Absence of voltage-dependent calcium channels delays photoreceptor degeneration in rd mice

Retinal degeneration results from the apoptotic cell death of photoreceptors. While mutations in a large number of genes give rise to retinal degeneration, the specific mechanisms are not well understood. One hypothesis involves mediation of apoptosis by high concentrations of intracellular Ca(2+). We used a mouse line that carries the rd mutation but also lacks the major L-type voltage-dependent Ca(2+) channel at the photoreceptor synapse to examine whether this route of Ca(2+) entry plays a role in photoreceptor degeneration. In both experimental and control mice, the photoreceptors degenerate. However, at postnatal days 16, 18, and 21 there is a delay in photoreceptor cell loss in the experimental mice, which lack L-type voltage-dependent Ca(2+) channels, compared to controls. These data indicate that Ca(2+) entry via the L-type voltage-dependent Ca(2+) channel contributes to the mechanisms responsible for photoreceptor cell death in this mouse model of retinitis pigmentosa.     

Light induces programmed cell death by activating multiple independent proteases in a cone photoreceptor cell line

PURPOSE: Although the apoptotic death of photoreceptor cells in retinal degenerative disorders is well documented, the molecular mechanism is not understood. The objective of this study was to determine the molecular events leading to the death of photoreceptor cells. METHODS: An assay was developed wherein 661W cells, a cone photoreceptor cell line, were stressed with light and percentage of surviving cells was determined. The degree of cell death was established using the MTT assay. Western blot analysis was used to confirm the activation of multiple proteases. Amounts of retinaldehydes were determined by extraction and HPLC. RESULTS: 661W cells were more susceptible to light stress only in the presence of the chromophore 9-cis retinal for 4 hours. On exposure to light, 9-cis retinal was converted to all-trans retinal, which was found to be toxic to cells in the presence of light. However, all-trans retinol, which is the product of action by the enzyme retinol dehydrogenase on all-trans retinal, was not toxic. The sensitivity to light increased with serum deprivation. Light stress activated caspases, calpain 2, and cathepsin D independently and led to the demise of the cell. The mitochondria-dependent apoptotic pathway was also activated after the truncation of Bid, the pre-proapoptotic protein. Truncation of Bid led to the release of cytochrome c from the mitochondria and the activation of caspase 9. CONCLUSIONS: The activation of multiple proteases by light-induced stress is a relevant finding for studies conducted to investigate the use of pharmaceutical agents to retard or cure the loss of cone photoreceptors observed in age-related macular degeneration and other degenerative retinal diseases.     

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.     

Oxidative Stress Resistance 1 Gene Therapy Retards Neurodegeneration in the rd1 Mutant Mouse Model of Retinopathy

PurposeOxidative stress is a major factor underlying many neurodegenerative diseases. However, antioxidant therapy has had mixed results, possibly because of its indiscriminate activity. The purpose of our study was to determine if the human OXR1 (hOXR1) antioxidant regulatory gene could protect neurons from oxidative stress and delay photoreceptor cell death.MethodsThe cone-like 661W cell line was transfected to stably express the hOXR1 gene. Oxidative stress was induced by the addition of hydrogen peroxide (H₂O₂). Intracellular levels of reactive oxygen species (ROS), caspase cleavage, and cellular resistance to oxidative stress were determined and compared between the control and hOXR1 cells. For in vivo analysis, AAV8-hOXR1 was injected subretinally into the rd1 mouse model of retinal degeneration. Functional and structural integrity of the photoreceptors were assessed using electroretinography (ERG), histology, and immunofluorescence analysis.ResultsExpression of hOXR1 increased cellular resistance and reduced ROS levels and caspase cleavage in the 661W cell line after H₂O₂-induced oxidative stress. Subretinal injection of AAV8-hOXR1 in the rd1 mice improved their photoreceptor light response, expression and localization of photoreceptor-specific proteins, and delayed retinal degeneration.ConclusionsOur results suggest that OXR1 is a potential therapy candidate for retinal degeneration. Because OXR1 targets oxidative stress, a common feature of many retinal degenerative diseases, it should be of therapeutic value to multiple retinal degenerative diseases.     

