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A. P. Surguchov, I. Surgucheva; Oxidative Stress-Induced Alterations in Transcriptome in Retinal Ganglion Cells. Invest. Ophthalmol. Vis. Sci. 2009;50(13):860.
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© ARVO (1962-2015); The Authors (2016-present)
Glaucomatous optic neuropathy implies a loss of retinal ganglion cells (RGC). Oxidative stress (OS) plays an important role in the pathophysiology of glaucomatous RGC death. The loss of RGC is also associated with the decrease of γ-synuclein - a protein which is highly expressed in these cells and may be considered an RGC marker (Mol Vis. 2008, 14:1540-8). Knock-down (KD) of γ-synuclein in RGC induces Bad dephosphorylation, its translocation to mitochondria and initiation of the apoptotic death cascade (JBC, 2008 Oct 20). In this study we investigated how OS affected the gene expression in RGC with normal and reduced levels of γ-synuclein.
OS was induced by the treatment of RGC-5 cells with hydrogen peroxide (300 mkM) which caused a loss of cell viability by the apoptosis. RNA was isolated from hydrogen peroxide treated cells with normal and KD γ-synuclein, as well as from control cells. RNA was converted to cDNA by reverse transcriptase and used for PCR and qRT-PCR. The following genes which are the targets of γ-synuclein were selected: periostin (ECM protein controlling cell adhesion and migration), potassium voltage-gated channel (Kcnh2) , axonal growth cone guidance protein semaphorin (Sema 3A), a nervous tissue-specific growth associated protein 43 (Gap43), Zn-finger transcription factor Wilms tumor 1 (Wt-1), oncogene serine/threonine kinase Pim-3, mitogen-activated protein kinase MAPK-8 , phosphoinositide 3-kinase (PI3K) class 1, protein phosphatases (PP2A regulatory -isoform, PP2A regulatory β-isoform), calmodulin 1, the ribosomal S6 protein kinase p70 S6 (p70 S6 kinase 1). β-Actin and 18S RNA were used as endogenous controls.
When the effect of OS and KD was analyzed separately, the expression of two genes was altered most significantly, i.e., Wt1 and periostin. However, their expression was changed in different directions. KD downregulated Wt1 (0.5) and upregulated periostin (5.2), whereas OS upregulated Wt1 (4.2) and downregulated periostin (0.19) in cells with both normal and decreased γ-synuclein level. When two stress conditions acted together (OS+KD), the upregulation of Wt-1 and downregulation of periostin persisted in OS-treated, but not in KD cells. The expression of - and β-synucleins was upregulated by stress conditions, which may reflect their properties as chaperones.
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