Calcineurin cleavage is triggered by elevated intraocular pressure, and calcineurin inhibition blocks retinal ganglion cell death in experimental glaucoma

Abstract

Increased intraocular pressure (IOP) leads, by an unknown mechanism, to apoptotic retinal ganglion cell (RGC) death in glaucoma. We now report cleavage of the autoinhibitory domain of the protein phosphatase calcineurin (CaN) in two rodent models of increased IOP. Cleaved CaN was not detected in rat or mouse eyes with normal IOP. In in vitro systems, this constitutively active cleaved form of CaN has been reported to lead to apoptosis via dephosphorylation of the proapoptotic Bcl-2 family member, Bad. In a rat model of glaucoma, we similarly detect increased Bad dephosphorylation, increased cytoplasmic cytochrome c (cyt c), and RGC death. Oral treatment of rats with increased IOP with the CaN inhibitor FK506 led to a reduction in Bad dephosphorylation and cyt c release. In accord with these biochemical results, we observed a marked increase in both RGC survival and optic nerve preservation. These data are consistent with a CaN-mediated mechanism of increased IOP toxicity. CaN cleavage was not observed at any time after optic nerve crush, suggesting that axon damage alone is insufficient to trigger cleavage. These findings implicate this mechanism of CaN activation in a chronic neurodegenerative disease. These data demonstrate that increased IOP leads to the initiation of a CaN-mediated mitochondrial apoptotic pathway in glaucoma and support neuroprotective strategies for this blinding disease.

Fig. 1.

CaN cleavage in experimental glaucoma. (A) Rats with elevated IOP in one eye for 5 or 10 days show the presence of the full-length CaN in all eyes and cleaved CaN (45 kDa) only in eyes with high IOP. Similar results were obtained in the DBA/2J mice. (B) Retinal protein from eyes of rats after ON crush. No cleaved CaN is observed in any eyes 3, 5, or 8 days after ON crush. (C) Rat summary data of the 45-kDa cleaved CaN band (n = 6 in all groups). Cleaved CaN was significantly increased in eyes with high IOP when compared with their fellow control eyes (†; 5 days, P < 0.02; 10 days, P < 0.005). In eyes with high IOP, the increase in cleaved CaN was significantly greater after 10 than after 5 days (^*, P < 0.02).

Fig. 2.

Cell loss in experimental glaucoma and protection by FK506. Summary data showing the percentage of cells lost compared to the fellow eye after 10 days of elevated IOP in control animals (n = 9) and animals receiving daily FK506 (n = 10; ^*, P = 0.00001).

Fig. 3.

ON injury in experimental glaucoma and protection by FK506. (A) ON cross section from a normal IOP rat eye. The architecture of the ON and the axons is normal. (B) ON cross section from a rat eye with high IOP for 10 days. There are widespread degenerative changes in the ON. (C) ON cross section from a rat with normal IOP given daily FK506 for 10 days with normal appearance. (D) ON cross section from a rat eye with high IOP for 10 days given daily FK506 with significant preservation of ON tissue.

Fig. 4.

Bad is dephosphorylated in eyes with high IOP, and this effect is blunted by FK506. (A) pBad levels are decreased in eyes with elevated IOP for 5 and 10 days. (B) Summary data showing pBad in eyes with normal IOP (open bars) and high IOP (black bars). There is significantly less pBad in eyes with high IOP, but this effect is blunted by the administration of FK506. (n = 6 in all groups; ^*, P < 0.01 comparing pBad in high IOP rats eyes after 10 days with and without daily FK506; †, comparing pBad levels between normal and high IOP eyes at 5 days, P < 0.02; 10 days, P < 0.005; 10 days with FK506, P < 0.01).

Fig. 5.

cyt c is released into the cytoplasm in eyes with high IOP, and this effect is blunted by FK506. (A) A decrease in mitochondrial cyt c and an increase in cytoplasmic cyt c in eyes with elevated IOP. FK506 significantly diminishes the amount of cyt c released from the mitochondria into the cytoplasm. (B) Summary data for mitochondrial and cytoplasmic levels of cyt c in eyes with normal and high IOP for 5 or 10 days (with and without FK506). (n = 6 in all groups). ^*, comparing cyt c levels between eyes with high IOP in rats with and without FK506 in both mitochondrial (P = 0.01) and cytoplasmic (P = 0.001) fractions. †, comparing cyt c levels between eyes with normal and high IOP in the mitochondrial fraction (5 days, P < 0.01; 10 days, P = 0.005; 10 days FK506, P = 0.03) and cytoplasmic fraction (5 days, P < 0.01; 10 days, P < 0.005; 10 days FK506, P < 0.02).