Induction of autophagy and cell death by tamoxifen in cultured retinal pigment epithelial and photoreceptor cells

Purpose. We investigated the mechanism of tamoxifen (TAM) retinotoxicity using human retinal pigment epithelial (RPE)-derived (ARPE-19) and photoreceptor-derived (661W) cells. Methods. Cultured ARPE-19 and 661W cells were treated with 5 to 10 μM TAM, and the resultant cell death was quantified using lactate dehydrogenase (LDH) release assay. Cellular oxidative stress was determined by measuring 5-(and-6)-carboxy-2',7'-dichlorohydrofluorescein diacetate (H(2)-DCFDA) fluorescence. Changes in intracellular free zinc levels were monitored using the zinc-specific fluorescent dye, FluoZin-3 AM. Autophagic vacuole formation was assessed morphologically in ARPE-19 and 661W cells transfected with the fluorescent protein-conjugated markers, RFP-LC3 or GFP-LC3. Results. Following exposure to TAM, both ARPE-19 and 661W cells had cytosolic vacuoles within 1 hour and underwent cell death within 18 hours. In both cell types, TAM-induced cell death was accompanied by increased oxidative stress and elevated zinc levels, and was attenuated by the antioxidant N-acetyl-L-cysteine (NAC) or the zinc chelator N,N,N'N'-tetrakis(-)(2-pyridylmethyl)-ethylenediamine (TPEN). The levels of LC3-II as well as the number of autophagic vacuoles (AVs) increased after TAM treatment. Double staining for lysosomes and AVs showed that autolysosome formation proceeded normally. Consistent with this, autophagy flux was increased. Finally, as shown in other cases of autophagic cell death, lysosomal membrane permeabilization (LMP) as well as caspase-dependent apoptosis contributed to TAM-induced cell death. Conclusions. ARPE-19 and 661W cells were vulnerable similarly to TAM-induced cytotoxicity. Increases in zinc levels and oxidative stress, excessive activation of autophagy flux, and ultimately the occurrence of LMP and consequent caspase activation may contribute to the well-established retinal cytotoxicity of TAM.     

Ceramide is a mediator of apoptosis in retina photoreceptors

PURPOSE: The precise mechanisms involved in photoreceptor apoptosis are still unclear. In the present study, the role of ceramide, a sphingolipid precursor that induces apoptosis on cellular stress, was investigated in relation to the activation of cell death in photoreceptors. METHODS: Rat retina neuronal cultures, with or without docosahexaenoic acid (DHA), were treated with the ceramide analogue acetylsphingosine (C2-ceramide), and with a glucosylceramide synthase inhibitor. Ceramide synthesis in cultures treated with the oxidant paraquat was evaluated with [3H]palmitate. The effect of inhibitors of ceramide de novo synthesis, fumonisin B1 and cycloserine, on photoreceptor apoptosis was investigated. Apoptosis, mitochondrial membrane potential, and Bcl-2 expression were determined. RESULTS: Addition of C2-ceramide induced photoreceptor apoptosis. Paraquat increased formation of [3H]ceramide in photoreceptors, compared with the control, whereas inhibition of ceramide synthesis, immediately before paraquat treatment, prevented paraquat-induced photoreceptor apoptosis. Fumonisin also reduced photoreceptor apoptosis during early development in vitro. DHA, the retina major polyunsaturated fatty acid, which protects photoreceptors from oxidative stress-induced apoptosis, completely blocked C2-ceramide-induced photoreceptor death, simultaneously increasing Bcl-2 expression. Inhibiting glucosylceramide synthase, which catalyzes ceramide glucosylation, before ceramide or paraquat treatment blocked DHA's protective effect. CONCLUSIONS: The results suggest that oxidative stress stimulated an increase in ceramide levels that induced photoreceptor apoptosis. DHA prevented oxidative stress and ceramide damage by upregulating Bcl-2 expression and glucosylating ceramide, thus decreasing its intracellular concentration. This shows for the first time that ceramide is a critical mediator for triggering photoreceptor apoptosis in mammalian retina and suggests that modulating ceramide levels may provide a therapeutic tool for preventing photoreceptor death in neurodegenerative diseases.     

