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Purpose: Retinal ischemic diseases primarily lead to damage of the inner retinal neurons. Electrophysiological studies also suggest impairment of the inner retinal neurons. Our recent studies with acute ocular hypertensive rats confirmed damage predominantly in the inner retinal layer along with the ganglion cell layer, changes that are ameliorated by the calpain inhibitor SNJ-1945. However, we do not know which specific neuronal cells in the inner retinal layer are damaged by calpains. Thus, the purpose of the present study was to identify specific calpain-damaged neuronal cells in the inner retina from acute ocular hypertensive rats., Methods: Intraocular pressure was elevated to 110 mm Hg for 40 min. One hour after ocular hypertension (OH), SNJ-1945 was administrated as a single oral dose of 50 mg/kg. Retinal function was assessed by scotopic electroretinography (ERG). Histological degeneration was evaluated by hematoxylin and eosin, terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick-end-labeling (TUNEL), and immunostaining in thin sections and flat mounts of the retina. Calpain activation was determined by proteolysis of the calpain substrate α-spectrin., Results: OH caused calpain activation, increased TUNEL-positive staining, decreased thickness of the inner nuclear layer (INL), and decreased amplitudes of the ERG a- and b-waves and oscillatory potentials (OPs). SNJ-1945 significantly inhibited calpain activation and the decrease in ERG values. Interestingly, the changes in the b-wave and OPs amplitudes were significantly correlated to changes in the thickness of the INL. In the inner retinal layer, the numbers of rod bipolar, cone-ON bipolar, and amacrine cells were decreased after OH. SNJ-1945 suppressed the loss of cone-ON bipolar and amacrine cells, but did not inhibit the loss of rod bipolar cells. We also observed increased glial fibrillary acid protein-positive staining in the Müller cells after OH and the treatment with SNJ-1945., Conclusions: Calpains may contribute to ischemic retinal dysfunction by causing the loss of cone-ON bipolar and amacrine cells and causing the activation of Müller cells. Calpain inhibitor SNJ-1945 may be a candidate compound for treatment of retinal ischemic disease.

Purpose: We previously showed involvement of calpains in neural retina degeneration induced by hypoxia and ischemia-reperfusion. Age-related macular degeneration (AMD) is one of the leading causes for loss of vision. AMD showed degeneration of neural retina due to dysfunction and degeneration of the retinal pigment epithelium (RPE). RPE performs critical functions in neural retina, such as phagocytosis of shed rod outer segments. The purpose of the current study was to determine the contribution of calpain-induced proteolysis to damage in cultured human RPE cells. Zinc chelator TPEN was used to induce cellular damage as zinc deficiency is a suspected risk factor for AMD., Methods: In RPE/choroid preparations from normal and AMD patients, calpain mRNAs were measured by qPCR, and calpain activity was assessed by casein zymography. Third- to fifth-passage cells from human RPE cells were cultured with TPEN. Cell damage was morphologically assessed under the phase-contrast microscope, and TUNEL staining was performed to detect apoptosis. Leakage of lactate dehydrogenase (LDH) into the medium was measured as a marker of RPE cell damage. Activation of calpains and proteolysis of the known calpain substrate alpha -spectrin were assessed by immunoblotting. To further confirm calpain-induced proteolysis, calpain in homogenized RPE was also activated directly by addition of calcium., Results: RPE/choroid from normal patients expressed mRNAs for calpain 1, calpain 2, and calpastatin moderately, and calpain 2 activity tended to be lower in AMD patients. TPEN caused RPE cell damage with positive TUNEL staining. TPEN also caused leakage of LDH into the medium from RPE cells, and calpain inhibitor SJA6017 inhibited the leakage. Caspase-3 inhibitors z-VAD and z-DEVD also showed inhibitory effects. Immunoblotting for calpain and alpha -spectrin showed activation of calpain in RPE cells cultured with TPEN. Proteolysis by activated calpain was confirmed by addition of calcium to homogenized RPE., Conclusions: These results suggested that activation of calpain contributed to cellular damage induced by TPEN in cultured human RPE cells.

