Mechanisms Underlying Membrane Androgen Receptor‐Induced Neurodegeneration
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A common characteristic of several neurodegenerative disorders is oxidative stress (OS). Many neurodegenerative disorders are more prevalent in men and postmenopausal women compared to premenopausal women, indicating the possible involvement of androgens (men > postmenopausal women > premenopausal women) in neurodegeneration. Our lab found testosterone can have either neuroprotective or neurodamaging effects depending on the presence of OS in the cellular environment. We have shown testosterone via a non‐genomic mechanism exacerbates OS damage in neurons. Indeed, our lab was the first to discover the presence of the androgen receptor (AR) splice variant, AR45, in the brain. Subcellular localization of AR45 is in the lipid rafts of the plasma membrane in several brain regions affected by neurodegenerative disorders (eg. substantia nigra, hippocampus). We found testosterone can initiate signaling cascades via this membrane associated AR (mAR), leading to increased OS. However, the mechanism for OS generation is unknown. NADPH Oxidase 1 and 2 (NOX 1/2) are major OS generators, and potential targets for androgen‐induced OS and cell death. Based on our studies showing protein‐protein interactions between NOX1/2, AR45, and Gα q, we hypothesize testosterone increases OS by activating mAR complexed with NOX 1/2, initiating IP 3 signaling. Using an immortalized neuronal cell line (N27 cells), we exposed cells to hydrogen peroxide (H 2 O 2 ) prior to testosterone (100 nM) or DHT‐BSA (500nM). Inhibitors were used to examine G protein, androgen receptor, IP 3 and NOX1/2 signaling. Cell viability and OS were quantified. In addition to in vitro experiments, we examined the effects of NOX 1/2 on DHT exacerbation of chronic intermittent hypoxia, CIH (AHI=10) induced OS by treating adult male Long Evans rats with the NOX1/2 inhibitor, apocynin (4mg/kg). Classical AR antagonists did not block testosterone's negative effects, indicating the classical AR does not mediate these effects. Since AR antagonists do not block mAR, we used an AR protein degrader, ASC‐J9 (5uM). Unlike AR antagonists, the AR degrader blocked testosterone's negative effects. Next, we examined signaling cascades associated with proteins complexed with mAR‐AR45, such as NOX1/2 and Gα q . To block NOX actions, we used apocynin (10 uM), a nonspecific NOX inhibitor. Apocynin did not alter H 2 O 2 ‐induced cell loss, indicating H 2 O 2 increases OS via a non‐NOX mechanism. However, apocynin completely blocked testosterone induced cell loss and OS, suggesting the involvement of NOX1/2. Consistent with our in vitro data, apocynin also decreased OS generation in DHT‐treated rats exposed to the oxidative stressor, CIH, during sleep phase for 7 days. Inhibition of Gα q or G protein activation did not alter testosterone's negative effects on cell viability. However, inhibition of IP 3 receptor blocked these effects. Interestingly, NOX can influence IP 3 receptor mediated signaling, indicating that testosterone may activate IP 3 signaling via the mAR‐NOX complex and not the mAR‐ Gα q complex localized in membrane lipid raft. Future studies will examine the mAR‐NOX complex as a therapeutic target for neurodegenerative diseases. Support or Funding Information NIH/NINDS R01 NS088514 to RLC This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal .Keywords:
NOX1
Dihydrotestosterone
Arterial hypertension is associated with increased levels of reactive oxygen species, which may scavenge endothelium-derived NO and thereby diminish its vasorelaxant effects. However, the quantitatively relevant source of reactive oxygen species is unclear. Thus, this potential pathomechanism is not yet pharmacologically targetable. Several enzymatic sources of reactive oxygen species have been suggested: uncoupled endothelial NO synthase, xanthine oxidase, and NADPH oxidases. Here we show that increased reactive oxygen species formation in aortas of 12- to 14-month-old spontaneously hypertensive rats versus age-matched Wistar Kyoto rats is inhibited by the specific NADPH oxidase inhibitor VAS2870 but neither by the xanthine oxidase inhibitor oxypurinol nor the NO synthase inhibitor N(G)-nitro-l-arginine methyl ester. NADPH oxidase activity, as well as protein expression of its catalytic subunits, NOX1 and NOX2, was increased in the aortas of spontaneously hypertensive rats, whereas the expression of NOX4 protein, the most abundant NOX isoform, was not significantly changed. Impaired acetylcholine-induced relaxation of spontaneously hypertensive rat aortas was significantly improved by VAS2870. In conclusion, NOX1 and NOX2 but not NOX4 proteins are increased in aged spontaneously hypertensive rat aortas. Importantly, these NOX isoforms, in particular, ectopic expression of NOX1 in endothelial cells, appear to affect vascular function in an NADPH oxidase inhibitor-reversible manner. NADPH oxidases may, thus, be a novel target for the treatment of systemic hypertension.