Characterization of Wnt signaling during photoreceptor degeneration

PURPOSE: The Wnt pathway is an essential signaling cascade that regulates multiple processes in developing and adult tissues, including differentiation, cellular survival, and stem cell proliferation. The authors recently demonstrated altered expression of Wnt pathway genes during photoreceptor death in rd1 mice, suggesting an involvement for Wnt signaling in the disease process. In this study, the authors investigated the role of Wnt signaling in retinal degeneration. METHODS: The Wnt signaling reporter mouse line Tcf-LacZ was crossed with retinal degeneration rd1 mice, and beta-galactosidase expression was used to localize Wnt signaling during photoreceptor death. To analyze the role of Wnt signaling activation, primary mixed retinal cultures were prepared, and XTT and TUNEL assays were used to quantify cell death. Luciferase reporter assays were used to measure Wnt signaling. RESULTS: The canonical Wnt signaling pathway was activated in Müller glia and the ganglion cell layer during rod photoreceptor degeneration in rd1/Tcf-LacZ mice. Wnt signaling was confirmed in cultured primary Müller glia. Furthermore, Wnt signaling activators protected photoreceptors in primary retinal cultures from H(2)O(2)-induced oxidative stress. The Wnt ligands Wnt5a, Wnt5b, Wnt10a, and Wnt13 were expressed in the degenerating retina and are candidate Wnt signaling activators in vivo. CONCLUSIONS: This study is the first demonstration that Wnt signaling is activated in the degenerating retina and that it protects retinal cultures from oxidative stress. These data suggest that Wnt signaling is a component of the glial protective response during photoreceptor injury. Therefore, inducing Wnt activation, alone or in combination with growth factors, may increase the threshold for apoptosis and halt or delay further photoreceptor degeneration.     

Regulation of ON bipolar cell activity

Synaptic transmission from photoreceptors to all types of ON bipolar cells is primarily mediated by the mGluR6 receptor. This receptor, which is apparently expressed uniquely in the nervous system by ON bipolar cells, couples negatively to a nonselective cation channel. This arrangement results in a sign reversal at photoreceptor/ON bipolar cell synapse, which is necessary in order to establish parallel ON and OFF pathways in the retina. The synapse is an important target for second messenger molecules that are known to modulate synaptic transmission elsewhere in the nervous system, second messengers that act on a time scale ranging from milliseconds to minutes. This review focuses on two of these molecules, Ca(2+) and cGMP, summarizing our current knowledge of how they modulate gain at the photoreceptor/ON bipolar cell synapse, as well as their proposed sites of action within the mGluR6 cascade. The implications of plasticity at this synapse for retinal function will also be examined.     

Sensitivity of photoreceptor-derived cell line (661W) to baculoviral p35, Z-VAD.FMK,and Fas-associated death domain

PURPOSE: Rod, cone, cone-rod, and macular dystrophies eventually bring about the death of cone photoreceptor cells. The present study explores means of inhibiting apoptosis in addition to inducing a specific apoptotic pathway within a photoreceptor-derived cell line. METHODS: Retinal cell culture of murine 661W photoreceptor-derived cells was used to assess the effect of both a synthetic peptide inhibitor of caspases (benzyloxycarbonyl-Val-Ala-DL-Asp-[Ome] fluoromethylketone [Z-VAD.FMK]) and a natural inhibitor, baculoviral p35. In addition, the effect of transfection of Fas-associated death domain (FADD), a cellular protein implicated in receptor-induced apoptosis, was assessed. Assays were performed by transient transfection of cell cultures, and results were recorded by cell counting, Western blot, and spectrophotometry. RESULTS: Western blot analysis and chromogenic caspase substrate cleavage analysis confirmed the activation of caspases within 661W cells. At a concentration of 80 micro M, Z-VAD.FMK, 72.36% +/- 0.93% of 661W cells survived cytotoxic insult compared with 6.99% +/- 1.35% of control cells. Transient transfection of 1200 ng baculoviral p35 conferred a protection of 75.30% +/- 4.23%, compared with 19.61% +/-1.84% of control cells, and it was additionally observed that as little as 50 ng transfection of FADD was capable of inducing the death of 53.21% +/- 1.33% of cells in 661W cultures. CONCLUSIONS: Apoptotic cell death in 661W cells is caspase dependent and may be inhibited with both a synthetic and natural inhibitor of caspase function. Furthermore, 661W cells are highly sensitive to the FADD protein, which may suggest a number of novel therapeutic approaches to halt photoreceptor cell apoptosis.     

