Cystathionine beta synthase expression in mouse retina

Shanu Markand, Amany Tawfik, Yonju Ha, Jaya Gnana-Prakasam, Srinivas Sonne, Vadivel Ganapathy, Nilkantha Sen, Ming Xian, Sylvia B. Smith

Research output: Contribution to journalArticle

11 Citations (Scopus)

Abstract

Purpose: Cystathionine β-synthase (CBS), a key enzyme in the transsulfuration metabolic pathway, converts homocysteine to cystathionine, which is converted to cysteine required for the synthesis of major retinal antioxidant glutathione (GSH). Enzyme activity assays suggest that CBS is present in human and pig retina, however recent studies reported that CBS is not expressed in mouse retina. We found this species difference puzzling. Given the plethora of studies using mouse retina as a model system, coupled with the importance of GSH in retina, we investigated CBS expression in mouse retina at the molecular and cell biological level. Methods: Wildtype (WT) mice or mice lacking the gene encoding CBS (cbs-/-) were used in these studies. RNA and protein were isolated from retinas and liver (positive control) for the analysis of cbs gene expression by RT-PCR and CBS protein expression by Western blotting, respectively. CBS was analyzed by immunofluorescence in retinal cryosections and primary retinal cells (ganglion, Müller, retinal pigment epithelial). CBS enzyme activity was measured in primary Müller cells. Results: RT-PCR revealed robust cbs expression in WT liver, brain and retina. Western blotting detected CBS in retina, brain and liver of WT mice, but not in cbs-/- mice liver. In immunohistochemical studies, CBS was present abundantly in the ganglion cell layer of retina; it was detected also in primary isolations of Müller, RPE and ganglion cells. CBS activity was detected in Müller cells by fluorescent detection of H2S. Conclusions: We have compelling molecular evidence that CBS is expressed in mouse retina at the gene and protein level. Our immunofluorescence data suggest that it is present in several retinal cell types and the data from the enzyme activity assay suggest activity in Müller cells. These findings set the stage to investigate the role of CBS and the transsulfuration pathway in the generation of GSH in mouse retina.

Original languageEnglish (US)
Pages (from-to)597-604
Number of pages8
JournalCurrent Eye Research
Volume38
Issue number5
DOIs
StatePublished - May 2013
Externally publishedYes

Fingerprint

Cystathionine beta-Synthase
Retina
Cystathionine
Liver
Enzyme Assays
Ganglia
Fluorescent Antibody Technique
Western Blotting
Polymerase Chain Reaction
Proteins
Retinal Pigments
Retinal Ganglion Cells
Brain
Homocysteine
Enzymes
Metabolic Networks and Pathways
Glutathione
Cysteine
Swine

Keywords

  • Antioxidant
  • Glutathione
  • Hydrogen sulfide
  • Primary cell culture
  • Retinal ganglion cells
  • Retinal Müller cells
  • Retinal pigment epithelial cells

ASJC Scopus subject areas

  • Ophthalmology
  • Sensory Systems
  • Cellular and Molecular Neuroscience

Cite this

Markand, S., Tawfik, A., Ha, Y., Gnana-Prakasam, J., Sonne, S., Ganapathy, V., ... Smith, S. B. (2013). Cystathionine beta synthase expression in mouse retina. Current Eye Research, 38(5), 597-604. https://doi.org/10.3109/02713683.2013.774024

Cystathionine beta synthase expression in mouse retina. / Markand, Shanu; Tawfik, Amany; Ha, Yonju; Gnana-Prakasam, Jaya; Sonne, Srinivas; Ganapathy, Vadivel; Sen, Nilkantha; Xian, Ming; Smith, Sylvia B.

In: Current Eye Research, Vol. 38, No. 5, 05.2013, p. 597-604.

