Excess homocysteine upregulates the NRF2-antioxidant pathway in retinal Müller glial cells

This study evaluated the effects of elevated homocysteine (Hcy) on the oxidative stress response in retinal Muller glial cells. Elevated Hcy has been implicated in retinal diseases including glaucoma and optic neuropathy, which are characterized by retinal ganglion cell (RGC) loss. To understand the mechanisms of Hcy-induced RGC loss, in vitro and in vivo models have been utilized. In vitro isolated RGCs are quite sensitive to elevated Hcy levels, while in vivo murine models of hyperhomocysteinemia (HHcy) demonstrate a more modest RGC loss (∼20%) over a period of many months. This differential response to Hcy between isolated cells and the intact retina suggests that the retinal milieu invokes mechanisms that buffer excess Hcy. Oxidative stress has been implicated as a mechanism of Hcy-induced neuron loss and NRF2 is a transcription factor that plays a major role in regulating cytoprotective responses to oxidative stress. In the present study we investigated whether HHcy upregulates NRF2-mediated stress responses in Muller cells, the chief retinal glial cell responsible for providing trophic support to retinal neurons. Primary Muller cells were exposed to L-Hcy-thiolactone [50μM–10mM] and assessed for viability, reactive oxygen species (ROS), and glutathione (GSH) levels. Gene/protein levels of Nrf2 and levels of NRF2-regulated antioxidants (NQO1, CAT, SOD2, HMOX1, GPX1) were assessed in Hcy-exposed Muller cells. Unlike isolated RGCs, isolated Muller cells are viable over a wide range of Hcy concentrations [50 μM – 1 mM]. Moreover, when exposed to elevated Hcy, Muller cells demonstrate decreased oxidative stress and decreased ROS levels. GSH levels increased by ∼20% within 24 h exposure to Hcy. Molecular analyses revealed 2-fold increase in Nrf2 expression. Expression of antioxidant genes Nqo1, Cat, Sod2, Hmox1, Gpx1 increased significantly. The consequences of Hcy exposure were evaluated also in Muller cells harvested from Nrf2−/− mice. In contrast to WT Muller cells, in which oxidative stress decreased upon exposure to Hcy, the Nrf2−/− Muller cells showed a significant increase in oxidative stress. Our data suggest that at least during early stages of Hhcy, a cytoprotective response may be in place, mediated in part by NRF2 in Muller cells.