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e]. Healthy cells maintain equilibrium between fissions and fusions, thus maintaining mitochondrial homeostasis [\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e]. The decrease in the phosphorylated DRP1 (Ser616)/total DRP1 ratio indicated impaired mitochondrial dynamics (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ed-e). When mild and transient stress induces mitochondrial autophagy, we call it \u0026lsquo;stress-induced mitophagy\u0026rsquo; [\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e]. Autophagy-related stress is primarily induced by ROS [\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e]. PINK1 and Parkin synergistically promote autophagic clearance of damaged mitochondria. activation of mitochondrial autophagy [\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e]. Upregulation of PINK1 and Parkin indicated activation of mitochondrial autophagy (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ef-i).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eCrosstalk between NOX and mitochondria-derived ROS with mutual feedback leads to an amplification of ROS in a vicious cycle, ultimately leading to mitochondrial dysfunction and neuronal damage [\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e]. However, current therapeutic approaches using ROS blockers are not always effective. Consequently, we need new therapeutic strategies for treating diseases resulting from oxidative stress, such as neurodegenerative diseases. Therefore, we focused on studying mito-apocynin, a mitochondrion-targeted NOX inhibitor, which may be an effective therapy against NOX and mitochondrial ROS crosstalk and help to investigate the role and mechanism of mitochondrial NOX in KA-mediated mitochondrial damage.\u003c/p\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003eMito-apocynin protects striatal neurons from KA-induced excitotoxicity\u003c/h2\u003e \u003cp\u003eAfter KA injection, the opening of glutamate receptors leads to massive calcium inward flow, causing seizure-like symptoms and motor dysfunction in the affected trunk during the acute phase. Importantly, neuronal death does not occur immediately but is secondary to a series of cascading responses triggered by calcium signaling, a progressive neurodegenerative process. Therefore, we chose to observe neuronal death on day 14 after KA modeling to fully assess the long-term effects of the injury. To explore the potential role of NOX inhibitors in neuroprotection, we used Mito-apocynin, a mitochondria-targeted NOX inhibitor. The NOX inhibitor Apocynin binds to the cationic portion of the mitochondria-targeting moiety triphenylphosphonic acid to generate the mitochondrion-targeted NOX inhibitor Mito-apocynin [\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e, \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e]. The blood-brain barrier is highly lipophilic, so mito-apocynin can cross through to reach the striatum [\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e]. Mito-apocynin was preadministered to mice one day before KA injection. Mito-apocynin was then administered once a day, and brain tissue was isolated for Nissl staining after 14 days (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ea). Exogenous supplementation with Mito-apocynin significantly ameliorated KA-induced neuronal crinkling and loss of Nisin vesicles (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eb). Additionally, Mito-apocynin reduced the number of neuronal deaths in a dose-dependent manner (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ec). Therefore, we chose the best protective effect of 75 \u0026micro;g/kg of Mito-apocynin for the follow-up study \u003cem\u003ein vivo\u003c/em\u003e.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003eMito-apocynin ameliorates KA-induced motor behavioral deficits in mice\u003c/h2\u003e \u003cp\u003eStriatal injury leads to altered motor and muscle control in mice, mainly in the forelimbs [\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e]. In a previous study, we found that KA-induced excitotoxicity leads to motor dysfunction in mice [\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e]. Therefore, we performed the cylinder test, the adhesive removal test and the inverted grid test as indicators to evaluate striatal damage [\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e]. Healthy mice tend to use both limbs to touch the container wall, can remove adhesive labels in a short time and have high muscle tone in the limbs. KA treatment resulted in an increased proportion of mice touching the wall unilaterally, a longer time to remove adhesive labels and a shorter time to maintain them on the grid. Treatment with Mito-apocynin promoted recovery of motor function in mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ea-d).