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M. Livia Bajenaru, Andrea Rachelle C. Santos, Betty A. Obeso, Ephraim Trakhtenberg, Dimitry Ivanov, Jamie Ponmattam, Sebastian Gutierrez, Eleut Hernandez, M Elizabeth Fini, Jeffrey L. Goldberg; β1 Integrin-Focal Adhesion Kinase (FAK) Signaling Modulates Retinal Ganglion Cells Survival. Invest. Ophthalmol. Vis. Sci. 2012;53(14):3482.
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Retinal ganglion cells (RGCs) survival and neurite outgrowth are promoted by neurotrophins and extracellular matrix (ECM) molecules, such as laminin. Integrins are the main receptors for ECM. Previously, we showed that rat retinal ischemic injury (RIRI) results in down-regulation of the integrin survival pathway in RGC as evidenced by decreased expression of β1 integrin, FAK, and Akt dephosphorylation, and a reduction in anti-apoptotic protein bcl-xL. In this study, we further investigate the role of β1 integrin, and FAK in RGC survival, and neurite growth.
β1 integrin activating (HUTS-21), or isotype control antibodies were added to purified RGCs, 12 hours after plating. In separate experiments, RGCs were preincubated with PP2, before HUTS-21 addition. RGCs viability was assessed 48, and 72 hours later by live cell imaging. To inhibit FAK expression, RGCs were electroporated with custom-made FAK, or control siRNA (Dharmacon, Inc) using Nucleofactor II (Amaxa). Knockdown of FAK in RGCs was tested 24-72 hours later by RT-PCR and immunohistochemistry. RGC survival and neurite outgrowth were evaluated by Cellomics (Thermo Scientific). RIRI was induced in Sprague-Dawley rats by unilateral elevation of intraocular pressure to 110 mm Hg for 1 hour (n=16). The animals were divided in 4 groups and HUTS-21, isotype control antibody, and PBS control were administered intravitreally into the rat eye 30 minutes, and 2 days post-RIRI. Animals were euthanized 5 days post-RIRI and fluorogold (FG) retrogradely labeled RGCs were counted in flat mounted retinas using AxioVision 4.7.
The activating antibody HUTS-21 mimicked the effect of laminin, and resulted in 70% RGC survival in vitro, compared to 50% by PDL. The FAK inhibitor PP2 abolished both laminin and HUTS-21 survival effect, suggesting that FAK is downstream of β1 integrin. siRNA treatment knocked-down FAK expression by 92% after 72 hrs, and resulted in 2-fold reduced survival, and 1.7-fold reduced neurite growth in RGC. RIRI resulted in 55% RGC death after 5 days, but intravitreal HUTS-21 administration in vivo significantly reduced RGC death to 20%, while the control antibody did not change RGC survival.
We demonstrated that β1 integrin and its downstream effector FAK are key regulators of the integrin survival pathway in RGCs. Intravitreal administration of the HUTS-21 antibody in the RIRI model has neuroprotective effect, and may be exploited to prevent RGCs death in ischemic optic neuropathy, and glaucoma.
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