NADPH and Mito-Apocynin Treatment Protects Against KA-Induced Excitotoxic Injury Through Autophagy Pathway

Abstract

AimPrevious research recognizes that NADPH can produce reduced glutathione (GSH) as a coenzyme and produce ROS as a substrate of NADPH oxidase (NOX). Besides, excessive activation of glutamate receptors results in mitochondrial impairment. The study aims at spelling out the effects of NADPH and Mito-apocynin, a NOX inhibitor which specifically targets the mitochondria, on the excitotoxicity induced by Kainic acid (KA) and its mechanism.MethodsThe in vivo neuronal excitotoxicity model was constructed by stereotypically injecting KA into the unilateral striatum of mice. Administrated NADPH (i.v, intravenous) 30 min prior and Mito-apocynin (i.g, intragastric) 1 day prior, respectively, then kept administrating daily until mice were sacrificed 14 days later. Nissl staining measured the lesion of striatum and survival status of neurons. Cylinder test of forelimb asymmetry and the adhesive removal test reflected the behavioral deficit caused by neural dysfunction. Determined Total superoxide dismutase (T-SOD), malondialdehyde (MDA), and GSH indicated oxidative stress. Western blot presented the expression levels of LC3-II/LC3-I, SQSTM1/p62, TIGAR, and NOX4. Assessed oxygen consumption rate using High-Resolution Respirometry. In vitro, the MitoSOX Indicator reflected superoxide released by neuron mitochondria. JC-1 and ATP assay Kit were used to detect mitochondrial membrane potential (MMP) and energy metabolism, respectively.ResultsIn this study, we have successfully established excitotoxic model by KA in vivo and in vitro. KA induced decreased SOD activity and increased MDA concentration. KA cause the change of LC3-II/LC3-I, SQSTM1/p62, and TIGAR expression, indicating the autophagy activation. NADPH plays a protective role in vivo and in vitro. It reversed the KA-mediated changes in LC3, SQSTM1/p62, TIGAR, and NOX4 protein expression. Mito-apocynin inhibited KA-induced increases in mitochondrial NOX4 expression and activity. Compared with NADPH, the combination showed more significant neuroprotective effects, presenting more neurons survive and better motor function recovery. The combination also better inhibited the over-activated autophagy. In vitro, combination of NADPH and Mito-apocynin performed better in restoring mitochondria membrane potential.ConclusionIn summary, combined administration of NADPH and NOX inhibitors offers better neuroprotection by reducing NADPH as a NOX substrate to generate ROS. The combined use of NADPH and Mito-apocynin can better restore neurons and mitochondrial function through autophagy pathway.

Various facets of excitotoxicity

“…The most frequently used indicators tetramethylrhodamine methyl ester (TMRM) [64,106,107] and rhodamine 123 [60,61,63,64,66] are accepted as the most reliable fluorescent indicators, with low interference rates in mitochondrial functions [105]. The fluorescent probe JC-1, also used to measure mitochondrial membrane potential in excitotoxicity studies [40,43,48], is the least indicated dye to evaluate these dynamics. The JC-1 dye emits fluorescence in the green light spectrum, and when aggregated within the mitochondrial matrix, shifts to a red fluorescence.…”

“…On the other hand, apoptosis is facilitated due to membrane rupture by mitochondrial swelling or by outer membrane permeabilization, causing the release of cytochrome c to the cytosol and activating the apoptotic caspase cascade pathway [108]. Mitochondrial activity in intact cells can be assessed by measuring oxygen consumption through high resolution respirometry [40,61]. The use of specific inhibitors allows us to collect important information about mitochondrial function during these assays.…”

“…While high resolution respirometry is crucial for analyzing mitochondria activity, it is important to combine it with the measurement of intracellular ATP concentration to provide reliable results. Luciferase-dependent assays are the most common for ATP quantification [40,48,51]. It is noteworthy to mention that prolonged mitochondrial depolarization can reverse ATP synthase activity, which then starts hydrolyzing ATP, contributing to bioenergetic collapse.…”

Disentangling Mitochondria in Alzheimer’s Disease

“…Importantly, MitoApo partially attenuated severe nigrostriatal degeneration, improved mitochondrial function, and inhibited NOX2 activation, oxidative damage, and neuroinflammation in MitoPark mice [ 223 ]. MitoApo was effective in this and other models of PD, as well as in a kainic acid-induced model of excitotoxicity [ 224 , 225 , 226 ]. Biodegradable polyanhydride-based nano-carriers can provide sustained delivery of diverse payloads to organelles with reduced toxicity and increased bioavailability [ 227 ].…”

Pretreatment with Methylene Blue Protects Against Acute Seizure and Oxidative Stress in a Kainic Acid-Induced Status Epilepticus Model

“…Previous clinical research has demonstrated that patients with SE had significantly lower concentrations of SOD and GSH and higher concentrations of MDA compared to controls [ 11 ]. KA triggered a decrease in SOD activity and increase in MDA concentration [ 12 ]. Therefore, we speculate that a large amount of MDA is produced and the activities of SOD and GSH are weakened during SE induced by KA, resulting in a serious oxidative stress response, a process that may be alleviated or even reversed by antioxidants.…”