The purpose of the present study was to determine if proteolysis by the calcium-dependent enzyme calpains (EC 3.4.22.17) contributed to retinal cell death in a rat model of photoreceptor degeneration induced by intraperitoneal injection of N-methyl-N-nitrosourea (MNU). Retinal degeneration was evaluated by H&E staining, and cell death was determined by TUNEL assay. Total calcium in retina was measured by atomic absorption spectrophotometry. Activation of calpains was determined by casein zymography and immunoblotting. Proteolysis of alpha-spectrin and p35 (regulator of Cdk5) were evaluated by immunoblotting. Calpain inhibitor SNJ-1945 was orally administrated to MNU-treated rats to test drug efficacy. MNU decreased the thickness of photoreceptor cell layer, composed of the outer nuclear layer (ONL) and outer segment (OS). Numerous cells in the ONL showed positive TUNEL staining. Total calcium was increased in retina after MNU. Activation of calpains and calpain-specific proteolysis of alpha-spectrin were observed after MNU injection. Oral administration of SNJ-1945 to MNU-treated rats showed a significant protective effect against photoreceptor cell loss, confirming involvement of calpains in photoreceptor degeneration. Conversion of p35 to p25 was well correlated with calpain activation, suggesting prolonged activation of Cdk5/p25 as a possible downstream mechanism for MNU-induced photoreceptor cell death. SNJ-1945 reduced photoreceptor cells death, even though MNU is one of the most severe models of photoreceptor cell degeneration. Oral calpain inhibitor SNJ-1945 may be a candidate for testing as a medication against retinal degeneration in retinitis pigmentosa.

The purpose of this study was to determine if calpain-induced proteolysis was associated with retinal degeneration or dysfunction in the rat acute ocular hypertensive model. Acute glaucoma was produced by elevation of IOP to 120 mm Hg for 1 hr. Retinal degeneration was evaluated by H&E staining and apoptosis was determined by TUNEL staining in histologic sections of retina. Electroretinogram (ERG) was carried out to evaluate changes in functionality. Activation of calpains was determined by casein zymography and immunoblotting. Total calcium in retina was measured by atomic absorption spectrophotometry. Proteolysis of alpha-spectrin, tau, cdk5, and p35 (a regulator of cdk5) were evaluated by immunoblotting. The thickness of inner plexiform layer (IPL) and inner nuclear layer (INL), and the number of cells in the ganglion cell layer (GCL) decreased after ocular hypertension. Numerous cells in the INL stained positive for TUNEL and some cells in the outer nuclear layer (ONL) showed TUNEL staining. The a-wave in ERG was temporarily decreased after ocular hypertension and then recovered to normal. In contrast, the b-wave was completely lost. Calpains were activated after ocular hypertension. Activation of calpains was associated with increased calcium in retina. Calpain-dependent proteolysis of alpha-spectrin, tau, and p35 were observed in retina after ocular hypertension. The results suggested that increased calcium and subsequent proteolysis by activated calpains was associated with the death of inner retinal cells due to acute ocular hypertension in the rat model. Calpain inhibitors may be candidate drugs for treatment of retinal degeneration and dysfunction resulting from glaucoma., (Copyright 2006 Wiley-Liss, Inc.)

To clarify the involvement of calpains in sarcolemmal remodeling, we examined the expression of calpains and their substrate, alpha-fodrin, in various disorders of muscle. Although immunohistological reactions for alpha-fodrin and calpains were weak in normal control muscles, intense immunoreactivity for alpha-fodrin at the sarcolemma and for calpains throughout the cytoplasm were detected in small muscle fibers from patients with inflammatory myositis (IM), rhabdomyolysis (Rhab), and Duchenne muscular dystrophy (DMD). Most of the calpain-alpha-fodrin double-positive muscle fibers in IM and Rhab also expressed the developmental form of myosin heavy chain. The sarcolemma of these small muscle fibers reacted with an antibody that specifically recognizes the 150-kDa fragments of alpha-fodrin (SBDP 150s) cleaved by calpain, but not caspase 3. Western blot analysis confirmed these results. These observations indicate that calpain is activated and reacts with alpha-fodrin as a substrate at the sarcolemma, and plays a key role in modulating sarcolemmal proteins to adapt to the specific conditions in each myopathy.

