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Yan ge, Zhiyong Yang, Jun Wan, Cindy Berlinicke, Jiang Qian, Harry A. Quigley, Donald J. Zack; Promoting Retinal Ganglion Cell Axonal Regeneration by Inhibition of Transforming Growth Factor Beta (TGFβ) Signaling. Invest. Ophthalmol. Vis. Sci. 2011;52(14):4617.
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Retinal ganglion cells (RGCs) and their axons are subject to injury in various diseases such as glaucoma and other optic neuropathies. We performed image-based high-content screening (HCS) with mouse primary RGCs to uncover the signaling pathway(s) that are important in regulating RGC survival and/or axon regeneration, and to identify candidate compound(s) that could serve as leads for development of novel treatment approaches for diseases involving RGC cell body and/or axonal injury.
RGCs from postnatal day 1-5 (P1-P5) C57BL/6 mice were isolated by immunopanning using a monoclonal antibody to Thy1.2. RGC viability and neurite growth were assessed after three days in culture using the Cellomics VTI imaging system combined with calcein, ethidium homodimer, and Hoescht staining. Neurite identities were determined by Microtubule-associated protein 2b (Map2b) and Tau immunohistochemistry. The axon-promoting activity of TGFβ receptor inhibitor (TGFβRI) was compared to those of Brain-derived neurotrophic factor (BDNF), Ciliary Neurotrophic factor (CNTF), and Glial cell-derived neurotrophic factor (GDNF) in cell culture and retinal explants. Intravitreal injections of a TGFβRI were performed after optic nerve crush in mice, and subsequent RGC axon regeneration was monitored by growth-associated protein 43(GAP43) immunostaining. Alterations in global gene expression induced by TGFβRI in the mouse primary RGCs were profiled with Affymetrix mouse exon 1.0ST arrays. Quantitative PCR (qPCR) was used to confirm the expression changes of candidate genes.
We identified a TGFβRI that can strongly stimulate RGC neurite outgrowth in vitro. The efficacy of the TGFβRI in promoting neurite growth is greater than that of BDNF, CNTF and GDNF in both cultured primary mouse RGCs and retinal explants. In addition, intravitreal delivery of TGFβRI can enhance RGC axon regeneration following optic nerve injury. Treatment of RGCs with TGFβRI resulted in significant changes in the expression of approximately 200 genes, including Calindin-28k and Sema3e.
Modulating the TGFβ signaling pathway can promote RGC axon regeneration both in vitro and in vivo, and may serve as a novel therapeutic approach for promoting RGC axon survival and regeneration.
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