The role of calcium-activated protease calpain in experimental retinal pathology

Abstract

The purpose of this review is to present the recent evidence linking the family of ubiquitous proteases called calpains (EC 3.4.22.17) to neuropathologies of the retina. The hypothesis being tested in such studies is that over-activation of calpains by elevated intracellular calcium contributes to retinal cell death produced by conditions such as elevated intraocular pressure and hypoxia. Recent x-ray diffraction studies have provided insight into the molecular events causing calpain activation. Further, x-ray diffraction data has provided details on how side chains on calpain inhibitors affect docking into the active site of calpain 1. This opens the possibility of testing calpain-specific inhibitors, such as SJA6017 and SNJ1945, for human safety and as a site-directed form of treatment for retinal pathologies.

Fig. 1

(A) Brown immunostaining for calpain 2 protein in layers and cells of retina. Layer designations are GCL = ganglion cells, IPL = inner plexiform, INL = inner nuclear, ONL = outer nuclear, OS = outer segment. (B) Red staining for calpain proteins in rat retinal ganglion cells (primary polyclonal antibody reactive with calpains 1 & 2, secondary antibody conjugated with Alexa fluo-594), (C) blue DNA staining in the cell nuclei (Hoechst 33342), and (D) merged images from (B) and (C), showing positive staining for calpain proteins 1 & 2 in the cytoplasm and nucleus.

Fig. 2

Canonical structure of a typical calpain showing the six domains found in the 80 and 30 kDa subunits of the ubiquitous, heterodimeric calpains 1 and 2. A deep, wide substrate-binding cleft separates the active site cysteine residue in subdomain IIa from the histidine and asparagine residues in subdomains IIb in the inactive molecule. The light green ovals represent EF-hand structures in domains IV and VI. The highly flexible domain V (N-terminal domain) of the 30 kDa regulatory domain contains clusters of non polar glycine residues, and is one of the sites for calcium-induced autolysis as is domain I of the 80 kDa subunit. Interactions between the two C-terminal EF structures stabilize the dimer.

Fig. 3

Casein zymogram with image inverted to show dark areas of lysis due to calpain activities in soluble proteins from normal monkey retinas (lane 1). The retinas were also cultured under normal oxygen conditions (lane 2) and under hypoxic treatment (lanes 3 & 4). Open arrowhead indicates calpain 1 activities, and solid arrowhead indicates calpain 2 activities. Retinas were incubated for ten hours under normoxia (O₂), hypoxia (N₂), or N₂ plus 100 μM calpain inhibitor SJA6017 (SJA) (modified with permission from Nakajima E et al27).

Fig. 4

Gene diagrams (lower) for calpain 3 variants found in retina compared to the parent protein molecule, muscle-preferred calpain 3 (upper). NS, novel sequence; IS1, insert region 1; IS2 insert region 2; IS3, insert region 3; AX1, alternative exon 1 coding region, dashed lines are deleted sequences. Due to a stop codon, AXI is not found in human or monkey retinas, where it is replaced with the NS region.

Fig. 5

Proteome map of the soluble proteins in normal rat retina on Coomassie blue-stained, two dimensional gel (2DE) showing more than 80 spots – many of which were identified by mass spectrometry. For example, one of the most concentrated retinal proteins was enolase, the protein spots labeled “ENOA.” The proteins with abbreviated labels marked on the gels were used as landmark proteins because their migration positions were not obviously altered by treatment with calpain 2 (Table 1). Treatment with activated calpain 2 lead to decreased amounts of proteins circled and numbered 1–27; identities are listed in Table 2. The horizontal axis of the 2DE gel is the first dimension with pH 3 on the left to pH 10 on the right. The second dimension was SDS-PAGE in the vertical axis with molecular mass markers in kDa on the left.

Fig. 6

Proposed pathways leading to cell death in hypoxic rat retina due to calpain-mediated modifications in α-spectrin and tau. Solid lines show confirmed pathways, and dotted lines show the pathways reported in the literature (reprinted with permission from Tamada Y et al57).

Fig. 7

Copy numbers for members of the calpain system in the retinas from rats, monkey and man (modified with permission from Oka T et al36).

Fig. 8

Histologic changes in rat retina from ischemia/reperfusion of the central retinal artery (A), and inhibition by SJA6017 on 7 days after ischemia/reperfusion (B) (modified with permission from Sakamoto YR et al45).

Fig. 9

Photomicrographs of H & E stained retinal sections from monkey with induced chronic ocular hypertension, showing decrease in GLC cells and disc retraction in the optic nerve head (ONH). (A) is ONH. (B) is superior mid-peripheral retina. Scale bars are 100 μm in (A) and 50 μm in (B)

Fig. 10

Histologic photograph of human glaucomatous retina showing loss of many ganglion cells. Arrows indicate the condensation of nucleus in the GCL. The NFL, GCL, and IPL were also involuted. The OPL, ONL, and photoreceptor layers were well maintained. NFL; Nerve Fiber Layer, GCL; Ganglion Cell Layer, IPL; Inner Plexiform Layer, INL; Inner Nuclear Layer, OPL; Outer Plexiform Layer, ONL; Outer Nuclear Layer, RL; Receptor Layer.

Fig. 11

Histologic changes (A) and thickness of photoreceptor (B) in retinas from rats treated with MNU (modified with permission from Oka T et al34).

Fig. 12

Three dimensional ribbon structure of calpain 2 without calcium (Reprinted with permission from Suzuki K et al54).

Fig. 13

Molecular modeling of SNJ-1945 docking in the active site (gold) of calpain 1 (based on PDB#: 1TL9 - calpain 1 with calcium and leupeptin). Purple indicates the calpain 1 residues in the active site cleft; dotted lines are hydrogen bonds. In the SNJ molecule, the carbon atoms are colored gray, nitrogen atoms are blue, and oxygen atoms are red. The black oval indicates the ketoamide warhead in SNJ-1945, in relation to the S1 and S2 subsites.