Multiple, parallel cellular suicide mechanisms participate in photoreceptor cell death

Photoreceptor degeneration in human photoreceptor dystrophies and in the relevant animal models has been thought to be executed by one common mechanism -- caspase-mediated apoptosis. However, recent experiments have challenged this concept. In previous experiments, analyzing gene expression in the degenerating rd/rd mouse retina, we have suggested that the gene defect leads to oxidative stress and altered metabolism, which may induce caspase-dependent and caspase-independent cell death mechanisms such as the activation of cystein-proteases, lysosomal proteases, autophagy and complement-mediated lysis. In this study we asked two questions. First, whether a temporal analysis of these different mechanisms during the course of degeneration would enable us to establish a causal relationship between these events; and second, whether photoreceptor degeneration in different models of photoreceptor dystrophies occurs by activating the same mechanisms. Three models of photoreceptor degeneration were chosen in which photoreceptor degeneration is caused by different events: the rd/rd mouse (calcium overload); the rds/rds mouse (structural defect); and light-damage (LD; oxidative stress). Marker genes were selected for the identified processes. PCR-analysis on laser capture microdissection samples was used to verify the expression of these genes in the rod photoreceptor layer. A temporal relationship between the processes was established at the mRNA level, using quantitative RT-PCR. The time course of gene expression was compared to that of cell loss (loss of rows of photoreceptor nuclei) and apoptosis (TUNEL labeling). Apoptosis and autophagy was analyzed using enzymatic assays. The time course of apoptosis and TUNEL labeling coincide in all three models. Complement-activated lysis was found to either parallel (rd/rd and rds/rds) or precede (LD) the development of TUNEL-positive cells. Autophagy was determined to parallel (rd/rd and LD) or lag (rds/rds) behind the development of TUNEL-positive cells. In all three models, glucose metabolism was found to be increased significantly prior to the onset of cell death, but then dropped in parallel with the loss of cells. The presence of the marker genes was verified by laser capture microdissection, and apoptosis (caspase activity) and autophagy (lysozyme and cathepsin activity) were verified in retina extracts. These results provide evidence that irrespective of whether photoreceptor degeneration is triggered by gene defects (lack of beta-PDE or rds/peripherin) or environmental stress (light-damage), a number of pro-apoptotic mechanisms are triggered leading to the degeneration of the photoreceptor cells. The temporal pattern of the different pathways suggests that the non-caspase-dependent mechanisms may actively participate in the demise of the photoreceptors, rather than represent a passive response of the retina to the presence of dying cells. Thus, unless the common upstream initiator for a given photoreceptor dystrophy is found, multiple rescue paradigms need to be used to target all active pathways.     

Inhibitory effects of retinoic acid receptor alpha stimulants on murine cataractogenesis through suppression of deregulated calpains

PURPOSE: To determine whether retinoic acid (RA)-mediated inhibition of deregulated calpains had any effect on the development of cataract given that accumulating evidence has demonstrated a possible relationship between cataractogenesis and inappropriate activation of calpains. METHODS: The authors examined for Ca(2+) influx and expression alteration of calpains in F9 cells with or without RAs, such as all-trans retinoic acid (ATRA), and specific stimulant of retinoic acid receptor alpha (RARalpha; Am580) in the presence of oxidative stress, such as mediated by H(2)O(2). They next examined the clinical relevance of RAs by applying these agents to a murine diabetic cataract and observed the development of the disease. RESULTS: F9 cells constitute a well-established autonomous cell model for investigating retinoid signaling, partially representing the lens epithelial phenotype, as determined by the expression of aquaporin 0, a specific differentiation marker for lens cells. Treatment with ATRA and Am580 significantly decreased the influx of Ca(2+) into the cells, causally resulting in decreased mRNA expression and inhibited activation of calpains. In addition, RARalpha agonists significantly abrogated the upregulation of calpain 2 induced by H(2)O(2), which is a potential etiological contributor to the diabetic cataract, whereas H(2)O(2) had no effect on calpain 1. Importantly, this RA-mediated gene-expression alteration was sufficient for dramatically inhibiting the development of lens opacity in mice with diabetes. CONCLUSIONS: Results showed that a certain type of RA inhibits Ca(2+) elevation and subsequent overactivation of calpains, suggesting the potential feasibility of calpain-targeting therapies mediated by RA for cataract.     

