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. 1a-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. 2a-c). NOX2 and NOX1 in mitochondria did not appear to be affected by KA (Fig. 2d-h). However, KA upregulated the expression of NOX2 in the cytoplasm (Fig. 2d-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. 3a). 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. 3b-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. 3d-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. 3f-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. 4a). Exogenous supplementation with Mito-apocynin significantly ameliorated KA-induced neuronal crinkling and loss of Nisin vesicles (Fig. 4b). Additionally, Mito-apocynin reduced the number of neuronal deaths in a dose-dependent manner (Fig. 4c). 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. 5a-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. 6a. In primary neurons, Mito-apocynin treatment reduced KA-induced neurotoxicity (Fig. 6b). 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. 6d-e). Mitochondrial defects lead to insufficient ATP production (Fig. 6c). 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. 6f-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. 7a-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. 7d-k). We conclude that the mitochondrion-targeted NOX inhibitor mito-apocynin may attenuate KA-mediated mitochondrial damage by inhibiting changes in mitochondrial NOX.