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. The pathological hallmark of glaucoma is atrophy of the optic nerve associated with retinal ganglion cell (RGC) death, leading to vision loss and blindness throughout decades. The major risk factor for developing glaucoma is increased intraocular pressure (IOP). RGCs have been shown to die by apoptosis in both human 1,2 and animal models 3-5 although the exact mechanism(s) by which RGC apoptosis occurs in response to increased IOP is unknown. Two distinct pathways of upstream, initiating caspases can each begin a cascade that leads to activation of downstream effector caspases resulting in apoptosis. 6 Caspase 8 is the initial caspase activated by cleavage of procaspase 8 to the active form in response to extrinsic cell signaling, such as binding of receptors with death domains that interact with Fas-associated death domains (FADD). 7 The mitochondrial stress pathway on the other hand, begins with the release of cytochrome c from mitochondria, which then interacts with Apaf-1, causing cleavage and activation of caspase 9. 8 The extrinsic pathway (through the death receptors) and the intrinsic pathway (through the mitochondria) for apoptosis are capable of operating independently leading to the activation of downstream effector caspases, caspase 3, 6, and 7. Poly (ADP-ribose) polymerase (PARP) is a 116-kd nuclear protein that is involved in the repair of DNA and in differentiation and in chromatin structure formation. During the late stages of apoptosis, downstream caspases, such as caspase 3, cleave PARP to yield 85-kd and 25-kd fragments. 9 It is unclear which of the specific upstream molecular events occur in the retina under conditions of elevated IOP. In one report, caspase 8 was shown by immunohistochemistry to be activated in RGCs in experimental glaucoma in the rat. 10 In another report, we showed that caspase 9 is activated by immunohistochemistry in RGCs and by Western blot analysis under conditions of elevated IOP also in the rat. ...

Efficient estimation of retinal ganglion cell number: A stereological approach

“…RGC counts in control eyes ranged from 78,000 to 93,600 with a mean (±s.d.) of 84,862 ± 5,753, as previously reported for some eyes (Huang et al, 2005a;Huang et al, 2005b). In eyes with elevated IOP for 10 days RGC counts ranged from 51,100 to 63,029 with a mean of 56,837 ± 4,272, indicating a 33% loss of RGCs (n=8; p < 0.05).…”

“…In addition to the savings involved in not having to prepare additional animals for such studies, importantly, all collected data can be directly correlated with RGC counts from the exact eye. For example, we have previously coupled cross-sectional stereological counting with laser capture microdissection (LCM) and qPCR analysis to demonstrate an upregulation of Caspase 8 and 9 mRNA in experimental glaucoma (Huang et al, 2005a). We also applied the method of cross-sectional stereological counting as a tool for assessing RGC survival and neuroprotection in experimental glaucoma as a result of drug treatment.…”

“…Two μl of a 3% Fluorogold (Fluorochrome LLC., Denver, CO) solution in PBS with 10% DMSO was then injected slowly over 10 minutes. The procedure was repeated on the contralateral side of the brain, the skin closed with a 4-0 silk suture, antibiotic ointment applied to the wound, and the animal allowed to recover from anesthesia in its home cage (Huang et al, 2005a). To ensure adequate backlabeling of RGCs, animals were allowed seven days for retrograde transport of Fluorogold before further experimental interventions (including induction of glaucoma).…”

“…RGCs have been shown to die by apoptosis in various models of cell death such as optic nerve crush (Berkelaar et al, 1994), optic nerve axotomy (Isenmann et al, 1997), ischemia (Selles-Navarro et al, 1996;Guo et al, 2003), anterior ischemic optic neuropathy (Bernstein et al, 2007) and glaucoma (Quigley, 1999;Huang et al, 2005a;Hanninen et al, 2002). Quantification of RGC numbers is essential for assessing pathologic damage, pharmacological effects and protection, and a better understanding of disease mechanisms in the eye and optic nerve.…”

Distal axonopathy with structural persistence in glaucomatous neurodegeneration

“…Downstream apoptotic mechanisms contribute late in glaucoma (25,37,38), and axonal pathology precedes somatic degeneration (14-16, 24, 39, 40). Early distal transport deficits and axonal dystrophies are relevant in Alzheimer disease, Parkinson disease, and amyotrophic lateral sclerosis (9)(10)(11).…”