In vivo administration of D609 leads to protection of subsequently isolated gerbil brain mitochondria subjected to in vitro oxidative stress induced by amyloid beta-peptide and other oxidative stressors: Relevance to Alzheimer’s disease and other oxidative stress-related neurodegenerative disorders

Abstract

Tricyclodecan-9-yl-xanthogenate (D609) has in vivo and in vitro antioxidant properties. D609 mimics glutathione (GSH), has a free thiol group, which upon oxidation forms a disulfide. The resulting dixanthate is a substrate for glutathione reductase, regenerating D609. Recent studies have also shown that D609 protects brain in vivo and neuronal cultures in vitro against the potential Alzheimer's disease (AD) causative factor, Aβ (1-42)-induced oxidative stress and cytotoxicity. Mitochondria are important organelles with both pro-and anti-apoptotic factor proteins. The present study was undertaken to test the hypothesis that i.p. injection of D609 would provide neuroprotection against free radical-induced, mitochondria-mediated apoptosis in vitro. Brain mitochondria were isolated from gerbils 1 h post injection intraperitonially (i.p.) with D609 and subsequently treated in vitro with the oxidants Fe 2+ /H 2 O 2 (hydroxyl free radicals), 2,2-azobis-(2-amidinopropane) dihydrochloride (AAPH, alkoxyl and peroxyl free radicals) and AD-relevant amyloid β-peptide 1-42 [Aβ (1-42)]. Brain mitochondria isolated from the gerbils previously injected i.p. with D609 and subjected to these oxidative stress inducers, in vitro, showed significant reduction in levels of protein carbonyls, protein-bound hydroxynonenal (HNE) [a lipid peroxidation product], 3-nitrotyrosine (3-NT), and cytochrome-c release compared to oxidant-treated brain mitochondria isolated from salineinjected gerbils. D609 treatment significantly maintains the GSH/GSSG ratio in oxidant-treated mitochondria. Increased activity of glutathione-S-transferase (GST), glutathione peroxidase (GPx) and glutathione reductase (GR) in brain isolated from D609-injected gerbils is consistent with the notion that D609 acts like GSH. These anti-apoptotic findings are discussed with reference to the potential use of this brain-accessible glutathione mimetic in the treatment of oxidative stress-related neurodegenerative disorders, including AD.

Oxidative stress and modification of synaptic proteins in hippocampus after traumatic brain injury

“…Determination of GSH was performed as previously described [8]. The reaction mixture containing 0.1 M sodium phosphate buffer (pH 8.0), 5.0 mM EDTA, 10 µl o-phthalaldehyde (1.0 mg/ml), and 10 µl of sample.…”

“…The estimation of GSSG was performed as previously described [8]. The samples were incubated first with 0.04 M N-ethylmaleimide (NEM) for 30 min to interact with GSH present in sample.…”

“…GPx (EC 1.11.1.9) activity was measured according to the procedure as previously described [7,8,24] with some modifications. The reaction mixture consisted of 0.05 M phosphate buffer (pH 7.0), 1.0 mM EDTA, 1.0 mM sodium azide, 1.4 U of 0.1 ml GR, 1.0 mM GSH, 0.2 mM NADPH, 0.25 mM H 2 O 2 , and 0.03 ml of PMS in a final volume of 0.3 ml.…”

“…GR (EC 1.6.4.2) activity was assayed as previously described [8,24] with some modifications. The assay system consisted of 0.1 M phosphate buffer (pH 7.6), 0.1 mM NADPH, 0.5 mM EDTA, 1.0 mM GSSG, and 0.03 ml PMS in a total volume of 0.3 ml.…”

“…Enhanced production of reactive oxygen/nitrogen species (ROS/RNS) cause oxidative/ nitrosative stress [5,6] leading to damage in lipids, proteins and nucleic acids [7,8]. Oxidative stress-related cascades resulting from TBI have been implicated in cytoskeletal damage, mitochondrial dysfunction [9] and altered signal transduction [10,11].…”