Our previous studies in retina on the mechanism for hypoxia-induced cell death suggested activation of a class of calcium-activated proteases known as calpains. This conclusion was based on data showing proteolysis of a calpain substrate alpha-spectrin, autolysis of activated calpain, and reduction of cell damage by calpain inhibitor SJA6017. Less is known about changes in downstream pathways after calpain activation. Thus, the purpose of the present investigation was to measure proteolysis of neuronal cytoskeletal proteins and apoptotic cell signaling factors during hypoxia-induced retinal cell death. Rat retinas were incubated in RPMI medium with glucose and 95% O2/5% CO2 to supply sufficient oxygen for retinal cell survival. Hypoxia was induced with 95% N2/5% CO2 without glucose. Immunoblotting was used to detect activation of calpain and proteolysis of substrates. Amounts of mRNA for calpain 1 and 2 were determined by quantitative PCR. Twelve times more calpain 2 mRNA than calpain 1 was present in retinas. Activation of calpain 2 and production of a calpain-specific alpha-spectrin breakdown product at 150 kDa were confirmed in hypoxic retinas. Further, pro-caspase-3 at 32 kDa was proteolyzed to a fragment at 30 kDa, tau protein was lost, and p35 was proteolyzed to p25 suggesting prolonged activation of cdk5. SJA6017 partially inhibited the production of these fragments. During hypoxia in rat retinas, calpains may be major proteases causing breakdown of neuronal proteins involved in apoptotic cell death. Calpain inhibitor SJA6017 may have potential for testing as a therapeutic agent against retinal pathologies such those caused by glaucoma, although future studies such as testing in in vivo animal models are required.

Results of our recent studies in rats suggested that calpains play an important role in retinal cell death induced by ischemia-reperfusion in vivo and by hypoxia in vitro. Study of spontaneous animal models could help determine the involvement of calpains in human retinopathy. The WBN/Kob rat is such a model for spontaneous retinal degeneration. The purpose of the study reported here was to determine the involvement of calpain isoforms during retinal degeneration in WBN/Kob rats. Histologic and functional retinal degeneration in WBN/Kob rats was observed by use of light microscopy and electroretinography, respectively. Proteolysis of alpha-spectrin in the retina was detected by use of immunoblot analysis in aging WBN/Kob rats. This proteolysis was associated with the increases of retinal calcium content and caseinolytic activity for calpains 1 and 2. Expression of calpain 1, calpain 2, and calpastatin mRNAs in the retina, as measured by use of reverse transcriptase-polymerase chain reaction (RT-PCR) analysis, were only slightly up-regulated at 24 weeks of age. In contrast, expression of retina-specific calpains, such as Rt88, Rt88', and Rt90 mRNA, was markedly down-regulated at 12 weeks of age. Expression of calpain 10 mRNA in the retina was only slightly down-regulated at 12 weeks of age. In contrast to mRNA expression, various expression patterns of calpain 10 proteins were observed. Increased retinal calcium content, leading to activation of calpains 1 and 2, may be an important event in the sequential changes leading to degeneration of the retina in WBN/Kob rats. Activated calpain causing proteolysis of alpha-spectrin and changes in Rt88, Rt88', Rt90 and calpain 10 may also contribute to retinal degeneration.

The purpose of the present study was to compare the susceptibility of crystallins proteolyzed by ubiquitous calpain 2 and by lens-specific calpain Lp82 to insolubilization. To test this, transgenic (TG) mice expressing a calpain 2, in which the active site cysteine 105 was mutated to alanine, were produced. Expression of mutated calpain 2 was driven in lens by coupling the mutated gene to the betaB1-crystallin promoter. Light scattering was measured in solutions of lens proteins after activation of endogenous calpain 2 and/or Lp82. Mass spectrometric analysis was performed to determine the cleavage sites and the calpain responsible for insolubilization of crystallins. Lens proteins from TG mice incubated in vitro with calcium showed higher light scattering compared to proteins from wild type (WT) mice. alphaA-crystallin from TG mice was proteolyzed by Lp82. In contrast, alphaA-crystallin in lenses from WT mice were proteolyzed by both calpain 2 and Lp82. These results suggested that Lp82-induced proteolysis of crystallins caused increased susceptibility of truncated crystallins to in vitro precipitation. Since Lp82 is highest in young animals, Lp82-induced proteolysis and precipitation may be one of the factors responsible for the cataract formation in young rodents.

To determine the involvement of calpains in human cataractogenesis, studies in aged animal models are needed. Aged, male WBN/Kob rats spontaneously develop cataract along with severe, persistent diabetes with hyperglycemia and nephropathy. The purpose of present experiments was to provide a biochemical mechanism for the involvement of ubiquitous calpains in cataractogenesis in WBN/Kob rats. Serum and urinary glucose were measured to confirm diabetes, and cataracts were observed by slit lamp biomicroscopy. Calcium determinations were performed on lens samples from several ages of WBN/Kob and Wistar rats. Casein zymography, immunoblot analysis for alpha-spectrin, calpain 2, and calpain 10 were performed to detect activation of calpain in lens samples. Serum glucose levels increased and cortical cataract developed in male WBN/Kob rats within 1 year, indicating diabetic cataract. Cataract was accompanied by several presumptive biochemical indicators of calpain activation, including increased calcium, proteolysis of alpha-spectrin, and decreased caseinolytic activity for calpains suggesting calpain activation followed by autolytic degradation. Activation of ubiquitous calpains may contribute to biochemical mechanism of cataractogenesis in spontaneously diabetic WBN/Kob rats. The WBN/Kob model may be useful for elucidating the roles of calpain 2 and calpain 10 in human cataractogenesis.

