Involvement of calpain isoforms in ischemia-reperfusion injury in rat retina

PURPOSE: Much evidence has accumulated suggesting that activation of calpain causes neuronal cell death in ischemic brain. However, little is known about the involvement of calpain in retinal cell death in ischemic injury. Thus, the purpose of present study was to investigate the involvement of calpain isoforms (m- and mu-calpain) in ischemia-reperfusion injury in retina from rat. METHODS: Retinal ischemia was produced by occlusion of the central retinal artery for one hour, and this was followed by reperfusion for seven days. Calpain mRNAs, calpain activities, total calcium content and proteolysis of alpha-spectrin were determined in retina. Effect of a calpain inhibitor SJA6017 was histologically tested in retinal injury after ischemia-reperfusion. RESULTS: Following retinal ischemia, most of cells in the ganglion cell layer were sloughed off by day 1 after reperfusion, followed by loss of cells in the inner plexiform layer on day 3 and loss of cells in the inner nuclear layer by day 5. These morphologic changes were accompanied by several presumptive biochemical indicators of calpain activation: increased calcium, proteolysis of alpha-spectrin (a sensitive substrate for calpains), decreased caseinolytic activity for both calpains (suggesting calpain activation followed by autolytic degradation), increased mRNA levels for mu-calpain and calpastatin - the endogenous inhibitor of calpains - and decreased mRNA levels for mu-calpain. Moreover, the calpain inhibitor SJA6017 protected the reduction of cell density in the ganglion cell layer after ischemia-reperfusion. CONCLUSION: These results suggest that calpain isoforms may play an important role in neuronal cell death induced by retinal ischemia-reperfusion injury in rat.     

Calpain may contribute to hereditary cataract formation in sheep

PURPOSE: To determine the involvement of calpain in ovine cataractogenesis by measuring calcium, calpain activity, proteolysis, and the effect of calpain inhibition. METHODS: Sheep with genetic cataracts were examined for cataract severity. Calcium in normal and cataract lenses was measured. The presence of calpain was detected by casein zymography and immunoblotting. Calpain activity was assayed using BODIPY-casein as a substrate. Degradation of calpain substrates spectrin and vimentin was assessed by immunoblotting. The calpain inhibitor SJA6017 was applied to the left eye of cataract lambs, leaving the right eye as an untreated control. Both eyes were monitored by slit-lamp microscopy for cataract progression. RESULTS: Cortical cataracts were first observed in lambs at 1 to 2 months of age. Lens calcium concentration increased in the early stages of cataract formation and was >10-fold higher in mature cataract than normal lenses. Three calpain isoforms were detected in young lamb lenses. Calpain activity decreased as cataracts progressed. Both spectrin and vimentin were degraded with cataract maturity, which could indicate calpain proteolysis. Cataract lambs treated with SJA6017 eyedrops over a period of 4 months showed significantly smaller cataracts in the left treated eye over the right untreated eye. CONCLUSIONS: The presence of calpains and calcium elevation during cataract formation suggests that proteolysis may play a role in opacification in ovine lens. This hypothesis is supported by the delay in opacification with SJA6017 treatment. The results also suggested that the ovine hereditary cataract is a useful nonrodent model to test the role of calpains in cataractogenesis.     

Comparison of various calpain inhibitors in reduction of light scattering, protein precipitation and nuclear cataract in vitro

PURPOSE: To compare effects of calpain inhibitors on in vitro light-scattering in rat lens soluble protein and calcium-ionophore (A23187)-induced cataract formation in cultured rat lenses. METHODS: Rat lens soluble protein was hydrolyzed for 24 hours by activation of endogenous lens calpain. Ten calpain inhibitors were tested in this model at 10 and 25 microM concentration. As an index of protein precipitation, light scattering was measured daily at 405 nm for 8 days. Lens proteins were analyzed by isoelectric-focussing. Subsequently, rat lenses were cultured for 5 days with 10 microM A23187. Calpain inhibitors (SJA6017, MDL28170, AK295 and PD150606), which inhibited light-scattering were tested at 100 microM concentration in this model. Cataract evaluation, isoelectric-focussing and calcium determinations were performed. RESULTS: At 25 microM concentration AK295, SJA6017, E-64, PD-150606 and MDL28170 produced greater than 25% inhibition of light-scattering. Isoelectric-focussing revealed that addition of Ca(2+) produced characteristic crystallin proteolysis and aggregation patterns. AK295, SJA6017, MDL28170 and E64c prevented these changes. Lenses cultured in A23187 exhibited nuclear cataract, elevated calcium and proteolysis and aggregation of crystallins. Co-culture with SJA6017, MDL28170 and E64c reduced A23187-induced nuclear opacities, proteolysis and aggregation of crystallins without affecting increased total calcium. CONCLUSIONS: Endogenous calpain-activation model and A23187-induced cataract model can be used sequentially to screen calpain inhibitors for potential anti-cataract activity. Proteolytic changes in lens cortex after exposure to A23187 are also due to calpain activation. AK295, SJA6017 and MDL28170 possess efficacy against calcium-induced models of rodent cataracts. Use of calpain inhibitors represents a promising approach to cataract therapy.     