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003eMito-apocynin ameliorates KA-induced cytotoxicity and mitochondrial dysfunction\u003c/h2\u003e \u003cp\u003eTo explore whether Mito-apocynin also has a restoring effect on KA-induced mitochondrial damage \u003cem\u003ein vitro\u003c/em\u003e, we examined the experiments shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ea. In primary neurons, Mito-apocynin treatment reduced KA-induced neurotoxicity (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eb). In excitotoxicity, mitochondrial membrane potential run-down is a key step in cell death [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e, \u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e]. The membrane potential of mitochondria was examined by staining with JC-1, and a decrease in mitochondrial membrane potential caused a decrease in JC-1 polymers (red fluorescence) and an increase in JC-1 monomers (green fluorescence) in the mitochondrial matrix, and fluorescence observations showed a significant decrease in the relative proportions of red and green fluorescence of KA-induced JC-1 (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ed-e). Mitochondrial defects lead to insufficient ATP production (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ec). Also produces too much superoxide [\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e, \u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e]. Mitochondrial superoxide levels are detected using the MitoSOX Red Mitochondrial Superoxide Indicator, with higher fluorescence intensity indicating more superoxide production (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ef-g). The above results suggest that Mito-apocynin treatment ameliorates KA-induced mitochondrial dysfunction in vitro.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eMito-apocynin inhibits KA-induced upregulation of mitochondrial NOX4 and promotes restoration of the mitochondrial quality control system\u003c/b\u003e \u003c/p\u003e \u003cp\u003eWe speculate that mitochondrial NOX4 may be responsible for mitochondrial damage in excitotoxicity. Mito-apocynin is mitochondria-selective and inhibits KA-induced upregulation of mitochondrial NOX4 (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003ea-c). The protective effect of Mito-apocynin may be dependent on its inhibition of mitochondrial NOX4 expression, thereby reducing mitochondrial ROS production. Immunoblotting data showed that Mito-apocynin treatment reversed the KA-induced decrease in the phosphorylated DRP1 (Ser616)/total DRP1 ratio and significantly reversed KA-induced upregulation of PGC-1α, PINK1 and Parkin (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003ed-k). We conclude that the mitochondrion-targeted NOX inhibitor mito-apocynin may attenuate KA-mediated mitochondrial damage by inhibiting changes in mitochondrial NOX.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eGlutamate is one of the most common neurotransmitters in the mammalian central nervous system, and it mediates excitatory neurotransmission [\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e]. However, high levels of glutamatergic input can cause excitotoxicity, leading to neuronal cell death after acute brain injury, such as stroke or trauma [\u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e]. An increasing amount of research has focused on understanding the mechanisms underlying excitotoxic injury and revealing potential therapeutic strategies targeting these mechanisms [\u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e, \u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e]. In the current study, we constructed an animal model of excitotoxicity by injecting the glutamate analog KA into the striatum. In the KA-mediated excitotoxicity animal models, upregulation of NOX4 and NOX2 was observed. Unlike NOX1 and NOX2, NOX4 activity is controlled by its expression level rather than cytosolic regulatory proteins [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]. NOX is also directly associated with mitochondria, and when NOX in mitochondria is activated, its interactions with electron transport chain elements may cause mitochondria to produce superoxide indirectly[\u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e56\u003c/span\u003e]. The expression of NOX4 is upregulated in mitochondrial fractions. NOX4 in mitochondria is thought to be a central mediator of reduced oxidative stress and may mediate an