Ian Rawe

Calpain Activation in Experimental Glaucoma

PURPOSE Glaucoma is a neurodegenerative disease in which elevated intraocular pressure (IOP) leads to progressive loss of retinal ganglion cells (RGCs) and blindness. Calcium dyshomeostasis has been suggested to play a role in the pathologic events that lead to RGC loss, though the details of these events are not well understood. Calcium-induced activation of calpain has been shown to contribute to neuronal death in a wide variety of neurodegenerative diseases. The authors hypothesize that similar events occur in glaucoma. METHODS The authors used a well-established rat model of experimental glaucoma. Retinal tissues were harvested after 5 or 10 days of elevated IOP and were subjected to immunoblot analysis, immunoprecipitation, and MALDI-ProTOF/MS peptide fingerprint mapping. Immunohistochemistry was used to localize calpain activation. RESULTS The authors present four independent lines of evidence that calpain is activated in experimental glaucoma. First, they showed that a 55-kDa autocatalytic active form of calpain is detected on immunoblot analysis. Second, they demonstrated the cleavage of two well-established calpain substrates, spectrin and calcineurin, only in eyes with elevated IOP. Third, they used MALDI-ProTOF to analyze cleaved calcineurin and immunoblot analysis of spectrin cleavage products and showed that both substrates were cleaved by calpain in experimental glaucoma. Fourth, they used immunohistochemistry to show that calpain-mediated spectrin cleavage occurs in RGCs under conditions of elevated IOP. CONCLUSIONS These data support the hypothesis that calpain is activated under conditions of elevated intraocular pressure and provide further details of the pathologic events leading to RGC loss in glaucoma.

Colocalization of increased transforming growth factor-β-induced protein (TGFBIp) and clusterin in Fuchs endothelial corneal dystrophy.

PURPOSE To investigate the differential expression of TGFBIp in normal human and Fuchs endothelial corneal dystrophy (FECD) endothelial cell-Descemets membrane (HCEC-DM) complex, and to asses the structural role of TGFBIp and clusterin (CLU) in guttae formation. METHODS HCEC-DM complex was dissected from stroma in normal and FECD samples. Proteins were separated by 2-D gel electrophoresis and subjected to proteomic analysis. N-terminal processing of TGFBIp was detected by Western blot analysis with two separate antibodies against the N- and C-terminal regions of TGFBIp. Expression of TGFBI mRNA was compared by using real-time PCR. Subcellular localization of TGFBIp and CLU in corneal guttae was assessed by fluorescence confocal microscopy. RESULTS A major 68-kDa fragment and a minor 39-kDa fragment of TGFBIp were identified on 2-D gels. Western blot analysis revealed an age-dependent proteolytic processing of the TGFBIp N terminus resulting in the increased formation of 57-kDa (P = 0.04) and 39-kDa (P = 0.03) fragments in older donors. FECD HCEC-DM showed a significant increase in the 68-kDa (P = 0.04), 57-kDa (P = 0.01), and 39- kDa (P = 0.03) fragments of TGFBIp. Real-time PCR analysis revealed that TGFBI mRNA was significantly increased (P = 0.04) in FECD samples. TGFBIp formed aggregates at the lower portions of guttae, next to Descemets membrane, whereas CLU localized mostly on top of the TGFBIp-stained areas at the level of the endothelial cell nuclear plane. CONCLUSIONS The overexpression of proaggregative protein CLU, and proadhesive protein TGFBIp, have been colocalized in the guttae. Such findings provide us with a better understanding of the major contributors involved in the aberrant cell-extracellular matrix interactions seen in the guttae of patients with FECD.