Search

Background: This paper seeks to investigate differences between the neonatal and adult retinal ganglion cell populations to apoptotic death stimuli. Design and Samples: In vitro and ex vivo paradigms involving P6 and P60 Sprague–Dawley rat retinal explants and retinal ganglion cells were employed. Methods: Postnatal day 6 (P6) and 60 (P60) Sprague–Dawley retinal ganglion cells and retinal explants were either serum starved or subjected to excitotoxicity using calcium ionophore A23187. Main Outcome Measures: Apoptosis was detected in both models using terminal dUTP nick end labelling. Expression of Apaf-1, active caspases-3 and 9 in P6 and P60 retinas, and in the ganglion cell layer was examined using Western blotting. Results: In both the dissociated retinal ganglion cell and retinal explant models, P60 retinal ganglion cells were significantly less susceptible to excitoxicity and serum starvation than their P6 counterparts. Western blotting indicated that active caspase-3 and Apaf-1 are downregulated in the Sprague–Dawley rat retina at P60 compared with P6 Conclusions: We demonstrate that neonatal Sprague–Dawley retinal ganglion cells are more susceptible to glaucoma-related death stimuli than their adult counterparts in dissociated retinal ganglion cells and axotomized retinal explant models. It is apparent that these different retinal ganglion cell populations are inherently designed to react differently to death stimuli. Thus caution should be exercised when noting the high susceptibility of neonatal retinal ganglion cells to glaucomatous death stimuli.

J. Neurochem. (2011) 116, 33–42. Abstract Programmed cell death is well established as a key factor in the development of the vertebrate nervous system of which the retina is a unique sensory component. However, it is of utmost importance for the survival of post-mitotic tissues such as the retina that the execution of the cell death program is kept under stringent control once development is complete. This is exemplified by the many retinal dystrophies where aberrant apoptosis results in loss of distinct cell layers in the mature retina and often culminates in blindness. In this study, we report that the extracellular signal-regulated kinase (ERK1/2) pathway plays a key role in the regulation of apoptosis during retinal development. We show that as the retina matures, the emphasis shifts towards survival and ERK1/2 is activated resulting in phosphorylation of the potent BH3-only protein BimEL and a dramatic decline in BimEL expression via proteasomal degradation. We find that activation of ERK1/2 also occurs in response to injury in retinal explants. However, this is a transient response and appears to be overcome by Jun N-terminal kinase activation resulting in induction of BimEL mRNA and photoreceptor apoptosis. Our findings provide new insights into the intracellular pathways responsible for regulating apoptosis during neuronal development and degeneration.

Apoptosis, a cellular process critical to retinal neurogenesis, has been implicated in several neurodegenerative disorders. As the retina matures the suppression of apoptosis occurs and the emphasis shifts towards survival. To identify the cellular changes that bring about this critical shift in the balance, we performed an expression analysis of pro- and anti-apoptotic mediators in the immature, post-natal day 6 (P6) and the post-mitotic adult P60 mouse retina. Laser capture microdissection (LCM) of the P6 and the P60 retina, followed by reverse transcriptase-polymerase chain reaction (RT-PCR) was employed to elucidate changes in the mRNA expression of Apaf-1, caspase-3 and caspase-9 in the individual retinal layers in the young and mature tissue. RT-PCR and Western blotting of whole P6 and P60 retinal preparations was carried out to determine changes in other caspases and key survival mediators at the mRNA and protein level, respectively. Our results demonstrate that each neuronal cell layer in the adult retina down-regulates the gene expression of Apaf-1 and caspase-3, and to a lesser extent, caspase-9. The protein expression levels of other executioner and initiator caspases are also reduced in the adult tissue. Interestingly, XIAP, a potent caspase inhibitor, increases in expression in the adult retina. Additionally, we demonstrate age-dependent increased expression and activation status of the components of the MAPK transduction cascade. Conversely, we observe decreased PI3-K and AKT expression and decreased activity of AKT (pAKT) in the adult retina. Furthermore, results from RNAi experiments demonstrate an additional mechanism of PI3-K regulation in photoreceptor cells. Our findings suggest that a survival strategy adopted by the post-mitotic retina involves a down-regulation of key pro-apoptotic factors concomitant with changes in expression and activation status of certain pro-survival mediators.

