Transcriptional up-regulation and activation of initiating caspases in experimental glaucoma

Abstract

In glaucoma, retinal ganglion cells (RGCs) die by apoptosis, generally attributed to an elevated intraocular pressure (IOP). We now describe the impact of elevated IOP in the rat on expression of caspase 8 and caspase 9, initiators of the extrinsic and intrinsic caspase cascades, respectively. Activation of both caspases was demonstrated by the presence of cleaved forms of the caspases and the detection of cleaved Bid and PARP, downstream consequences of caspase activation. Surprisingly, the absolute level of procaspase 9 was also elevated after 10 days of increased IOP. To examine the cause of increased levels of the procaspase, we used laser capture microdissection to capture Fluorogold back-labeled RGCs and real-time polymerase chain reaction to measure mRNA changes of initiating caspases. The mRNA levels of both caspase 8 and caspase 9 were increased specifically in RGCs. These data suggest that elevated IOP activates a transcriptional up-regulation and activation of initiating caspases in RGCs and triggers apoptosis through both extrinsic and intrinsic caspase cascades.

Figure 1

A: Procaspase 8, cleaved caspase 8, and tBid protein expression in whole retina after experimentally elevated IOP for 10 days. Elevated IOP resulted in an increase in caspase 8 cleavage and the presence of truncated Bid (lanes 2 and 4) compared to normal retinas (lanes 1 and 3). B: Quantification of the Western blots further confirmed that cleaved caspase 8 and tBid levels increase in elevated IOP retinas, however, procaspase 8 levels did not change significantly (n = 21, *P < 0.05).

Figure 2

A: Procaspase 9 and cleaved caspase 9 protein expression in whole retina after experimentally elevated IOP for 10 days. Elevated IOP resulted in an increase in procaspase 9 and cleaved caspase 9 (lanes 2 and 4) compared to normal retinas (lanes 1 and 3). B: Quantification of the Western blots further confirmed that procaspase 9 and cleaved caspase 9 levels increase in elevated IOP retinas significantly (n = 21, *P < 0.05).

Figure 3

A: Cleaved PARP protein expression in whole retina after experimentally elevated IOP for 10 days. Cleaved PARP is only present in retinas with elevated IOP (lanes 2 and 4). B: Quantification of the Western blots further confirmed that PARP cleavage increase in elevated IOP retinas significantly (n = 21, *P < 0.05).

Figure 4

PARP immunohistochemical staining of inner retina after 10 days of elevated IOP. A: Normal IOP retina. B: Elevated IOP retina. Arrows indicate baseline perinuclear staining and arrowheads indicate increased nuclear PARP immunoreactivity. GCL, ganglion cell layer; IPL, inner plexiform layer; INL, inner nuclear layer. Original magnifications, ×200.

Figure 5

LCM of RGCs in the rat retina. Top: Intact retinal tissue back-labeled with Fluorogold. RGCs indicated by arrows. Center: Retinal tissue after LCM of the back-labeled RGCs using 7.5-μm diameter laser pulses. Tissue surrounding the extracted cells remains intact after the procedure. Arrows indicate prior positions of back-labeled RGCs. Bottom: Highly enriched population of LCM-captured back-labeled RGCs on the capture cap. GCL, ganglion cell layer; IPL, inner plexiform layer; INL, inner nuclear layer; ONL, outer nuclear layer.

Figure 6

Proposed effects of elevated IOP on RGCs.