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

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.     

Contribution of calpain to cellular damage in human retinal pigment epithelial cells cultured with zinc chelator

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.     

Decreased sensitivity of lens-specific calpain Lp82 to calpastatin inhibitor.

The purpose of the present investigation was to test three calpain inhibitors (recombinant calpastatin domain I, E64, and SJA6017) against Lp82 calpain in rat lenses. Lp82 is a lens-specific isoenzyme from the calpain super family of calcium-activated, cysteine proteases (EC 34.22.17). Lp82 and m-calpain proteolytic activities and protein levels were measured by casein zymography and immunoblotting. Activity of endogenous Lp82 against vimentin was also tested by in vitro incubation of rat lens soluble and insoluble fractions with calcium. Most of the Lp82 activity could be inhibited by irreversible inhibitor E64 and reversible inhibitor SJA6017. However, a major finding of the present investigation was that Lp82 in the soluble and insoluble fractions of the lens was less sensitive to inhibition by recombinant domain I from the endogenous tissue inhibitor of ubiquitous calpains, calpastatin, than m-calpain. By using recombinant calpastatin to inhibit endogenous lens m-calpain, we were able to demonstrate the first example of a substrate for Lp82, vimentin. These data suggest that Lp82-induced proteolysis in rodent lenses may occur even in the presence of calpastatin.     

Activation of the mitochondrial caspase pathway and subsequent calpain activation in monkey RPE cells cultured under zinc depletion

Purpose

Decreased zinc levels in the macula are reported in patients with age-related macular degeneration, and the zinc chelator N,N,N'',N′-tetrakis (2- pyridylmethyl) ethylenediamine) (TPEN) causes death of human retinal pigment epithelial (RPE) cells. The purpose of the present study was to investigate signal transduction pathways during cell death initiated by TPEN, using monkey RPE cells.

Methods

RPE cells were cultured with TPEN. Activation of calpains and caspases, and proteolysis of their substrates were detected by immunoblotting. Incubation of calpain inhibitor SNJ-1945 or caspase inhibitor z-VAD-fmk was used to confirm activation of specific proteases.

Results

TPEN caused a time-dependent decrease in viable RPE cells. Cell death was accompanied by activation of calpain-1, caspase-9, and caspase-3. SNJ-1945 inhibited calpain activation and slightly inhibited caspase-9 activation. z-VAD-fmk inhibited caspases and calpain-1 activation. TPEN did not activate caspase-12.

Conclusions

Relative zinc deficiency in RPE cells causes activation of cytosolic calpain and mitochondrial caspase pathways without ER stress.     

Retinal penetration of calpain inhibitors in rats after oral administration.

Calpain-mediated proteolysis has been involved in neuronal cell death of retinal neurological degeneration. An aldehyde-based calpain inhibitor, SJA6017 (1), was effective following oral administration in a rat retinal ischemia model but had low oral bioavailability. The aim of this study was to identify calpain inhibitors with good retinal penetration after oral dosing. The orally bioavailable inhibitors, hemiacetal 3 (SNJ-1715), amphipathic ketoamide 5 (SNJ-1945), and pyridine ketoamide 6 (SNJ-2008), were evaluated for their retinal pharmacokinetic (PK) profiles. The retinal drug exposure of these inhibitors was more than tenfold higher than 1. Among these compounds, 5 exhibited the most favorable retinal PK properties, such as good penetration and long half-life. Comparisons of 5 and the structurally related ketoamide 6 suggested that the presence of a methoxy diethylene glycol moiety resulted in the inhibitor with high penetration into the retina and the sustained high retinal levels. Ketoamide 5 was selected as the development candidate for the treatment of retinal diseases.     