Apoptosis of photoreceptor cells in the early postnatal period is a normal feature of mammalian retinal development. The role of mitochondria and caspases in the process has been well established; however, the identification of key apoptotic mediators still remains elusive. Here we report that BIM(EL), a pro-apoptotic BCL-2 family member, may be one such molecule. Following growth factor deprivation, BIM(EL) was up-regulated in mouse 661W cone photoreceptors. This event correlated with the release of mitochondrial apoptogenic factors into the cytosol, the activation of caspases and apoptosis. Moreover, a similar behaviour was observed in response to UV radiation, ionomycin or H(2)O(2) treatments. We identified the PI3K-Akt-FKHRL1 signalling cascade as the main regulatory pathway of BIM(EL) expression in these cells. Finally, using RNA interference, we were able to silence BIM(EL) expression and subsequently suppress caspase-3 activation. In conclusion, we propose BIM(EL) as a critical factor in mitochondria-dependent apoptosis of 661W photoreceptors.

Apoptosis is the mode of photoreceptor cell death in many retinal dystrophies. Exposure of Balb/c mice to excessive levels of light induces photoreceptor apoptosis and represents an animal model for the study of retinal degenerations. Caspases have emerged as central regulators of apoptosis, executing this tightly controlled death pathway in many cells. Previously we have reported that light-induced photoreceptor apoptosis occurs independently of one the key executioners of apoptosis, caspase-3. This present study extends these results reporting on the lack of activation of other caspases in this model including caspases-8, -9, -7, and -1. Furthermore, photoreceptor apoptosis cannot be inhibited with the broad range caspase inhibitor zVAD-fmk indicating that light-induced retinal degeneration is caspase-independent. We demonstrate that cytochrome c does not translocate from mitochondria to the cytosol during photoreceptor apoptosis. We also show that during retinal development apoptotic protease activating factor (Apaf-1) protein levels are markedly decreased and this is associated with the inability to activate the mitochondrial caspase cascade in the mature retina. In addition, there is also a significant reduction in expression of caspases-3 and -9 during retinal maturation and these levels do not increase following light exposure. Finally, we show that the calcium-dependent proteases calpains are active during light-induced retinal degeneration and establish that the calcium channel blocker D-cis-diltiazem completely inhibits photoreceptor apoptosis.

Apoptosis is the mode of photoreceptor cell death in inherited and induced retinal degeneration. However, the molecular mechanisms of photoreceptor cell death in human cases and animal models of retinal dystrophies remain undefined. Exposure of Balb/c mice to excessive levels of white light results in photoreceptor apoptosis. This study delineates the molecular events occurring during and subsequent to the induction of retinal degeneration by exposure to white light in Balb/c mice. We demonstrate an early increase in intracellular calcium levels during photoreceptor apoptosis, an event that is accompanied by significant superoxide generation and mitochondrial membrane depolarization. Furthermore, we show that inhibition of neuronal nitric-oxide synthase (nNOS) by 7-nitroindazole is sufficient to prevent retinal degeneration implicating a key role for neuronal nitric oxide (NO) in this model. We demonstrate that inhibition of guanylate cyclase, a downstream effector of NO, also prevents photoreceptor apoptosis demonstrating that guanylate cyclase too plays an essential role in this model. Finally, our results demonstrate that caspase-3, frequently considered to be one of the key executioners of apoptosis, is not activated during retinal degeneration. In summary, the data presented here demonstrate that light-induced photoreceptor apoptosis in vivo is mediated by the activation of nNOS and guanylate cyclase and is caspase-3-independent.

This paper seeks to investigate differences between the neonatal and adult retinal ganglion cell populations to apoptotic death stimuli. DESIGN AND SAMPLES: In vitro and ex vivo paradigms involving P6 and P60 Sprague-Dawley rat retinal explants and retinal ganglion cells were employed. Postnatal day 6 (P6) and 60 (P60) Sprague-Dawley retinal ganglion cells and retinal explants were either serum starved or subjected to excitotoxicity using calcium ionophore A23187.Apoptosis was detected in both models using terminal dUTP nick end labelling. Expression of Apaf-1, active caspases-3 and 9 in P6 and P60 retinas, and in the ganglion cell layer was examined using Western blotting.In both the dissociated retinal ganglion cell and retinal explant models, P60 retinal ganglion cells were significantly less susceptible to excitoxicity and serum starvation than their P6 counterparts. Western blotting indicated that active caspase-3 and Apaf-1 are downregulated in the Sprague-Dawley rat retina at P60 compared with P6.We demonstrate that neonatal Sprague-Dawley retinal ganglion cells are more susceptible to glaucoma-related death stimuli than their adult counterparts in dissociated retinal ganglion cells and axotomized retinal explant models. It is apparent that these different retinal ganglion cell populations are inherently designed to react differently to death stimuli. Thus caution should be exercised when noting the high susceptibility of neonatal retinal ganglion cells to glaucomatous death stimuli.

