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Soumya Navneet, Jing Wang, Xuezhi Cui, Sylvia B Smith; Elevated homocysteine (Hcy) upregulates the NRF2 antioxidant pathway in retinal Müller cells. Invest. Ophthalmol. Vis. Sci. 2017;58(8):5391.
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© ARVO (1962-2015); The Authors (2016-present)
Hcy, a sulfur containing, non-proteinogenic amino acid, is implicated in some retinal neurovascular diseases (e.g. glaucoma, diabetic retinopathy); whether Hcy is a biomarker or pathogenic is unknown. Studies of two hyperhomocysteinemic (HHcy) mouse strains revealed modest ganglion cell (RGC) loss, diminished nerve fiber layer thickness & mild-moderate vasculopathy (Markand et al, 2015; Tawfik et al, 2014). Interestingly, when primary RGCs are exposed to slightly elevated Hcy [50µM] for 18h, 50% of RGCs die. Clearly, the intact retinal milieu invokes mechanisms that buffer excess Hcy. Given that Müller glial cells (MCs) maintain retinal homeostasis & NRF2, which regulates >500 antioxidant genes, plays a key role in cellular antioxidant responses, we hypothesized that HHcy upregulates the NRF2-mediated stress response in MCs.
1°MCs isolated from C57BL/6J mice were exposed 3-24h to L-Hcy-thiolactone [50µM – 10mM] and assessed for: (a) viability using a resazurin-based reagent; (b) gliosis detected by measuring GFAP; (c) reactive oxygen species (ROS) using DCFDA & CellRox; (d) glutathione (GSH) using the Enzo calorimetric assay. Gene/protein levels of Nrf2 & levels of NRF2-regulated antioxidants (NQO1, CAT, SOD2, HMOX1, GPX1) were assessed by qRT-PCR & immunoblotting in 1°MCs exposed to [50µM] Hcy. Experiments were performed in triplicate & data analyzed by ANOVA.
Unlike 1°RGCs, 1°MCs are viable when exposed to [50µM-1mM] Hcy, though significant dose-dependent cell death occurs at high (>2mM) Hcy concentrations. GFAP levels increased 2 fold in 1°MCs exposed to 50µM Hcy compared to controls, indicating gliosis. Interestingly, ROS levels decreased 10-15% in 1°MCs exposed 3h to [50µM or 1mM] Hcy and GSH levels increased 20% within 24h exposure to [50µM or 1mM] Hcy. Molecular analyses of 1°MCs exposed 3-8h to 50µM Hcy revealed 2 fold increase in Nrf2 gene expression. Expression of antioxidant genes Nqo1, Cat, Sod2, Hmox1, Gpx1 increased significantly as did protein levels of HMOX1 & CAT.
The apparent buffering within retinas of HHcy mice (versus marked vulnerability of primary RGCs) may reflect a ‘beneficial’ gliosis induced by Hcy. Our data indicate that at least short term, mild HHcy exposure induces a cytoprotective response mediated by NRF2 in Müller cells, the duration of which in vivo is unknown, but is being investigated.
This is an abstract that was submitted for the 2017 ARVO Annual Meeting, held in Baltimore, MD, May 7-11, 2017.
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