Protective effect of docosahexaenoic acid on oxidative stress-induced apoptosis of retina photoreceptors

PURPOSE: In a recent study, it was demonstrated that docosahexaenoic acid (DHA) promotes the survival of retinal photoreceptors in vitro, delaying apoptosis. However, lipid enrichment in DHA is known to contribute to retina vulnerability to oxidative stress. In this study, the effect of oxidative damage on rat retina neurons in vitro and whether DHA enhances or diminishes this damage were investigated. METHODS: Rat retina neurons in 3-day cultures, with or without DHA, were treated with the oxidant paraquat. After 24 hours, apoptosis, mitochondrial membrane integrity, and Bcl-2 and Bax expression were immunocytochemically determined. RESULTS: Paraquat induced apoptosis in amacrine and photoreceptor neurons, major neuronal types in the culture. Neuronal apoptosis was accompanied by mitochondrial membrane depolarization, an increase in the amount of photoreceptors expressing Bax, and a decrease in those expressing Bcl-2. Addition of DHA reduced photoreceptor apoptosis by almost half, simultaneously preserving their mitochondrial membrane integrity. DHA blocked the paraquat-induced increase in Bax expression and remarkably upregulated Bcl-2 expression. Glia-derived neurotrophic factor, a photoreceptor trophic factor, only slightly increased Bcl-2 expression and did not protect photoreceptors from oxidative damage. Similarly, other fatty acids tested did not prevent photoreceptor apoptosis. CONCLUSIONS: These results show that oxidative damage induces apoptosis in retinal neurons during their early development in culture and suggest that the loss of mitochondrial membrane integrity is crucial in the apoptotic death of these cells. DHA activates intracellular mechanisms that prevent this loss and by modulating the levels of pro- and antiapoptotic proteins of the Bcl-2 family selectively protect photoreceptors from oxidative stress.     

Transgenic Bcl-2 expressed in photoreceptor cells confers both death-sparing and death-inducing effects.

To examine its potential role within the retina as a modulator of cell death and photoreceptor degeneration, bcl-2 expression was targeted to the photoreceptors of transgenic mice by the human IRBP promoter. Three transgenic families were established, with levels of transgene expression between 0.2 and two-fold relative to that of endogenous bcl-2. The effect of bcl-2 expression on genetically programmed photoreceptor degeneration was evaluated by crossing these transgenic mice with mice that develop a rapid degeneration of rod photoreceptors due to expression of a distinct transgene, SV40 T antigen (Tag). Transgenic Bcl-2 was localized to photoreceptor inner segments and was capable of abrogating the activation of caspase activity and the resulting cell death associated with ectopic expression of Tag. However, Bcl-2 itself ultimately caused photoreceptor cell death and retinal degeneration. Several proteins not expressed normally in Tag or other transgenic retinas undergoing photoreceptor degeneration were induced in the Bcl-2 transgenic retinas. Analysis by mass spectroscopy identified one of these proteins as alphaA-crystallin, a member of a protein family that associates with cellular stress. Since Bcl-2 can promote as well as spare cell death in the same photoreceptor population, its potential utility in ameliorating photoreceptor death in human hereditary blinding disorders is compromised.     

Calcium/calmodulin-stimulated phosphorylation of photoreceptor proteins in Limulus

Calcium (Ca2+) is thought to play a major role in the photoresponse of both vertebrates and invertebrates, but the mechanisms through which Ca2+ exerts its effects are unclear. In many systems, some effects of Ca2+ on cellular processes are thought to be mediated via activation of calcium/calmodulin protein kinase (CaCAM-PK) and the phosphorylation of specific proteins. Thus, protein substrates for CaCAM-PK in photoreceptor cells may be important in mediating the effects of Ca2+ on the photoresponse. In this study, we identify eight substrates for CaCAM-PK found in both the ventral and lateral eyes of Limulus. We focus on a characterization of one of these, a 46-kD substrate. We show that its subcellular distribution in ventral photoreceptors and its isoelectric forms are identical to the 46-kD light-stimulated phosphoprotein (46A) described by Edwards et al. (1989). Furthermore, we present evidence that 46A is unique to photoreceptor cells, and that it is present throughout the cell. Based on the results of this study, and the previous study by Edwards et al. (1989), we propose that 46A is involved in mediating the effects of Ca2+ on Limulus photoreceptor cell function, and that it may be involved in dark adaptation.     