Tumour stroma gene expression in biopsy specimens may obscure the expression of tumour parenchyma, hampering the predictive power of microarrays. We aimed to assess the utility of fluorescence-activated cell sorting (FACS) for generating cell populations for gene expression analysis and to compare the gene expression of FACS-purified tumour parenchyma to that of whole tumour biopsies. Single cell suspensions were generated from colorectal tumour biopsies and tumour parenchyma was separated using FACS. Fluorescence-activated cell sorting allowed reliable estimation and purification of cell populations, generating parenchymal purity above 90%. RNA from FACS-purified and corresponding whole tumour biopsies was hybridised to Affymetrix oligonucleotide microarrays. Whole tumour and parenchymal samples demonstrated differential gene expression, with 289 genes significantly overexpressed in the whole tumour, many of which were consistent with stromal gene expression (e.g., COL6A3, COL1A2, POSTN, TIMP2). Genes characteristic of colorectal carcinoma were overexpressed in the FACS-purified cells (e.g., HOX2D and RHOB). We found FACS to be a robust method for generating samples for gene expression analysis, allowing simultaneous assessment of parenchymal and stromal compartments. Gross stromal contamination may affect the interpretation of cancer gene expression microarray experiments, with implications for hypotheses generation and the stability of expression signatures used for predicting clinical outcomes.

Programmed cell death (PCD) during development of the mouse retina involves activation of the mitochondrial pathway. Previous work has shown that the multidomain Bcl-2 family proteins Bax and Bak are fundamentally involved in this process. To induce mitochondrial membrane permeabilization, Bax and Bak require that prosurvival members of the family be inactivated by binding of “BH3-only” members. We showed previously that the BH3-only protein BimEL is highly expressed during postnatal retinal development but decreases dramatically thereafter. The purpose of this study was to investigate a possible role for Bim, in retinal development and degeneration, upstream of Bax and Bak.Bim−/−mice analyzed for defective retinal development exhibit an increase in retinal thickness and a delay in PCD, thereby confirming a role for Bim. We also demonstrate that in response to certain death stimuli,bim+/+retinal explants upregulate BimEL leading to caspase activation and cell death, whereasbim−/−explants are resistant to apoptosis. Finally, we analyzed Bim expression in the retinal degeneration (rd) mouse, anin vivomodel of retinal degeneration. Bim isoforms, which decrease during development, are not reexpressed during retinal degeneration and ultimately photoreceptor cells die by a caspase-independent mechanism. Thus, we conclude that in cases in which BimEL is reexpressed during pathological cell death, developmental cell death pathways are reactivated. However, the absence of BimEL expression correlates with caspase-independent death in the rd model.

The intrinsic apoptotic pathway is essential for murine development. We have previously shown that key mediators of this pathway, such as Bim, Apaf-1 and caspase-3, are down-regulated during the postnatal development of the retina. In this study, we demonstrate that this expression pattern is a feature of other distinct tissues such as the brain, heart and skeletal muscle. Caspase-9 expression is also examined and is shown to follow a different pattern in each tissue. Interestingly, we show that peripheral cells of the internal granular layer of the cerebellum do not down-regulate the intrinsic apoptotic pathway proteins Bim, Apaf-1 or caspase-3. Furthermore, Bim expression is also detectable in the brain stem and the CA3 region of the hippocampus in the adult cerebrum. Finally, we demonstrate that the incidence of TUNEL positive cells in the selected tissues decreases during postnatal development in correlation with the general down-regulation of key apoptotic pathway proteins. In contrast, we also demonstrate that apoptosis persists in the adult thymus and that this tissue continues to express Bim, Apaf-1 and caspase-3 at the same levels as the neonate. In summary, this study shows that a selection of post-mitotic tissues down-regulate key apoptotic proteins, in contrast to the thymus, which requires apoptosis for normal function in early adulthood.