Calpains are activated by light but their inhibition has no neuroprotective effect against light-damage

Calpain had been shown to be highly activated at one day after exposure to the damaging light (Perche et al. (2007)Caspase-dependent apoptosis in light-induced retinal degeneration. Invest Ophthalmol Vis Sci 48:2753–2759.), suggesting that they might play a critical role in photoreceptor apoptosis induced by light. Therefore in the present study we investigate the role of calpain in light-induced photoreceptor cell death. In a first set of experiments, untreated albino Wistar rats were sacrificed at 0, 2, 4, 6, 12, 24 h of light exposure and at one day after the light was turned off (D1) to measure retinal calpain activity and to study calpain expression. In a second set of experiments, after control electroretinograms (ERGs), rats were uninjected or injected intravitreally with DMSO or the calpain inhibitor Mu-Phe-hPhe-FMK, before being exposed to the damaging light for 24 h. ERGs were then recorded at one day (D1) and fifteen days (D15) after the end of light exposure. Rats were sacrificed at D1 for apoptotic cell detection or D15 for histological analysis (ONL thickness). Calpain activity and expression significantly increased in Untreated retinas, from 0 h to D1. DMSO has no effect on calpain activity. Mu-Phe-hPhe-FMK significantly inhibited retinal calpain activity by 85% at 2 h of light exposure and still 48% at D1. However, Mu-Phe-hPhe-FMK has no effect on light-induced retinal degeneration as evidence by equivalent loss of function, equivalent loss of photoreceptor cells and an equivalent number of apoptotic cells in Mu-Phe-hPhe-FMK and DMSO retinas. Therefore, calpains are up-regulated by light stress but they do not have a pivotal role in photoreceptor apoptosis.     

Calpains are activated by light but their inhibition has no neuroprotective effect against light-damage

Calpain had been shown to be highly activated at one day after exposure to the damaging light (Perche et al. (2007) Caspase-dependent apoptosis in light-induced retinal degeneration. Invest Ophthalmol Vis Sci 48:2753–2759.), suggesting that they might play a critical role in photoreceptor apoptosis induced by light. Therefore in the present study we investigate the role of calpain in light-induced photoreceptor cell death. In a first set of experiments, untreated albino Wistar rats were sacrificed at 0, 2, 4, 6, 12, 24 h of light exposure and at one day after the light was turned off (D1) to measure retinal calpain activity and to study calpain expression. In a second set of experiments, after control electroretinograms (ERGs), rats were uninjected or injected intravitreally with DMSO or the calpain inhibitor Mu-Phe-hPhe-FMK, before being exposed to the damaging light for 24 h. ERGs were then recorded at one day (D1) and fifteen days (D15) after the end of light exposure. Rats were sacrificed at D1 for apoptotic cell detection or D15 for histological analysis (ONL thickness). Calpain activity and expression significantly increased in Untreated retinas, from 0 h to D1. DMSO has no effect on calpain activity. Mu-Phe-hPhe-FMK significantly inhibited retinal calpain activity by 85% at 2 h of light exposure and still 48% at D1. However, Mu-Phe-hPhe-FMK has no effect on light-induced retinal degeneration as evidence by equivalent loss of function, equivalent loss of photoreceptor cells and an equivalent number of apoptotic cells in Mu-Phe-hPhe-FMK and DMSO retinas. Therefore, calpains are up-regulated by light stress but they do not have a pivotal role in photoreceptor apoptosis.     

Calpain-induced light scattering in young rat lenses is enhanced by UV-B.