Our previous study suggested that calpain isoforms played an important role in retinal ganglion cell death induced by ischemia-reperfusion in rats [Curr. Eye Res. 21 (2000) 571]. The purpose of the present study was to further establish the direct involvement of calpain in hypoxia-induced damage by administering calpain inhibitor SJA6017 to oxygen-starved, cultured retinas. Retinas were incubated in RPMI medium with glucose and 95% O2/5% CO2 to supply sufficient oxygen for retinal cell survival. To induce a hypoxic condition, retinas were incubated with 95% N2/5% CO2. Leakage of LDH in the medium was measured to assess retinal cell damage. Activation of calpain and proteolysis of calpain substrate alpha-spectrin were analyzed by casein zymography and immunoblotting. Large amounts of LDH leaked into the medium from retinas under hypoxic conditions for 12 h, and SJA6017 significantly reduced LDH leakage. Caseinolytic activity of mu- and m-calpains decreased with hypoxia for 5 and 12 h, suggesting calpain activation followed by autolytic degradation. SJA6017 partially inhibited decreased calpain activities. Proteolysis of 230 kDa alpha-spectrin to 150 and 145 kDa breakdown products was observed in retinas with hypoxia. SJA6017 completely inhibited production of the 145 kDa breakdown product and partially inhibited production of the 150 kDa breakdown product. These results confirm the direct involvement of calpains in retinal cell damage induced by hypoxia in vitro.

Purpose: 1) To measure the amount of calpain inhibitor SJA6017 taken up by lenses of young rats after administration; and 2) To test efficacy of SJA6017 against selenite cataract in regard to amelioration of proteolysis of lens protein and prevention of lens nuclear opacity., Methods: Selenite nuclear cataracts were produced by subcutaneous injection of an overdose of sodium selenite to 16-day-old rats. SJA6017 was administered daily using intraperitoneal injections at 100 mg/kg body weight/day for 4 days. Lenses were observed and photographed by slit lamp biomicroscopy, and scored into one of three stages. Enucleated lenses were also scored into one of four stages and lens opacities in the nuclear region were quantified by image analysis. Proteolysis of crystallins was detected by SDS-PAGE. The amount of SJA6017 taken up by the lens was detected with a column switching HPLC system., Results: Nuclear cataracts were visible in 31% of the animals receiving only selenite, while the frequency of nuclear cataract in the Se+SJA6017 group was reduced to only 16%. This effect of SJA6017 was confirmed by densitometric analysis as a reduction in the density of the nucleus. Similar proteolytic changes of crystallins occurred at all stages of selenite cataract formation. The amount of SJA6017 in the lens was detected at the level of 0.03 microM., Conclusions: Systemic SJA6017 was taken up by the lens, and SJA6017 ameliorated in vivo selenite cataract formation. These studies are important because they partially validate the biochemical rationale for developing non-surgical, drug treatments for cataract prevention in man.

Purpose: To compare calcium ionophore-induced cataract formation and in vitro light scattering in cultured lenses from guinea pig and rabbit., Methods: Lenses from guinea pig and rabbit were cultured for 5 or 6 days with calcium ionophore A23187. To assess the involvement of calpain in cataract formation; SDS-PAGE, immunoblotting and calcium determinations were performed. For in vitro light scattering, lens soluble proteins from rabbit were hydrolyzed for 24 h by either endogenous lens calpain, or by addition of purified m-calpain and then further incubated for up to 10 days. Light scattering was measured daily at 405 nm., Results: Lenses from younger guinea pigs cultured in A23187 first developed outer cortical opacities followed by nuclear cataract. Total calcium was markedly increased by A23187 in lenses of all ages. Proteolysis of crystallins and alpha-spectrin were observed in nuclear cataract in younger guinea pigs. This was attenuated with age, in association with the attenuation of cataract formation with age. Calpain 80 kDa subunit in the lenses cultured with A23187 was also decreased. Co-culture with SJA6017 or E64d (reversible and irreversible inhibitors of calpain, respectively) reduced A23187-induced nuclear opacities, proteolysis of crystallins and alpha-spectrin, and loss of calpain without affecting increased total calcium. In contrast, rabbit lenses cultured in A23187 did not develop nuclear cataract, although biochemical changes in cultured rabbit lenses were similar to those in cultured guinea pig lenses. Furthermore, no appreciable in vitro light scattering occurred in soluble proteins from rabbit lenses after activation of endogenous m-calpain, or after addition of exogenous purified m-calpain, although crystallins were partially hydrolyzed by calpain., Conclusions: Both rabbit and guinea pig lenses undergo calpain-induced proteolysis upon elevation of lenticular calcium. However, factors in intact guinea pig lenses may promote light scattering and insolubilization after proteolysis by calpain, but these factors were not functional in rabbit lenses. Discovery of the factors promoting light scatter and insolubilization after proteolysis will help to explain the role of certain crystallin polypeptides in cataract formation.

