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

Soumya Navneet, Xuezhi Cui, Jing Zhao, Jing Wang, Navneet Ammal Kaidery, Bobby Thomas, Kathryn Elizabeth Bollinger, Yisang Yoon, Sylvia B Smith

Research output: Contribution to journalArticle

3 Citations (Scopus)

Abstract

This study evaluated the effects of elevated homocysteine (Hcy) on the oxidative stress response in retinal Müller 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 Müller cells, the chief retinal glial cell responsible for providing trophic support to retinal neurons. Primary Müller 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 Müller cells. Unlike isolated RGCs, isolated Müller cells are viable over a wide range of Hcy concentrations [50 μM – 1 mM]. Moreover, when exposed to elevated Hcy, Müller 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 Müller cells harvested from Nrf2 −/− mice. In contrast to WT Müller cells, in which oxidative stress decreased upon exposure to Hcy, the Nrf2 −/− Müller 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 Müller cells.

Original languageEnglish (US)
Pages (from-to)228-237
Number of pages10
JournalExperimental eye research
Volume178
DOIs
StatePublished - Jan 1 2019

Fingerprint

Homocysteine
Neuroglia
Up-Regulation
Antioxidants
Oxidative Stress
Retinal Ganglion Cells
Hyperhomocysteinemia
Reactive Oxygen Species
Cats
Retinal Neurons
Retinal Diseases
Optic Nerve Diseases
Glaucoma
Glutathione
Retina
Buffers
Transcription Factors

Keywords

  • Exfoliative glaucoma
  • Hyperhomocysteinemia
  • Mouse
  • Optic neuropathy
  • Oxidative stress
  • Retinal glial cells

ASJC Scopus subject areas

  • Ophthalmology
  • Sensory Systems
  • Cellular and Molecular Neuroscience

Cite this

Excess homocysteine upregulates the NRF2-antioxidant pathway in retinal Müller glial cells. / Navneet, Soumya; Cui, Xuezhi; Zhao, Jing; Wang, Jing; Kaidery, Navneet Ammal; Thomas, Bobby; Bollinger, Kathryn Elizabeth; Yoon, Yisang; Smith, Sylvia B.

In: Experimental eye research, Vol. 178, 01.01.2019, p. 228-237.

Research output: Contribution to journalArticle

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abstract = "This study evaluated the effects of elevated homocysteine (Hcy) on the oxidative stress response in retinal M{\"u}ller 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 M{\"u}ller cells, the chief retinal glial cell responsible for providing trophic support to retinal neurons. Primary M{\"u}ller 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 M{\"u}ller cells. Unlike isolated RGCs, isolated M{\"u}ller cells are viable over a wide range of Hcy concentrations [50 μM – 1 mM]. Moreover, when exposed to elevated Hcy, M{\"u}ller 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 M{\"u}ller cells harvested from Nrf2 −/− mice. In contrast to WT M{\"u}ller cells, in which oxidative stress decreased upon exposure to Hcy, the Nrf2 −/− M{\"u}ller 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 M{\"u}ller cells.",
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author = "Soumya Navneet and Xuezhi Cui and Jing Zhao and Jing Wang and Kaidery, {Navneet Ammal} and Bobby Thomas and Bollinger, {Kathryn Elizabeth} and Yisang Yoon and Smith, {Sylvia B}",
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AU - Kaidery, Navneet Ammal

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AB - This study evaluated the effects of elevated homocysteine (Hcy) on the oxidative stress response in retinal Müller 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 Müller cells, the chief retinal glial cell responsible for providing trophic support to retinal neurons. Primary Müller 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 Müller cells. Unlike isolated RGCs, isolated Müller cells are viable over a wide range of Hcy concentrations [50 μM – 1 mM]. Moreover, when exposed to elevated Hcy, Müller 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 Müller cells harvested from Nrf2 −/− mice. In contrast to WT Müller cells, in which oxidative stress decreased upon exposure to Hcy, the Nrf2 −/− Müller 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 Müller cells.

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