imbalance in mitochondrial function and cellular injury in a variety of diseases. Therefore, mitochondrial NOX4 is considered a major target for designing new therapeutic strategies to uncover effective therapies for various diseases characterized by oxidative stress, including neurodegenerative diseases[\u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e56\u003c/span\u003e, \u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e57\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eCalcium overload is caused by excessive glutamate receptor activation, which leads to disruption of mitochondrial morphology and mitochondrial dysfunction. Exposure of mitochondria to high levels of KA leads to disturbances in mitochondrial dynamics and metabolism and mitochondrial autophagy. An important factor that determines how neurons respond to glutamate is their bioenergetic state. This suggests that to counter excitotoxicity, mitochondrial function must be targeted[\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]. Although new therapies may target glutamate receptors or glutamate uptake, pharmacological strategies that promote mitochondrial function may also prove beneficial. The NOX family is a new class of pharmacological targets for the treatment of neurodegenerative diseases because of the roles of NOXs in oxidant production and their possible role in the regulation of neurons. Mito-apocynin has higher mitochondrial selectivity than other NOX inhibitors. Mito-apocynin, which can reach the striatum, was administered orally. Supplementation with Mito-apocynin attenuated excitotoxic damage.\u003c/p\u003e \u003cp\u003eHere, we confirmed the protective effect of mito-apocynin against KA-induced excitotoxicity. However, apocynin is a nonspecific NOX inhibitor, and even though mito-apocynin was able to inhibit KA-induced upregulation of mitochondrial NOX4, it is still difficult to exclude its effect on other NOX isoforms in mitochondria. However, due to the lack of animal models that can be used to elucidate the subtype- and subcellular-specific function of NOX4 \u003cem\u003ein vivo\u003c/em\u003e, the role of NOX4 in excitotoxicity-mediated mitochondrial injury and neurodegenerative diseases requires further in-depth study.\u003c/p\u003e \u003cp\u003eIn summary, in KA-induced excitotoxicity, mitochondrial NOX expression is upregulated, and this change is accompanied by mitochondrial dysfunction and impairment of the mitochondrial quality control system. Mito-apocynin, an exogenous mitochondrion-targeted NOX inhibitor, can exert neuroprotective effects by inhibiting mitochondrial NOX expression and ameliorating mitochondrial dysfunction and mitochondrial quality control system impairment, thereby reversing KA-induced mitochondrial damage (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e \u003ch2\u003eEthics Approval\u003c/h2\u003e \u003cp\u003eThe study has been examined and certified by the Ethics Committee of Soochow University, and informed consent was obtained from all participants included in the study, in agreement with institutional guidelines.\u003c/p\u003e \u003c/p\u003e \u003cp\u003e \u003cstrong\u003eConsent for Publication\u003c/strong\u003e \u003cp\u003e All authors have approved for publication.\u003c/p\u003e \u003c/p\u003e \u003cp\u003e \u003cstrong\u003eCompeting interests\u003c/strong\u003e \u003cp\u003eThe authors declare no competing interests.\u003c/p\u003e \u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eMML and YW designed the study. XRW, HCW,NL, YYJ, YCS and JW assisted with the experiments. MML, YW, HDX, JX and ZHQ contributed to the drafting of the manuscript and figures. All authors have approved the final article.\u003c/p\u003e\u003ch2\u003eAcknowledgments\u003c/h2\u003e \u003cp\u003eThis work was supported by the Natural Science Foundation of Jiangsu Province (No. BK20221360), the National Natural Science Foundation of China (No. 81671252, 81730092, 81773768) and the Priority Academic Program Development of the Jiangsu Higher Education Institutes (PAPD).\u003c/p\u003e\u003ch2\u003eData Availability\u003c/h2\u003e \u003cp\u003eThe authors declare the availability of data and material.\u003c/p\u003e\n"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eHou L, Zhang L, Hong JS, Zhang D, Zhao J, Wang Q (2020) Nicotinamide Adenine Dinucleotide Phosphate Oxidase and Neurodegenerative Diseases: Mechanisms and Therapy. 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Biomed Pharmacother 111:1478\u0026ndash;1498. