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J. Y. Koh, R. M. Sappington, D. J. Calkins; Effects of TRPV1 Overexpression and Silencing on Retinal Ganglion Cell Survival in vitro. Invest. Ophthalmol. Vis. Sci. 2007;48(13):4358.
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© ARVO (1962-2015); The Authors (2016-present)
TRPV1, a transient receptor potential (TRP) receptor, is expressed in an array of central nervous system cells including neurons, astrocytes, and microglia. It is a non-selective cation channel characterized by a robust Ca2+ conductance that contributes to pressure, heat sensitivity and to Ca2+ -dependent apoptosis when activated. These properties led us to propose that activation of TRPV1 could contribute to Ca2+-dependent degeneration of RGCs and their axons in response to a pressure stimulus, as in glaucoma. We have now characterized the in vivo expression of TRPV1 in the DBA2J glaucoma mouse model and the effects of TRPV1 overexpression in the RGC-5 transformed retinal ganglion cell line.
TRPV1 rat cDNA was cloned into an over-expression plasmid and transfected into the RGC-5 cell line. Transfected cells were positively identified by the co-transcribed GFP. To study the effects of TRPV1 suppression, RGC-5 cells were transfected with an AAV2 virus containing 21 base pairs of DNA engineered to silence TRPV1 expression by RNA interference. Transfected cells were positively identified by the coexpression of dsRED2. To test whether cell survival was influenced by Ca2+ conductance, transfected RGC-5 cells were cultured in the presence or absence of ruthenium red, a broad spectrum TRPV antagonist. Cells were fixed, permeabilized, and subjected to TUNEL staining in response to TRPV1 overexpression, gene silencing or antagonism.
The in vivo expression levels of TRPV1 in the retina of aged, high IOP DBA2J animals by quantitative PCR show that TRPV1 gene expression is upregulated by nearly 20-fold compared to 3 month, low IOP DBA2J retina. Overexpression of TRPV1 in RGC-5 cells resulted in a 50% loss of cells due to apoptosis. This was suppressed by the addition to the culture of 7.0mM ruthenium red. Similarly, cotransfection of RGC-5 cells with a TRPV1 siRNA AAV2 virus promoted survival by decreasing gene expression of TRPV1.
These results suggest that TRPV1 plays an important role in maintaining normal calcium homeostasis in RGCs. TRPV1 overexpression apparently results in a critical inability to properly gate calcium in the cell; whether this fault occurs at the endoplasmic reticulum, an important calcium storage vessel, or at the plasma membrane is currently being determined. Furthermore, we have constructed a TRPV1 siRNA containing AAV2 virus that reduces TRPV1 expression in vitro. We are determining its effects in an in vivo acute pressure rat model to assess whether regulation of TRPV1 expression in RGCs in vivo may have potent therapeutic effects in the treatment of glaucoma.
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