The purpose of this study is to determine if UV-B enhances light scattering after proteolysis of crystallins by calpains, and to determine if lens-specific calpain Lp82 is involved, along with m-calpain, in the mechanism of in vitro precipitation. Lens soluble proteins from young rats were hydrolyzed for 24 hr by endogenous lens calpains, and the proteins were further incubated for up to 7 days with periodic irradiation by UV-B. Light scattering was measured daily at 405 nm. SDS-PAGE and immunoblotting assessed proteolysis of crystallins, activation of calpains, and formation of high molecular weight aggregations. Appreciable light scattering occurred in lens soluble proteins after proteolysis of crystallins by m-calpain and Lp82. UV-B markedly enhanced this light scattering and the formation of higher molecular weight aggregates consisting of proteolyzed alpha- and beta- and intact gamma-crystallins. Calpain inhibitor E64 and antioxidants DTE or GSH prevented the light scattering. These results show that calpain-induced light scattering is enhanced by the natural oxidant UV-B. Activation of Lp82, along with m-calpain, contributed to the light scattering. The linkage between proteolysis and oxidation is important because both oxidation and truncation of crystallins are found in aged human lenses, which are constantly exposed to UV irradiation.     

Calpain inhibitor, SJA6017, reduces the rate of formation of selenite cataract in rats.

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.     

Expression of phosphorylated c-Jun N-terminal protein kinase (JNK) in experimental glaucoma in rats

To examine the expression of phosphorylated c-Jun N-terminal kinase (JNK) in cells in the retinal ganglion cell layer of glaucoma, intraocular pressure (IOP) of adult Wistar rats was elevated unilaterally by repeated trabecular argon laser photocoagulation 5 days after intracameral injection of India ink. Animals were euthanized after 3 days, and 1, 2, and 5 weeks of IOP elevation. Immunohistochemistry with specific antibodies against phosphorylated JNK was performed on retinas. Retrograde labeling using Fluorogold and fluorescence immunohistochemistry was performed on retinas 5 weeks after IOP elevation. TdT-mediated biotin-dUTP nick end labeling (TUNEL) was performed on the retinal sections to determine the rate of cell death. There was increased IOP (52.3%) from 3 days to 5 weeks after repeated trabecular laser photocoagulation. Mean number of TUNEL-positive cells in the retinal ganglion cell layer of eyes with experimental glaucoma was 0.43, 0.36, 0.57, and 0.19 per retinal section at 3 days, and 1, 2 and 5 weeks, respectively. No TUNEL-positive cells were noted in controls. In parallel to TUNEL, significantly increased numbers of phosphorylated JNK-labeled cells in the retinal ganglion cell layer were noted at 3 days (9.95 versus 4.15; P=0.005), 1 week (7.65 versus 4.00; P=0.006), 2 weeks (9.13 versus 4.48; P=0.032), and 5 weeks (8.06 versus 4.96; P=0.017) of IOP elevation when compared with contralateral control eyes. Fluorogold labeled RGCs were co-localized with increased phosphorylated JNK immunoreactivity. Some TUNEL-positive cells were phosphorylated JNK immuno-positive. Phosphorylation of JNK occurs in experimental glaucoma and may play a role in retinal ganglion cell death.     

Involvement of cysteine proteases in bFGF-induced angiogenesis in guinea pig and rat cornea.

Overexpression and activation of matrix metalloprotease (MMP) have been implicated in angiogenesis. However, the involvement of cysteine proteases, such as calpains (EC 34.22.17), is obscure. Thus, the purpose of this experiment was to study the involvement of cysteine proteases in angiogenesis induced by basic fibroblast growth factor (bFGF) in guinea pig and rat corneas using cysteine protease inhibitors. Sustained-release polymers containing bFGF were implanted into guinea pig and rat corneas to induce angiogenesis. For treatment of corneal angiogenesis, polymers containing cysteine protease inhibitors, leupeptin or SJA6017, were also implanted into corneas. Using the slit lamp, the corneas were observed for nine days after polymer implantation. Soluble proteins and albumin levels were used as markers of corneal injury by angiogenesis. bFGF induced angiogenesis in guinea pig and rat corneas. In guinea pig cornea, wet weight, the amount of soluble protein and albumin was highest at four days after bFGF-containing pellet implantation. In rat cornea, the amount of soluble protein and albumin was highest at six days, and wet weight increased within four days. One hundred nmole of leupeptin showed a tendency to reduce bFGF-induced angiogenesis in guinea pig cornea, and 10 nmole of SJA6017 was effective in reducing bFGF-induced angiogenesis in rat cornea, although SJA6017 showed a stronger effect than leupeptin. Ten nmole of SJA6017 significantly reduced the number of new blood vessels. These data suggested involvement of cysteine proteases in angiogenesis in guinea pig and rat cornea.     

