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N. Marsh-Armstrong, E. Cox, I. Soto; Gamma Synuclein Changes Cellular Distribution in Glaucomatous DBA/2J Mice. Invest. Ophthalmol. Vis. Sci. 2008;49(13):5490.
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We have recently shown that gamma synuclein (Sncg) mRNA is expressed specifically in retinal ganglion cells (RGCs) of the adult mouse and its expression declines prior to RGC loss in a mouse model of glaucoma, the DBA/2J mouse. In order to determine whether a corresponding decline occurs for the Sncg protein, we generated an Sncg antibody and used it to study Sncg protein expression in the context of RGC loss in DBA/2J mice.
Retinas and optic nerves of 3month (m) and 10m DBA/2J and 2m and 22m C57Bl/6J mice were compared using an antibody generated against the mouse Sncg C-terminus. Most retinas were processed as flatmounts for Sncg mRNA in situ hybridization, Sncg and phosphorylated neurofilament antibody labeling, and a modified TUNEL assay to label all nuclei in the ganglion cell layer. We examined the number of RGCs, the correspondence of Sncg protein and mRNA, the somatic versus nuclear localization of Sncg protein, and the amount of axonal Sncg protein. Optic nerves were embedded in OCT and sectioned longitudinally or transversely, and processed for in situ hybridization for Sncg mRNA as well as PLP and Vimentin mRNAs, markers for oligodendrocytes and optic nerve astrocytes respectively, as well as antibodies for Sncg, phosphorylated neurofilament, Iba1 and NG2.
In young and old C57Bl/6J and young DBA/2J miced, gamma synuclein is found in multiple compartments of RGCs, including dendrites, soma, and axons. While some RGCs have detectable Sncg protein in the nucleus, the majority of Sncg protein is found outside the nucleus in the majority of RGCs. In contrast, in old DBA/2J retinas, large numbers of RGCs with strong nuclear expression of Sncg are found in discrete clusters. In the optic nerve, the majority of the gamma synuclein protein expression is lost as the axons are lost, but now Sncg protein and mRNA are found in discrete groups of glial cells.
Dramatic changes in the distribution of gamma synuclein protein within retinal ganglion cells occur specifically in aged DBA/2J mice. Nuclear accumulation of gamma synuclein precedes loss of many if not most RGCs. Understanding this nuclear accumulation and other changes in gamma synuclein expression will likely yield insight into other molecular changes occurring within RGCs during glaucoma.
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