The purpose of the present investigation was to compare in vitro light scattering in the soluble proteins from rodent lenses after hydrolysis by the calcium-activated protease, m-calpain (EC 3.4.22.17). Light scattering was measured in solutions of lens proteins from mice, rats, and guinea pigs after activation of endogenous m-calpain or after addition of purified m-calpain. We found for the first time that, in addition to rat, crystallins from another rodent lens, young mouse, were susceptible to calpain-induced light scattering. As in rats, aging of mouse lens prevented calpain-induced light scattering. Although crystallins from guinea pig lens were also partially hydrolysed by calpain, appreciable light scattering did not occur. Limited proteolysis may cause common changes in the biophysical properties of mouse and rat crystallins to decrease their solubility. Discovery of the nature of these biophysical changes may help our understanding as to why crystallins precipitate under cataractous conditions.

The purposes of this experiment were to: (1), characterize the peptide aldehyde SJA6017, N-(4-fluorophenylsulfonyl)-L-valyl-L-leucinal, a newly synthesized inhibitor of calpain, and (2) test the effect of SJA6017 in preventing calcium ionophore-induced cataract in cultured rat lenses. In vitro, SJA6017 strongly inhibited purified m-calpain from porcine kidney. Casein zymography confirmed that SJA6017 reversibly bound to the active site of m-calpain. SJA6017 was also confirmed to be a cell-permeable inhibitor in Molt-4 cells. In cultured lenses, SJA6017 reduced nuclear opacity and proteolysis of crystallins and alpha-spectrin caused by calcium ionophore A23187. These results suggested that SJA6017 is a reversible and cell-permeable calpain inhibitor which may possess great efficacy against calcium-induced models of cataract.

Abstract: Background: Our recent study suggested involvement of calpain-induced proteolysis in retinal degeneration and dysfunction in acute ocular hypertensive rats. The purpose of the present study was to determine if an orally available form of calpain inhibitor, ((1S)-1-((((1S)-1-benzyl-3-cyclopropylamino-2,3-di-oxopropyl)amino)carbonyl)-3-methylbutyl)carbamic acid 5-methoxy-3-oxapentyl ester (SNJ-1945), ameliorated retinal degeneration induced by acute hypertension in rats. To help extrapolate the effect of SNJ-1945 from the rat model to the human glaucomatous patient, in vitro inhibition of calpain-induced proteolysis by SNJ-1945 in monkey and human retinal proteins was compared with proteolysis in rat proteins. Methods: Intraocular pressure (IOP) in rats was elevated to 110 mm Hg for 50 min. SNJ-1945 was administrated i.p. or orally before ocular hypertension. Retinal degeneration was evaluated by hematoxylin and eosin (H&E) staining and cell counting. Transcripts for calpains and calpastatin in rat, monkey, and human retinas were measured by quantitative RT-PCR. Calpain activities were determined by casein zymography. Soluble retinal proteins from rat, monkey, and humans were incubated with calcium to activate calpains, with or without SNJ-1945. Proteolysis of calpain substrate α-spectrin was analyzed by immunoblotting. Results: Elevated IOP caused retinal degeneration and proteolysis of α-spectrin. Both i.p. and oral administration of SNJ-1945 inhibited proteolysis of α-spectrin and ameliorated retinal degeneration. Transcript levels for calpain 1 and calpastatin were similar in rat, monkey, and human retinas. Calpain 2 transcript levels were higher in rats compared with monkey and human. Appreciable caseinolytic activities due to calpains were observed in monkey and human retinas. Incubation of retinal soluble proteins with calcium led to proteolysis of α-spectrin due to calpains in rat, monkey, and human samples. SNJ-1945 similarly inhibited proteolysis in all species. Conclusion: Our results suggested that orally available calpain inhibitor SNJ-1945 might be a possible candidate drug for testing in preventing progression of glaucomatous retinal degeneration. [Copyright &y& Elsevier]