Apoptosis plays a crucial role in the sculpture of the mammalian retina during development. However, once the retina is fully differentiated, the emphasis must shift towards survival and mechanisms have to be put in place to prevent inappropriate cell death. In this study, we identify a potential control point at the level of mitochondrial permeability. We show that pro-apoptotic Bcl-2 family members known to be involved in the regulation of permeability transition and physiological cell death in the retina are down regulated during postnatal retinal development. In addition, we demonstrate an age-dependent susceptibility to retinal cell death induced by various stimuli known to target mitochondrion. These results potentially explain why retinal cells employ different death pathways depending on their stage of development. In contrast to developmental apoptosis, pathological retinal cell death in several animal models has been reported to occur independently of caspase activation. Here, we show that not only is cytochrome c release precluded from degenerating retinas but other pro-death molecules such as Omi/HtrA2 and AIF also remain in the mitochondrion. Our results indicate that transcriptional regulation of ‘death genes’ such as pro-apoptotic Bcl-2 family members during retinal development affords protection in adult post-mitotic neurons by preventing execution of the archetypal mitochondrial death pathway.

During development of the mammalian retina, neurons that do not succeed in establishing functional synaptic connections are eliminated by apoptosis, allowing the formation of a finely tuned network. Growth factors play a crucial role in controlling the balance between apoptosis and survival signals not only at developmental stages but also in long-term preservation of retinal functions. In the present work, we explore the apoptotic mechanisms triggered by growth factor deprivation of retina-derived 661W cells. Under serum starvation conditions, these cone photoreceptors underwent cell death with participation of caspase-9, -3 and -12. Interestingly, inhibition of caspases did not prevent apoptosis but only resulted in a temporary delay. We show m-calpain activation in parallel with caspases, indicating that more than one execution pathway is available to cone photoreceptors. Moreover, crosstalk of the caspase and calpain pathways was detected, suggesting a loop that may act to amplify the apoptotic cascade.

Apoptosis is the mode of cell death in retinitis pigmentosa, a group of retinal degenerative disorders primarily affecting rod photoreceptors. Although caspases have been demonstrated to play a central role in many incidences of apoptosis, accumulating evidence suggests that they may not be required for all forms of apoptotic cell death. The present study examined the mechanism of cell death in twoin vivomodels of photoreceptor apoptosis: the retinal degeneration (rd) mouse, a naturally occurring mutant model, andN-methyl-N-nitrosourea-induced retinal degeneration. Specifically, we examined the activation status of caspase-9, -8, -7, -3, and -2 and determined the caspase requirements for cytochromecrelease, DNA fragmentation, and apoptosis-associated proteolysis of specific caspase substrates. We show that apoptosis in bothin vivomodels is independent of caspase-9, -8, -7, -3, and -2 activation. DNA fragmentation occurs in the absence of caspase-mediated ICAD (inhibitor of caspase-activated DNase) proteolysis, suggesting that an alternative endonuclease is responsible for DNA cleavage in these models. Importantly, we show that apoptosome activation is prevented because of an absence of mitochondrial cytochromecrelease. Experiments performed using a cell-free system indicate that cytochromec-dependent proteolysis and activation of caspase-9 can be restored in a neonatal cell-free system. However, we found that cytochromec-dependent proteolysis and activation of caspase-9 could not be restored in an adult cell-free system because of an age-related decrease in the expression of Apaf-1 in the normal developing mouse retina. In the rd mouse, however, this age-related downregulation of apoptotic proteins was not observed, highlighting a critical feature of this model and the prevention of cytochromecrelease as an apical event in caspase-independent apoptosis in this system.

Cells are constantly generating reactive oxygen species (ROS) during aerobic metabolism. As a consequence, each cell is equipped with an extensive antioxidant defence system to combat excessive production of ROS. Oxidative stress occurs in cells when the generation of ROS overwhelms the cell's natural antioxidant defences. There is a growing consensus that oxidative stress and the redox state of a cell plays a pivotal role in regulating apoptosis, a tightly controlled form of cell death in which a cell partakes in its own demise. More recently, a role for reactive nitrogen species (RNI) as both positive and negative regulators of cell death has been established. This review describes the major sources of ROS and RNI in a cell, the control of cell death by these species and the role of antioxidants as regulators of oxidative stress and apoptosis. Finally, the various methods that can be employed in establishing a role for both ROS and RNI in apoptosis will be discussed with particular emphasis on their intracellular detection.