Research output: Contribution to journalArticle

Markand, S, Tawfik, A, Ha, Y, Gnana-Prakasam, J, Sonne, S, Ganapathy, V, Sen, N, Xian, M & Smith, SB 2013, 'Cystathionine beta synthase expression in mouse retina', Current Eye Research, vol. 38, no. 5, pp. 597-604. https://doi.org/10.3109/02713683.2013.774024
Markand S, Tawfik A, Ha Y, Gnana-Prakasam J, Sonne S, Ganapathy V et al. Cystathionine beta synthase expression in mouse retina. Current Eye Research. 2013 May;38(5):597-604. https://doi.org/10.3109/02713683.2013.774024
Markand, Shanu ; Tawfik, Amany ; Ha, Yonju ; Gnana-Prakasam, Jaya ; Sonne, Srinivas ; Ganapathy, Vadivel ; Sen, Nilkantha ; Xian, Ming ; Smith, Sylvia B. / Cystathionine beta synthase expression in mouse retina. In: Current Eye Research. 2013 ; Vol. 38, No. 5. pp. 597-604.
@article{e4b36c3c2ba246269c2eda98ddac97ac,
title = "Cystathionine beta synthase expression in mouse retina",
abstract = "Purpose: Cystathionine β-synthase (CBS), a key enzyme in the transsulfuration metabolic pathway, converts homocysteine to cystathionine, which is converted to cysteine required for the synthesis of major retinal antioxidant glutathione (GSH). Enzyme activity assays suggest that CBS is present in human and pig retina, however recent studies reported that CBS is not expressed in mouse retina. We found this species difference puzzling. Given the plethora of studies using mouse retina as a model system, coupled with the importance of GSH in retina, we investigated CBS expression in mouse retina at the molecular and cell biological level. Methods: Wildtype (WT) mice or mice lacking the gene encoding CBS (cbs-/-) were used in these studies. RNA and protein were isolated from retinas and liver (positive control) for the analysis of cbs gene expression by RT-PCR and CBS protein expression by Western blotting, respectively. CBS was analyzed by immunofluorescence in retinal cryosections and primary retinal cells (ganglion, M{\"u}ller, retinal pigment epithelial). CBS enzyme activity was measured in primary M{\"u}ller cells. Results: RT-PCR revealed robust cbs expression in WT liver, brain and retina. Western blotting detected CBS in retina, brain and liver of WT mice, but not in cbs-/- mice liver. In immunohistochemical studies, CBS was present abundantly in the ganglion cell layer of retina; it was detected also in primary isolations of M{\"u}ller, RPE and ganglion cells. CBS activity was detected in M{\"u}ller cells by fluorescent detection of H2S. Conclusions: We have compelling molecular evidence that CBS is expressed in mouse retina at the gene and protein level. Our immunofluorescence data suggest that it is present in several retinal cell types and the data from the enzyme activity assay suggest activity in M{\"u}ller cells. These findings set the stage to investigate the role of CBS and the transsulfuration pathway in the generation of GSH in mouse retina.",
keywords = "Antioxidant, Glutathione, Hydrogen sulfide, Primary cell culture, Retinal ganglion cells, Retinal M{\"u}ller cells, Retinal pigment epithelial cells",
author = "Shanu Markand and Amany Tawfik and Yonju Ha and Jaya Gnana-Prakasam and Srinivas Sonne and Vadivel Ganapathy and Nilkantha Sen and Ming Xian and Smith, {Sylvia B.}",
year = "2013",
month = "5",
doi = "10.3109/02713683.2013.774024",
language = "English (US)",
volume = "38",
pages = "597--604",
journal = "Current Eye Research",
issn = "0271-3683",
publisher = "Informa Healthcare",
number = "5",

}

TY - JOUR

T1 - Cystathionine beta synthase expression in mouse retina

AU - Markand, Shanu

AU - Tawfik, Amany

AU - Ha, Yonju

AU - Gnana-Prakasam, Jaya

AU - Sonne, Srinivas

AU - Ganapathy, Vadivel

AU - Sen, Nilkantha

AU - Xian, Ming

AU - Smith, Sylvia B.

