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Ning Tian, Li Jiang, Ping Wang, Wolfgang Baehr; The Immune Protein CD3 Directly Regulates The Dendritic Morphology Of Rgcs In Mouse Retina. Invest. Ophthalmol. Vis. Sci. 2011;52(14):4559.
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Recent study suggests that a key component of T-cell receptor (TCR), CD3ζ, is expressed by retinal ganglion cells (RGCs) and that mice with a null mutation of CD3ζ have impaired RGC dendritic morphology. The goal of current study is to determine whether CD3ζ directly regulates the dendritic morphology of RGCs.
To determine whether expressing CD3ζ by AAV2 virus in RGCs reverses the dendritic defects of RGCs of CD3ζ-/- mice, we transfected retinas of Thy1-YFP/CD3ζ-/- mice with self-complementary (sc) double-strained AAV2-CD3ζ-mCherry vector at P3-5 and quantitatively examined the dendritic morphology of RGCs expressing both YFP and CD3ζ-mCherry at P13-14. To determine whether CD3ζ is intrinsically active in RGCs and regulates RGC dendrites directly, we knocked down CD3ζ in retinas of Thy1-YFP mice with AAV2-virus expressing CD3ζ specific shRNA and mCherry as a reporter at P3-5 and quantitatively examined the dendritic morphology of RGCs expressing both YFP and mCherry at P13-14.
Our data showed that intraocular injection of AAV2 packed with sc CD3ζ-mCherry or CD3ζ specific shRNA-mCherry (3X108 virus particles/eye) at P3-5 could effectively transfect a large number of retinal neurons at P13-14. The expression of CD3ζ-mCherry fusion protein in RGCs of Thy1-YFP/CD3ζ-/- mice significantly reduces the density of filopodia of RGCs at P13-14. However, RGCs not expressing CD3ζ-mCherry fusion protein in transfected eyes have significantly higher density of filopodia in comparison with WT mice and mCherry-expressing RGCs of Thy1-YFP/CD3ζ-/- mice. In addition, CD3ζ specific shRNA-mCherry transfected RGCs in Thy1-YFP mice have much increased density of filopodia while the mCherry-negative RGCs in CD3ζ specific shRNA-mCherry transfected eyes are not different from that of WT mice.
These results demonstrated that CD3ζ-mCherry fusion constructs and CD3ζ specific shRNA-mCherry constructs can be expressed by AAV2 virus in vivo in RGCs and the expressed CD3ζ protein and CD3ζ specific shRNA are functional in the regulation of RGC dendrites. In addition, the dendritic defects of RGCs in CD3ζ-/- mice are reversed by AAV2-induced expression of CD3ζ. Furthermore, the effectiveness of CD3ζ specific shRNA only in transfected RGCs of WT mice suggests that CD3ζ is intrinsically active in RGCs and regulates RGC dendrites directly.
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