Selective failure of long-term survival of isolated photoreceptors from both homozygous and heterozygous rd (retinal degeneration) mice

Retinas from homozygous rdle/rdle and heterozygous rdle/++ C57BL/6J mice were dissected and dissociated on postnatal day 2, when they are still essentially indistinguishable. The resulting cell suspensions were seeded on highly adhesive substrata, to which the cells attach as individual units, and grown in vitro for 2 weeks in serum-free, chemically defined media. The behavior of neurons and photoreceptors in vitro was investigated with several techniques; essentially no differences were found between rdle/rdle and rdle/++ cells. Three distinctive cell types could be recognized in cultures of both genotypes towards the end of the first week in vitro: process-free cells, multipolar neurons and rod photoreceptors. There were similarities between rdle/rdle and rdle/++ cultures in the number and morphology of photoreceptor cells, to include the presence of a cilium and a short neurite terminating in a spherule-like body. Moreover, in cultures of both genotypes, only photoreceptors showed opsin immunoreactivity and the antigen recognized by the rod-specific monoclonal antibody RET-P1. Biochemical and autoradiographic studies demonstrated that rdle/rdle and rdle/++ cells also showed similar uptakes of the putative amino acid neurotransmitters glutamate and aspartate (associated with most of the photoreceptors and only some neurons), and gamma-aminobutyric acid (associated with neurons but absent in photoreceptors). Thus, according to several parameters, the properties shown by photoreceptor cells were similar in rdle/rdle and rdle/++ cultures during the first week in vitro. Massive photoreceptor cell death was observed in both genotypes during the second week in vitro, coinciding with the time when photoreceptor degeneration occurs in vivo in rd/rd, but not in rd/+ retinas. Photoreceptor death in culture appeared to be specific, since approx. 80% of the non-photoreceptor neurons survived normally during the period when photoreceptor degeneration took place. Several reports from the literature suggest that the period around postnatal days 8-10 represents a critical stage for rd/rd photoreceptors, since they survive until this time but degenerate thereafter. Genetically normal photoreceptors apparently undergo a comparable crisis during maintenance in primary culture, suggesting the involvement of cell-cell contacts and/or retina-derived environmental signals in the survival or rod visual cells.     

Intracellular organelles and calcium homeostasis in rods and cones

The role of intracellular organelles in Ca2+ homeostasis was studied in salamander rod and cone photoreceptors under conditions that simulate photoreceptor activation by darkness and light. Sustained depolarization evoked a Ca2+ gradient between the cell body and ellipsoid regions of the inner segment (IS). The standing pattern of calcium fluxes was created by interactions between the plasma membrane, endoplasmic reticulum (ER), and mitochondria. Pharmacological experiments suggested that mitochondria modulate both baseline [Ca2+]i in hyperpolarized cells as well as kinetics of Ca2+ entry via L type Ca2+ channels in cell bodies and ellipsoids of depolarized rods and cones. Inhibition of mitochondrial Ca2+ sequestration by antimycin/oligomycin caused a three-fold reduction in the amount of Ca2+ accumulated into intracellular organelles in both cell bodies and ellipsoids. A further 50% decrease in intracellular Ca2+ content within cell bodies, but not ellipsoids, was observed after suppression of SERCA-mediated Ca2+ uptake into the ER. Inhibition of Ca2+ sequestration into the endoplasmic reticulum by thapsigargin or cyclopiazonic acid decreased the magnitude and kinetics of depolarization-evoked Ca2+ signals in cell bodies of rods and cones and decreased the amount of Ca2+ accumulated into internal stores. These results suggest that steady-state [Ca2+]i in photoreceptors is regulated in a region-specific manner, with the ER contribution predominant in the cell body and mitochondrial buffering [Ca2+] the ellipsoid. Local [Ca2+]i levels are set by interactions between the plasma membrane Ca2+ channels and transporters, ER and mitochondria. Mitochondria are likely to play an essential role in temporal and spatial buffering of photoreceptor Ca2+.     

Photoreceptor mitochondrial oxidative stress in experimental autoimmune uveitis

In experimental autoimmune uveitis (EAU), the macrophages infiltrate the retina during the late phase, 10-14 days after immunization with uveitogenic antigen, causing photoreceptor damage. However, prior to inflammatory cell infiltration, during the early phase (5-7 days after immunization), increased generation of reactive oxygen and nitric oxide species was observed in the photoreceptor mitochondria indicating oxidative stress. The oxidative-stress-induced nitration of photoreceptor mitochondrial proteins and peroxidation of membrane lipids led to activation and migration of microglia toward the photoreceptors. These observations suggest oxidative stress could be an initial pathologic event leading to amplification of inflammation inducing photoreceptor damage, thereby causing clinical and histologic expression of uveitis in the form of inflammatory cell infiltration.     