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://doi.rog/10.1016/j.biopha.2018.11.128\u003c/span\u003e\u003cspan address=\"http://doi.rog/10.1016/j.biopha.2018.11.128\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\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":"molecular-neurobiology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"moln","sideBox":"Learn more about [Molecular Neurobiology](https://www.springer.com/journal/12035)","snPcode":"12035","submissionUrl":"https://submission.nature.com/new-submission/12035/3","title":"Molecular Neurobiology","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"Mito-apocynin, KA, Excitotoxicity, Mitochondrial dysfunction, NADPH oxidase","lastPublishedDoi":"10.21203/rs.3.rs-4537012/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4537012/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eNeurodegenerative diseases are major global health problems with increasing incidence rates. A large amount of data suggests that excitotoxicity is a potential target of neurodegenerative diseases. However, effective pharmacological interventions against excitotoxicity are lacking. We aimed to elucidate the neuroprotective effect and mechanism of the mitochondrion-targeted NOX inhibitor mito-apocynin on kainic acid (KA)-induced excitotoxicity. We found that KA impaired mitochondrial morphology and led to impaired mitochondrial energy metabolism and dysfunction. In Western blotting experiments, KA disrupted mitochondrial quality control. In Nissl staining and CCK8 experiments, Mito-apocynin attenuated the death of neurons due to excitotoxic damage induced by KA both \u003cem\u003ein vivo\u003c/em\u003e and \u003cem\u003ein vitro\u003c/em\u003e. Mito-apocynin ameliorated neurobehavior induced by KA deficits \u003cem\u003ein vivo\u003c/em\u003e and mitochondrial dysfunction \u003cem\u003ein vitro\u003c/em\u003e. Mito-apocynin significantly reversed the increase in NOX4 levels caused by KA in the mitochondria of the striatum, decreased phosphorylated DRP1 (Ser616)/total DRP1 and increased PGC-1α, PINK1 and Parkin protein expression in the total striatum. In summary, Mito-apocynin alleviated oxidative stress, maintained normal mitochondrial function and energy metabolism levels, and promoted the balance of mitochondrial quality control by regulating the expression of NOX in mitochondria, thus reducing KA-induced excitatory toxic damage.\u003c/p\u003e","manuscriptTitle":"Mito-apocynin protects against kainic acid-induced excitotoxicity by ameliorating mitochondrial impairment","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-06-24 03:27:18","doi":"10.21203/rs.3.rs-4537012/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-01-02T04:30:40+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-01-02T04:27:24+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"179053236502281604513073757069788501015","date":"2024-12-26T10:18:55+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"228534965805312803234476881308128779023","date":"2024-12-24T08:37:06+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-10-01T08:17:29+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"328348897553409684139258400969942791019","date":"2024-09-09T05:41:13+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"320965650378660752696848123637411738501","date":"2024-09-08T18:21:18+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2024-06-16T13:26:41+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2024-06-10T01:47:00+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2024-06-10T01:46:10+00:00","index":"","fulltext":""},{"type":"submitted","content":"Molecular Neurobiology","date":"2024-06-06T03:23:10+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"molecular-neurobiology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"moln","sideBox":"Learn more about [Molecular Neurobiology](https://www.springer.com/journal/12035)","snPcode":"12035","submissionUrl":"https://submission.nature.com/new-submission/12035/3","title":"Molecular Neurobiology","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"cbdedc46-d536-495a-8e5c-742f1f6ad0d9","owner":[],"postedDate":"June 24th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2025-03-24T16:08:08+00:00","versionOfRecord":{"articleIdentity":"rs-4537012","link":"https://doi.org/10.1007/s12035-025-04827-3","journal":{"identity":"molecular-neurobiology","isVorOnly":false,"title":"Molecular Neurobiology"},"publishedOn":"2025-03-17 15:57:36","publishedOnDateReadable":"March 17th, 2025"},"versionCreatedAt":"2024-06-24 03:27:18","video":"","vorDoi":"10.1007/s12035-025-04827-3","vorDoiUrl":"https://doi.org/10.1007/s12035-025-04827-3","workflowStages":[]},"version":"v1","identity":"rs-4537012","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4537012","identity":"rs-4537012","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}