Oxidative stress figures prominently in retinal diseases including diabetic retinopathy and glaucoma. Ligands for σ1R, a unique transmembrane protein localized to the ER, mitochondria, nuclear and plasma membrane, have profound retinal neuroprotective properties in vitro and in vivo. Studies to determine the mechanism of σ1R-mediated retinal neuroprotection have focused mainly on neurons. Little is known about effects of σ1R on Müller cell function, yet these radial glial cells are essential for homeostatic support of the retina. Here we investigated whether σ1R mediates the oxidative stress response of Müller cells using wildtype (WT) and σ1R knockout (σ1RKO) mice. We observed increased endogenous ROS levels in σ1RKO Müller cells compared to WT, which was accompanied by decreased expression of Sod1, Catalase, Nqo1, Hmox1, Gstm6 and Gpx1. The protein levels of SOD1, CAT, NQO1 and GPX1 were also significantly decreased. The genes encoding these antioxidants contain an antioxidant response element (ARE), which under stress is activated by NRF2, a transcription factor that typically resides in the cytoplasm bound by KEAP1. In the σ1RKO Müller cells Nrf2 expression was decreased significantly at the gene (and protein) level, while Keap1 gene (and protein) levels were markedly increased. NRF2-ARE binding affinity was decreased markedly in σ1RKO Müller cells. We investigated system xc−, the cystine-glutamate exchanger important for synthesis of GSH, and observed decreased function in σ1RKO Müller cells compared to WT as well as decreased GSH and GSH/GSSG ratios. This was accompanied by decreased gene and protein levels of xCT, the unique component of system xc−. We conclude that Müller glial cells lacking σ1R manifest elevated ROS, perturbation of antioxidant balance, suppression of NRF2 signaling and impaired function of system xc−. The data suggest that the oxidative stress-mediating function of retinal Müller glial cells may be compromised in the absence of σ1R. The neuroprotective role of σ1R may be linked directly to the oxidative stress-mediating properties of supportive glial cells.
Neuronal cell death contributes significantly to the pathology of traumatic brain injury (TBI) irrespective of the mode or severity of the injury. Activation of a pro-survival protein, Akt, is known to be regulated by an E3 ligase TRAF6 through a process of ubiquitination-coupled phosphorylation at its T308 residue. Here we show that upregulation of a pro-apototic protein, GADD34, attenuates TRAF6-mediated Akt activation in a controlled cortical impact model of TBI in mice. TBI induces the expression of GADD34 by stimulating binding of a stress inducible transcription factor, ATF4, to the GADD34 promoter. GADD34 then binds with TRAF6 and prevents its interaction with Akt. This event leads to retention of Akt in the cytosol and prevents phosphorylation at the T308 position. Finally, in vivo depletion of GADD34 using a lentiviral knockdown approach leads to a rescue of Akt activation and markedly attenuates TBI-induced cell death.
Mild to moderate hyperhomocysteinemia is prevalent in humans and is implicated in neurovascular diseases, including recently in certain retinal diseases. Herein, we used hyperhomocysteinemic mice deficient in the Cbs gene encoding cystathionine-β-synthase (Cbs(+/-)) to evaluate retinal vascular integrity. The Cbs(+/+) (wild type) and Cbs(+/-) (heterozygous) mice (aged 16 to 52 weeks) were subjected to fluorescein angiography and optical coherence tomography to assess vasculature in vivo. Retinas harvested for cryosectioning or flat mount preparations were subjected to immunofluorescence microscopy to detect blood vessels (isolectin-B4), angiogenesis [anti-vascular endothelial growth factor (VEGF) and anti-CD105], gliosis [anti-glial fibrillary acidic protein (GFAP)], pericytes (anti-neural/glial antigen 2), blood-retinal barrier [anti-zonula occludens protein 1 (ZO-1) and anti-occludin], and hypoxia [anti-pimonidazole hydrochloride (Hypoxyprobe-1)]. Levels of VEGF, GFAP, ZO-1, and occludin were determined by immunoblotting. Results of these analyses showed a mild vascular phenotype in young mice, which progressed with age. Fluorescein angiography revealed progressive neovascularization and vascular leakage in Cbs(+/-) mice; optical coherence tomography confirmed new vessels in the vitreous by 1 year. Immunofluorescence microscopy demonstrated vascular patterns consistent with ischemia, including a capillary-free zone centrally and new vessels with capillary tufts midperipherally in older mice. This was associated with increased VEGF, CD105, and GFAP and decreased ZO-1/occludin levels in the Cbs(+/-) retinas. Retinal vein occlusion was observed in some Cbs(+/-) mouse retinas. We conclude that mild to moderate elevation of homocysteine in Cbs(+/-) mice is accompanied by progressive alterations in retinal vasculature characterized by ischemia, neovascularization, incompetent blood-retinal barrier, and vascular occlusion.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.