To explore the possible influence of defined genetic backgrounds on photoreceptor viability and function in mice carrying a targeted disruption of the rhodopsin gene, the severities of retinopathies in Rho-/- mice on C57BL/6J and 129Sv congenic backgrounds were compared by light microscopy and electroretinography and qualitatively by in situ end labeling of DNA in apoptotic photoreceptor nuclei of retinal sections. Cone photoreceptor viability and function were shown to deteriorate more slowly on the C57BL/6J background in comparison to that of the 129Sv, with significantly greater numbers of outer nuclear layer nuclei in the retinas of C57BL/6J mice at 3 and 4 months of age. Both amplitude and waveform features of the ERG were shown to be remarkably different in the two strains, indicating an approximately 6-fold difference in C57BL/6J Rho-/- mice compared to 129Sv Rho-/- mice at 80 days. Thus, in comparison with the 129Sv strain, genetic modifiers appear to constitute a component of the C57BL/6J background, the expression of which significantly protects cone photoreceptors from apoptotic death in a mutation-induced murine retinopathy. The differences in phenotype revealed in this study are sufficient in principle to provide a basis for comparisons to be made between QTLs in light-induced and mutation-induced systems.

PURPOSE. Gliosis is the response of glial cells within retinal tissue to injury. It can be beneficial in the short term, but if the response is extended it can lead to scar formation, which contributes to blindness. Phosphorylation of extracellular signal regulated kinase 1/2 (ERK1/2) is considered to be a hallmark event of gliosis, but the factors involved throughout its associated signaling pathway remain poorly understood, particularly in the retina. Because reactive oxygen species (ROS) can inhibit phosphatases, thereby altering the phosphorylation of proteins, this study tested the hypothesis that ROS regulate the phosphorylation of ERK1/2 (pERK1/2) in gliosis. METHODS. Increases in pERK1/2 were detected using Western blotting and immunofluorescence in three models of retinal stress, specifically the in vivo light induction, the rd1 disease, and the ex vivo retinal explant models. Explanted murine retinas were used to identify the signaling partners of pERK1/2 via Western blotting, in conjunction with inhibitors. The effect of this pathway on cell death was measured with terminal dUTP nick end labeling. RESULTS. It was demonstrated that several inhibitors of ROS greatly reduce the levels of pERK1/2 in the somata of M¨ cells and furthermore decrease two other downstream signaling events: the phosphorylation of STAT3 and the upregulation of basic fibroblast growth factor. Using the specific inhibitor of ERK1/2, UO126, the resultant outcomes of this signaling pathway were determined to contribute significantly to cell survival. CONCLUSIONS. The novel finding of this study that ROS contribute to a prosurvival signaling pathway in retinal M¨ cell gliosis indicates that some degree of caution should be used when considering antioxidants as therapeutics. (Invest Ophthalmol Vis Sci. 2012;53:6645‐6654) DOI:10.1167/ iovs.12-10525

* University of Pittsburgh Cancer Institute, Center for Environmental Oncology, 5150 Centre Avenue, Suite 435, Pittsburgh, Pennsylvania, 15232, USA. University of Pittsburgh Graduate School of Public Health. Department of Epidemiology, Department of Environmental and Occupational Health, Pittsburgh, Pennsylvania, USA. International Agency for Research on Cancer and Institut National de la Sante et de la Recherche Medicale, Lyon, France, now with University of Bordeaux, Department of Preventive Medicine, France. Correspondence to D.L.D. e-mail: davisdl@upmc.edu

Purpose Apoptosis is a form of programmed cell death essential for both tissue development and maintenance of tissue homeostasis. Apoptosis protease activating factor (Apaf)-1 and caspase 3 are downregulated in the retina during postnatal development. The decreased expression of these genes is potentially a critical survival strategy adopted to protect against accidental cell death. The purpose of this study was to investigate the transcriptional mechanism involved in the downregulation of Apaf-1 and caspase 3. Methods SDS-polyacrylamide gel electrophoresis and semiquantitative PCR were used to examine Apaf-1 and caspase 3 expression levels during development. TdT-mediated dUTP nick-end labeling (TUNEL) and DNA laddering were used to identify cells undergoing apoptosis. Results A decrease in expression of Apaf-1 and caspase 3 during retinal development correlated with a decreased susceptibility to an apoptotic stimulus. Furthermore, treatment with a histone deacetylase (HDAC) inhibitor, trichostatin A (TSA), resulted in widespread hyperacetylation in the retina, coinciding with transcriptional activation of Apaf-1 and caspase 3 and subsequent induction of apoptosis in postnatal day (P)5 and P15 retinas. However, inhibition of HDAC activity is not sufficient to induce apoptosis in the mature retina (P60). Conclusions Overall, these results elicit the conclusion that downregulation of Apaf-1 and caspase 3 in the developing retina correlates with a decreased susceptibility to apoptotic stimuli and ensures the survival of the retina. Furthermore, the authors propose that, in the early postnatal retina, HDAC activity governs the transcriptional regulation of these genes. Upregulation of Apaf-1 and caspase 3 coincides with an induction of apoptosis. In the mature retina transcriptional activation of these genes or induction of apoptosis was not observed.

