Neuron-Glia interaction and role of Nrf2 in hyperhomocysteinemic retina

Abstract

Elevated level of the excitatory amino acid homocysteine (Hcy) or hyperhomocysteinemia (Hhcy) has been reported in patients with glaucoma, a disease characterized by increased oxidative stress and retinal ganglion cell (RGC) degeneration. Whether Hhcy is causative or merely a biomarker for glaucoma is not known. Primary RGCs exhibit acute sensitivity to Hcy exposure, while in vivo murine models of Hhcy demonstrate a more modest RGC loss (∼20%) over a period of several months. This differential response to Hhcy in isolated cells and the intact retina suggests the presence a buffering mechanism invoked by the retinal milieu. Oxidative stress has been implicated as a mechanism of Hcy-induced neuronal loss. Owing to the key role of Müller glial cells (MCs) in retinal antioxidant defense we hypothesized that MCs protect RGCs under conditions of Hhcy via the NRF2 antioxidant pathway. Compared to RGCs, MCs were less sensitive to Hcy. Hcy exposure increased oxidative stress and induced apoptosis in RGCs, whereas in MCs Hcy evoked several cytoprotective responses including reduced oxidative stress, increased antioxidant levels and improved mitochondrial function. Hcy upregulated the expression of Nrf2 and several downstream antioxidant targets including glutathione in MCs. To investigate the role of NRF2 in Hcy-induced RGC degeneration, we crossed Nrf2-/- mice with two mouse models of chronic Hhcy (Cbs+/- and Mthfr+/- mice) and generated Cbs+/-Nrf2-/- and Mthfr+/-Nrf2-/- mouse models and analyzed their retinas. Absence of NRF2 reduced inner retinal thickness and visual acuity, accelerated RGC loss and increased gliosis in Hhcy mice. To understand the role of Müller glial specific NRF2 in RGC survival, we established an ex-vivo indirect co-culture system using primary RGCs and MCs. Apoptosis induced by Hcy exposure in primary RGCs were abrogated when the RGCs were co-cultured with wild type (WT) MCs but not with Nrf2-/- MCs. Hhcy induced robust mitochondrial and glycolytic response in WT MCs, but not in Nrf2-/- MCs. Altogether, the in vivo and in vitro data here suggest that the deleterious effects of Hhcy on RGCs are likely dependent upon the health of retinal glial cells and the availability of retinal antioxidant response mechanism.