PY - 2013/5

Y1 - 2013/5

N2 - Purpose: Cystathionine β-synthase (CBS), a key enzyme in the transsulfuration metabolic pathway, converts homocysteine to cystathionine, which is converted to cysteine required for the synthesis of major retinal antioxidant glutathione (GSH). Enzyme activity assays suggest that CBS is present in human and pig retina, however recent studies reported that CBS is not expressed in mouse retina. We found this species difference puzzling. Given the plethora of studies using mouse retina as a model system, coupled with the importance of GSH in retina, we investigated CBS expression in mouse retina at the molecular and cell biological level. Methods: Wildtype (WT) mice or mice lacking the gene encoding CBS (cbs-/-) were used in these studies. RNA and protein were isolated from retinas and liver (positive control) for the analysis of cbs gene expression by RT-PCR and CBS protein expression by Western blotting, respectively. CBS was analyzed by immunofluorescence in retinal cryosections and primary retinal cells (ganglion, Müller, retinal pigment epithelial). CBS enzyme activity was measured in primary Müller cells. Results: RT-PCR revealed robust cbs expression in WT liver, brain and retina. Western blotting detected CBS in retina, brain and liver of WT mice, but not in cbs-/- mice liver. In immunohistochemical studies, CBS was present abundantly in the ganglion cell layer of retina; it was detected also in primary isolations of Müller, RPE and ganglion cells. CBS activity was detected in Müller cells by fluorescent detection of H2S. Conclusions: We have compelling molecular evidence that CBS is expressed in mouse retina at the gene and protein level. Our immunofluorescence data suggest that it is present in several retinal cell types and the data from the enzyme activity assay suggest activity in Müller cells. These findings set the stage to investigate the role of CBS and the transsulfuration pathway in the generation of GSH in mouse retina.

AB - Purpose: Cystathionine β-synthase (CBS), a key enzyme in the transsulfuration metabolic pathway, converts homocysteine to cystathionine, which is converted to cysteine required for the synthesis of major retinal antioxidant glutathione (GSH). Enzyme activity assays suggest that CBS is present in human and pig retina, however recent studies reported that CBS is not expressed in mouse retina. We found this species difference puzzling. Given the plethora of studies using mouse retina as a model system, coupled with the importance of GSH in retina, we investigated CBS expression in mouse retina at the molecular and cell biological level. Methods: Wildtype (WT) mice or mice lacking the gene encoding CBS (cbs-/-) were used in these studies. RNA and protein were isolated from retinas and liver (positive control) for the analysis of cbs gene expression by RT-PCR and CBS protein expression by Western blotting, respectively. CBS was analyzed by immunofluorescence in retinal cryosections and primary retinal cells (ganglion, Müller, retinal pigment epithelial). CBS enzyme activity was measured in primary Müller cells. Results: RT-PCR revealed robust cbs expression in WT liver, brain and retina. Western blotting detected CBS in retina, brain and liver of WT mice, but not in cbs-/- mice liver. In immunohistochemical studies, CBS was present abundantly in the ganglion cell layer of retina; it was detected also in primary isolations of Müller, RPE and ganglion cells. CBS activity was detected in Müller cells by fluorescent detection of H2S. Conclusions: We have compelling molecular evidence that CBS is expressed in mouse retina at the gene and protein level. Our immunofluorescence data suggest that it is present in several retinal cell types and the data from the enzyme activity assay suggest activity in Müller cells. These findings set the stage to investigate the role of CBS and the transsulfuration pathway in the generation of GSH in mouse retina.

KW - Antioxidant

KW - Glutathione

KW - Hydrogen sulfide

KW - Primary cell culture

KW - Retinal ganglion cells

KW - Retinal Müller cells

KW - Retinal pigment epithelial cells

UR - http://www.scopus.com/inward/record.url?scp=84875891588&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=84875891588&partnerID=8YFLogxK

U2 - 10.3109/02713683.2013.774024

DO - 10.3109/02713683.2013.774024

M3 - Article

VL - 38

SP - 597

EP - 604

JO - Current Eye Research

JF - Current Eye Research

SN - 0271-3683

IS - 5

ER -