PURPOSE. The purpose of this study was to determine the susceptibility of the retinal ganglion cell layer (GCL) to apoptosis after optic nerve transection and excitotoxic stimulus and to investigate the regulation of apoptosis in the GCL during development. The authors also sought to determine the role played by caspases in cell death and their expression during development. METHODS. TdT-mediated dUTP nick end labeling (TUNEL) was used to identify cells undergoing apoptosis during mouse retinal development from postnatal day (P)3 to P5 and in retinal explant sections under various conditions. The expression of active caspases was determined by immunohistochemistry (IHC) using an antibody that detects the cleaved large subunit. IHC was also used to detect the expression levels of procaspase-3, procaspase-9, and Apaf-1 in P6 and P60 whole eye sections. Retinal ganglion cells at ages P6 and P60 were purified by immunopanning, total RNA was extracted, and mRNA levels of the above proteins were determined by semiquantitative PCR. RESULTS. After optic nerve transection, a significant number of TUNEL-positive cells were seen 24 hours after lesion in P6 retinas. This death was caspase dependent, as shown by IHC and caspase inhibition with zVAD-fmk. In contrast, adult GCL was resistant to apoptosis under these conditions. Similarly, after excitotoxic stimulus, the GCL of the P6 retinas underwent apoptosis at 6 hours and was caspase dependent, whereas adult GCL was resistant. Developmental apoptosis in the GCL between P2 and P6 was shown to involve caspase-3 and caspase-9. Significant downregulation of Apaf-1 and caspase-3 was detected in the P60 GCL at both the mRNA and the protein levels. CONCLUSIONS. Adult GCL is more resistant to apoptosis than neonatal GCL after ON transection and excitotoxic stimulus. The expression of caspase-3 and Apaf-1 is significantly reduced in adult GCL. The authors suggest that age-dependent susceptibility to apoptosis may be caused by this reduced expression. (Invest Ophthalmol Vis Sci. 2006;47:807‐814) DOI:10.1167/ iovs.05-0520

Purpose The primary purpose of this study was to characterize photoreceptor apoptosis in the rd mouse. Given that apoptosis is the final common pathway in many cases of retinal degeneration, the ability to retard or even arrest this process may ameliorate retinal disorders such as retinitis pigmentosa (RP). The absence of any recognized therapy emphasizes the fact that a detailed knowledge of the molecular events involved is necessary to identify rational targets for therapeutic intervention. Methods Flow cytometry was used to measure physical and chemical characteristics in the photoreceptor population. Individual cells flow in suspension past one or more lasers, scattering light and emitting fluorescence. Western blot techniques demonstrated cleavage of calpain-specific substrates. Retinal explant cultures were used for inhibitor studies. Postnatal day 10 (P(10)) rd retinas were cultured without retinal pigment epithelium (RPE) attached up to P(17). Results This study demonstrated calcium overload in the cytosol and subsequently in mitochondria. Mitochondrial membrane depolarization and reactive oxygen species (ROS) were detected later, during the peak of cell death. Analysis of downstream events indicated early activation of calcium-activated calpains. Treatment of rd retinal explants with the calpain inhibitor N-acetyl-Leu-Leu-Nle-CHO (ALLN) successfully inhibited calpain-induced alpha-fodrin cleavage, yet it did not protect against photoreceptor degeneration. Finally, the results demonstrate an increase in the levels of both precursor and processed forms of the aspartate protease cathepsin D. Conclusions Excessive calcium influx is an early event that initiates the activation of calcium-activated proteases. However, these proteases are not singularly the cause of death, because their inhibition does not prevent apoptosis. Indeed, the results presented herein suggest that multiple pathways are involved and that each of these components may have to be addressed for cell death to be successfully inhibited.