Caspase activation and amyloid precursor protein cleavage in rat ocular hypertension

PURPOSE: Retinal ganglion cell (RGC) death in glaucoma involves apoptosis. Activation of caspases and abnormal processing of amyloid precursor protein (APP) are important events in other chronic neurodegenerations, such as Alzheimer's disease (AD). The retinal expression and activation of caspases and the patterns of caspase-3-mediated APP processing in ocular hypertensive models of rat glaucoma were investigated. METHODS: RGC death was produced in one eye by chronic exposure to increased intraocular pressure (IOP) or by optic nerve transection. Elevated IOP was produced by obstruction of aqueous humor outflow with laser coagulation or limbal hypertonic saline injection. Caspase activity and APP processing in the retina were examined by RNase protection assay (RPA), immunocytochemistry, immunoblot assay, and colorimetric assay. RESULTS: RPA revealed elevations of caspase-3 mRNA, as well as other apoptosis-related mRNAs. Immunocytochemistry showed caspase-3 activation in RGCs damaged by ocular hypertension. The generation of the caspase-3-mediated APP cleavage product (DeltaC-APP) was also increased in ocular hypertensive RGCs. Western immunoblot assay and colorimetry revealed significantly more activated caspase-3 in ocular hypertensive retinas than in control retinas. The activated form of caspase-8, an initiator caspase, and amyloid-beta, a product of APP proteolysis and a component of senile plaques in AD, were detected in RGCs by immunohistochemistry significantly more often in ocular hypertensive than in control retinas. The amounts of full-length APP were reduced and amyloid-beta-containing fragments were increased in ocular hypertensive retinas by Western immunoblot assay. CONCLUSIONS: Rat RGCs subjected to chronic ocular hypertension demonstrate caspase activation and abnormal processing of APP, which may contribute to the pathophysiology of glaucoma.     

Activation of caspase 9 in a rat model of experimental glaucoma

PURPOSE: We investigated retinal ganglion cell (RGC) death and activation of caspase 9 in rats with experimental glaucoma. METHODS: Elevated intraocular pressure (IOP) was induced in rats using the Morrison model. Surviving backlabeled RGC were counted and TUNEL staining detected apoptosis. Procaspase 9 expression and activated caspase 9 were studied by immunoblot and immunohistochemistry. RESULTS: IOP correlated with surviving RGC. TUNEL-positive RGC were observed in animals with elevated IOP. Procaspase 9 levels increased with IOP intensity. Cleaved caspase 9 was detected by immunoblot only in rats with peak IOP above 35 mm Hg for > or =6 days. Cleaved caspase 9 staining was seen only in the ganglion cell layer of retinas from rats with peak IOP > or =32 mm Hg. CONCLUSIONS: RGC loss is correlated with IOP in experimental glaucoma. These results support activation of caspase 9, the intrinsic caspase cascade, in RGC death in experimental glaucoma.     

Purification of calpain II from rat lens and determination of endogenous substrates

Calpain II (EC 3.4.22.17), a calcium-dependent neutral protease, was purified approximately 7000-fold from the soluble of rat lens. The estimated molecular weight of rat lens calpain II was 120,000, and the enzyme was composed of 80,000 and 28,000 MW subunits. Calpain II required 400 microM calcium, a reducing agent, and pH = 7.5 for maximal activity. The enzyme could not be activated by magnesium, and was inhibited by leupeptin and iodoacetate, but not by phenylmethylsulfonyl fluoride. Purified calpain II degraded rat alpha, beta H-, and beta L-crystallins, insoluble proteins, and intrinsic membrane proteins, gamma-Crystallin was not degraded. The proteolysis caused by purified calpain II was similar to proteolysis occurring during the formation of several experimental cataracts in rodents; this suggested that the enzyme may play a role in cataract formation.     

