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I. G. Surgucheva, A. D. Weisman, J. L. Goldberg, A. Surguchov; Is Gamma-Synuclein a Specific Marker of the Retinal Ganglion Cells?. Invest. Ophthalmol. Vis. Sci. 2008;49(13):1650. doi: https://doi.org/.
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Earlier we showed that three members of the synuclein family, i.e., alpha, beta, and gamma-synucleins are differentially expressed in the retina. The localization of gamma-synuclein in ocular tissues is altered in glaucoma and other diseases. Here we investigated whether gamma-synuclein is a specific marker of retinal ganglion cells (RGCs) using human retina and primary cultures of RGCs.
Cultures of RGC-5 cells was kindly provided by Neeraj Agarwal (UNT Health Science Center, Fort Worth, Tx). For primary cultures of RGCs from Sprague-Dawley rats, cells were purified by sequential immunopanning using a monoclonal antibody to Thy1.1. The culture of immortalized optic nerve astrocytes A7 from newborn rats was received from Dr. H. Geller (UMDNJ). Immunofluorescence staining was performed as described previously (Cell Motility and Cytoskeleton, 63, 447-458, 2006). Images were taken using a Leica DMI 6000B inverted microscope. RNA was isolated from 4 x 106 cells using RNeasy Protect Mini Kit (Qiagen) using spin columns. The RNA was converted into cDNA using High-Capacity cDNA Reverse Transcription Kits with random primers. Relative quantification was performed using Quantitative Real-Time PCR (Applied Biosystems; 7300 instrument) with TaqMan reagents starter kit for two steps RT-qPCR. The samples were amplified in triplicate and three independent isolations of RNA/cDNA were used.
We found that gamma-synuclein is highly expressed in the RGC layer of human retina. Immunohistochemical staining of human retina revealed its presence in the cell body and in axons. Axons of RGCs are immunopositive for gamma-synuclein in both the lamina cribrosa and the retrobulbar optic nerve. In the optic nerve of glaucoma patients, axon swellings are immunopositive for gamma-synuclein. Changes in gamma-synuclein localization were also observed in the retina of patients with retinoblastoma. gamma-Synuclein is also highly expressed in immortalized RGC-5 cultures and in primary rat RGC cultures by RT-qPCR and immunostaining. In these cells, gamma-synuclein is localized in the cytoplasm and in cell processes.
gamma-Synuclein is extensively expressed in RGC in the retina, primary cultures of RGCs and in immortalized RGC-5 cells. It can be used for the studies of normal physiology of these cells and for the characterization of different pathological conditions. In pathologic states, gamma-synuclein abundance may vary between RGC somas and axons.
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