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W. Huang, A.P. Dobberfuhl, T. Filippopoulos, J.B. Fileta, G.P. Kwon, C.L. Grosskreutz; Neuroprotection From Calcineurin–Mediated Retinal Ganglion Cell Apoptosis by FK506 in Experimental Glaucoma . Invest. Ophthalmol. Vis. Sci. 2005;46(13):4726.
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Purpose: We have previously shown that the autoregulatory domain of calcineurin (CaN) is cleaved in experimental glaucoma eyes leading to a constitutively active, potentially toxic form. The purpose of this study was to examine the intracellular signaling pathways involved in CaN–mediated RGC apoptosis and to test if CaN inhibition would be neuroprotective. Methods: Unilateral experimental glaucoma was induced in Brown Norway rats (Morrison model) treated with and without FK506 (5mg/kg/day). Cell loss was estimated with unbiased stereology using the optical fractionator. Immunoblot analysis of CaN, phospho–Bad, cytochrome c, and PARP were performed following 10 days of elevated intraocular pressure (IOP). Immunohistochemistry (IHC) of PARP was also performed. Results: Following 10 days of elevated IOP, the average integrated pressure–time difference between control and experimental eyes was 320.26 ± 54.23mmHg–days. The cleavage product of CaN appeared in all elevated IOP eyes. In elevated IOP eyes, the levels of cleaved CaN were significantly increased, while phospho–BAD levels were significantly reduced (p < 0.01). Cyt c was detected in the cytosol of elevated IOP eyes at a much greater level than controls, but a decreased level in the mitrochodrial fraction. PARP cleavage was significantly increased in elevated IOP eyes. In control retinas, only moderate cleaved PARP expression was observed by IHC in the ganglion cell layer (GCL). With elevated IOP, there was a significant increase in PARP immunoreactivity (IR) in the GCL and there was also more IR in the nucleus compared with control retinas where most of the IR was confined to the cytoplasm. FK506 treatment reduced RGC loss from 33 ± 2% (n=6) to 16 ± 5% (n=4), a 50% protection (p<0.01). Conclusions: These data support our hypothesis that IOP elevation leads to CaN activation triggering Bad dephosphorylation and translocation to the mitochondria with subsequent cyt c release, initiation of apoptosis, and PARP cleavage. Pharmacologic inhibition of CaN activity provided substantial neuroprotection.
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