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NOX4
Endothelial Dysfunction
NAD(P)H oxidase
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NOX1
Endothelial Dysfunction
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NOX1
Mitochondrial ROS
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Vascular tissue
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Androgens secreted by the testes bind the androgen receptor in developing target tissues to induce the expression of genes required for male sexual differentiation and development. Androgen concentration and androgen receptor levels vary in male reproductive target tissues during development. Exposure to environmental androgen antagonists during critical windows of fetal and postnatal development can inhibit male sexual development by blocking transcription of androgen-dependent genes. As the sensitivity to androgen antagonists under conditions of varying androgen concentrations and varying androgen receptor levels is unknown, we used a luciferase reporter assay to investigate the transcriptional effects of a known androgen antagonist (the vinclozolin metabolite M2) at different androgen concentrations and different androgen receptor levels. The ability of M2 to inhibit transcription was greater at lower concentrations of androgen (5α-dihydrotestosterone) and androgen receptor. The data were modeled to determine the dose-response surface of M2 and androgen receptor concentrations at different 5α-dihydrotestosterone levels and the relationship of the 3 components to the response. The model and hypothesis testing results suggest that, at 0.01 and 0.1 nM 5α-dihydrotestosterone concentrations within the expected in vivo range of free androgen levels during development, the response-surface shapes were similar and the interaction of the androgen receptor and M2 concentrations to the response were similarly antagonistic. Thus, two components of the developmental stage, androgen and androgen receptor concentrations, are critical for sensitivity to the inhibitory effects of an androgen antagonist.
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The involvement of reactive oxygen species (ROS) in psychiatric disorders has been reported. However, the source of ROS has not been identified yet. NADPH oxidase is a superoxide-generating enzyme composed of multiple subunits including a membrane-spanning catalytic subunit, NOX. We investigated the role of NOX1/NADPH oxidase in the anxiety-like behavior using mice deficient in Nox1(NOX1-KO).
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The Noxes (NADPH oxidases) are a family of ROS (reactive oxygen species)-generating enzymes. Of the seven family members, four have been identified as important sources of ROS in the vasculature: Nox1, Nox2, Nox4 and Nox5. Although Nox isoforms can be influenced by the same stimulus and co-localize in cellular compartments, their tissue distribution, subcellular regulation, requirement for cofactors and NADPH oxidase subunits and ability to generate specific ROS differ, which may help to understand the multiplicity of biological functions of these oxidases. Nox4 and Nox5 are the newest isoforms identified in the vasculature. Nox4 is the major isoform expressed in renal cells and appear to produce primarily H2O2. The Nox5 isoform produces ROS in response to increased levels of intracellular Ca2+ and does not require the other NADPH oxidase subunits for its activation. The present review focuses on these unique Noxes, Nox4 and Nox5, and provides novel concepts related to the regulation and interaction in the vasculature, and discusses new potential roles for these isoforms in vascular biology.
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Objective(s) Increased reactive oxygen species (ROS) production is implicated in the pathogenesis of arterial hypertension and the development of endothelial dysfunction. NADPH oxidase type enzyme family has been suggested to form ROS and to interfere with endothelium-dependent relaxation. However, the specific isoform of NADPH oxidases that may predominantly contribute to these events remains to be clarified. Materials and Methods Here we investigated the expressional regulation of NADPH oxidase isoforms (NOX1, NOX2 and NOX4) in aorta of aged spontaneously hypertensive rats (SHR) in comparison to age matched Wistar Kyoto rats (WKY). Moreover, we examined the effect of in vitro inhibition of NADPH oxidase by apocynin or the novel NADPH oxidase inhibitor, VAS2870 on the vascular reactivity and ROS production. Results Our results showed that ROS formation was largely increased in aorta of SHR as measured by dihydroethidine (DHE) fluorescence and inhibited by apocynin or VAS2870. NADPH oxidase activity, measured by lucigenin-enhanced chemiluminescence and of NOX1 and NOX2 protein levels were increased in aortic homogenates from SHR compared to WKY. However, NOX4 protein expression was not significantly changed. Furthermore, the impaired acetylcholine-induced relaxation of SHR aorta was significantly improved in the presence of either apocynin or VAS2870. Conclusion Collectively, our data suggest that NADPH oxidases, particularly NOX1 and NOX2 are relevant sources of ROS in the aorta of aged SHR thereby cause endothelial dysfunction, and VAS2870 is effective as apocynin in reversing these consequences.
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NOX1
NOX4
Endothelial Dysfunction
P22phox
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