Sustained elevation of intracellular cGMP causes oxidative stress triggering calpain-mediated apoptosis in photoreceptor degeneration

Sustained elevation in cGMP and a concomitant increase in intracellular Ca(2+) levels in the rd1 photoreceptors are followed by a rapid loss of photoreceptors. In a murine-derived photoreceptor cell line, 661W, treated with the phosphodiesterase inhibitor IBMX or the cyclic GMP-gated channel agonist 8-bromo-cGMP, it was previously found that the induced cell death was mediated by calpain and caspase-3. Because oxidative stress is a common product of ionic imbalance or elevated Ca(2+), we tested the role of oxidative stress in cGMP-induced photoreceptor cell death. In the rd1 mouse retina, oxidative stress was found to precede calpain and caspase-3 activation. In 661W cells, the increase in intracellular cGMP and Ca(2+) resulted in the generation of reactive oxygen species (ROS), the activation of oxidative stress enzymes, and the activation of calpain, followed by apoptosis mediated by the effector caspase-3. All these events, including calpain activation, were ameliorated by docosahexanoic acid (DHA). The cell-permeable inhibitor of calpain, SJA6017, while inhibiting cell death, had no effect on the generation of oxidative stress. These results establish a central role for oxidative stress in cGMP-induced cell death and suggest a ROS-mediated sequential activation of signal transduction events, which provide targets for future treatment strategies.     

Human neural progenitor cells promote photoreceptor survival in retinal explants

Different types of progenitor and stem cells have been shown to provide neuroprotection in animal models of photoreceptor degeneration. The present study was conducted to investigate whether human neural progenitor cells (HNPCs) have neuroprotective properties on retinal explants models with calpain- and caspase-3-dependent photoreceptor cell death. In the first experiments, HNPCs in a feeder layer were co-cultured for 6 days either with postnatal rd1 mouse or normal rat retinas. Retinal histological sections were used to determine outer nuclear layer (ONL) thickness, and to detect the number of photoreceptors with labeling for calpain activity, cleaved caspase-3 and TUNEL. The ONL thickness of co-cultured rat and rd1 retinas was found to be almost 10% and 40% thicker, respectively, compared to controls. Cell counts of calpain activity, cleaved caspase-3 and TUNEL labeled photoreceptors in both models revealed a 30-50% decrease when co-cultured with HNPCs. The results represent significant increases of photoreceptor survival in the co-cultured retinas. In the second experiments, for an identification of putative survival factors, or a combination of them, a growth factor profile was performed on conditioned medium. The relative levels of various growth factors were analyzed by densitometric measurements of growth factor array membranes. Following growth factors were identified as most potential survival factors; granulocyte colony stimulating factor (G-CSF), granulocyte-macrophage colony stimulating factor (GMCSF), insulin-like growth factor II (IGF-II), neurotrophic factor 3 (NT-3), placental growth factor (PIGF), transforming growth factors (TGF-β1 and TGF-β2) and vascular endothelial growth factor (VEGF-D). HNPCs protect both against calpain- and caspase-3-dependent photoreceptor cell death in the rd1 mouse and against caspase-3-dependent photoreceptor cell death in normal rat retinas in vitro. The protective effect is possibly achieved by a variety of growth factors secreted from the HNPCs.     

A key role for calpains in retinal ganglion cell death

PURPOSE: The purpose of this study was to examine the importance of calpains in retinal ganglion cell (RGC) apoptosis and the protection afforded by calpain inhibitors against cell death. METHODS: Two different models of RGC apoptosis were used, namely the RGC-5 cell line after either intracellular calcium influx or serum withdrawal and retinal explant culture involving optic nerve axotomy. Flow cytometry analysis with Annexin V/PI staining was used to identify RGC-5 cells undergoing apoptosis after treatment. TdT-mediated dUTP nick end labeling (TUNEL) was used to identify cells undergoing apoptosis in retinal explant sections under various conditions. Serial sectioning was used to isolate the cell population of the ganglion cell layer (GCL). Western blotting was used to demonstrate calpain cleavage and activity by detecting cleaved substrates. RESULTS: In the RGC-5 cell line, the authors reported the activation of mu-calpain and m-calpain after serum starvation and calcium ionophore treatment, with concurrent cleavage of known calpain substrates. They found that the inhibition of calpains leads to the protection of cells from apoptosis. In the second model, after a serial sectioning method to isolate the cells of the ganglion cell layer (GCL) on a retinal explant paradigm, protein analysis indicated the activation of calpains after axotomy, with concomitant cleavage of calpain substrates. The authors found that inhibition of calpains significantly protected cells in the GCL from cell death. CONCLUSIONS: These results suggest that calpains are crucial for apoptosis in RGCs after calcium influx, serum starvation, and optic nerve injury.