Minocycline protects photoreceptors from light and oxidative stress in primary bovine retinal cell culture

PURPOSE: To determine whether minocycline, a compound known to protect the retina against light-induced damage in rodent models, and its structurally related analogues would protect photoreceptor cells in primary bovine retinal cell culture against light and oxidative stress. METHODS: Minocycline and its analogues were tested in primary retinal cell culture to see whether they would inhibit light or oxidative stress-induced cell death. Primary cell cultures composed of photoreceptors, bipolar cells, and glial cells were prepared from bovine retinas. The extent of cell death induced by light or oxidative stress was assessed by using Sytox Green (Invitrogen-Molecular Probes, Eugene, OR) a nucleic acid dye uptake assay. Differential protection of photoreceptor cells from stress were examined using immunocytochemistry. RESULTS: Minocycline and methacycline were cytoprotective against light- or oxidative stress-induced damage of bovine primary photoreceptors in culture with an EC(50) < 10 microM. In contrast, structurally related analogues such as demeclocycline, meclocycline, and doxycycline were phototoxic at >3 to >10 microM. Though demeclocycline was found to be phototoxic, it was cytoprotective (EC(50) = 5 microM) against oxidative stress in the absence of exposure to light. CONCLUSIONS: The protective action of minocycline against light-induced damage in the cell-based assays agrees with earlier reports in animal models and suggests that the in vitro assay using bovine primary retinal cell culture is a suitable model for evaluating compounds for retinal protection. Cellular protection or toxicity produced by structurally related compounds show that minor structural modifications can alter the function of minocycline and lead to potent retinal protective compounds.     

Activation of caspase-3 during degeneration of the outer nuclear layer in the rd mouse retina

Caspase activation has been implicated in apoptosis, and the nature of the apoptotic stimulus determines the specific caspase activation during apoptosis. In this study, we examined the activation of caspase-3 during photoreceptor degeneration in the rd mouse, which has a mutation on a gene encoding cyclic GMP phosphodiesterase. The outer nuclear layer of the rd mouse retina was observed using light and electron microscopy. The progress of degeneration was determined chronologically and correlated with the activation of caspase-3 and the fragmentation of poly-ADP-ribose polymerase. Additionally, the active form of caspase-3 was detected during photoreceptor degeneration in the outer nuclear layer of the rd mouse. The chronological observation of the caspase-3 activation pattern correlates with the pattern of photoreceptor degeneration. As a result of this study, we present here our findings that caspase-3 was activated in photoreceptor cells of the rd mouse.     

Developmental death of photoreceptors in the C57BL/6J mouse: association with retinal function and self-protection

We have investigated the relationship between cell death among photoreceptors and the expression of function- and stress-related proteins during the development of the retina of the C57BL/6J mouse. Retinas from mice aged P(postnatal day)4 to P63 (adult) were examined for cell death using the TUNEL technique, and for the expression of basic fibroblast growth factor (bFGF), cytochrome oxidase (CO), rod opsin and glial fibrillary acidic protein (GFAP), using immunocytochemistry. At P4, cell death is most prominent in the inner layers of retina, declining to near-zero levels by P16. Cell death among photoreceptors occurs in a discrete wave commencing at approximately P12 and remaining elevated into the 4th postnatal week, beginning, peaking and declining later than in inner retina. The onset of photoreceptor death correlates with the expression of function-related molecules, such as CO and opsin. The decline in photoreceptor death correlates with the expression of the protective factor bFGF in photoreceptors. At the anterior edge of the retina photoreceptor death and the expression of bFGF are accelerated, and the expression of bFGF and GFAP is upregulated, by an edge-specific stress. We conclude that in the mouse photoreceptors undergo a wave of death which culls the neonatal population to adult levels. The onset of photoreceptor death is related to the acceleration of photoreceptor metabolism and function between P12 and P20. The decline of photoreceptor death to the very low levels found in the adult may be mediated by the upregulation of protective factors such as bFGF. Photoreceptor death and the expression of bFGF and GFAP at the edge of the retina are regulated by a still-unidentified, edge-specific stress, from as early as P16.