Interleukin‐4 blocks thapsigargin‐induced cell death in rat rod photoreceptors: Involvement of cAMP/PKA pathway

Although the photoreceptors cell death is the main cause of some retinopathies diseases, the mechanisms involved in this process are poorly understood. The neuroprotective effects of interleukin‐4 (IL‐4) have been shown in several tissues, including retina. We demonstrate that treatment of rat retinal explants with IL‐4 completely inhibited the thapsigargin‐induced rod photoreceptor cell death after 24 hr in culture. We also showed that IL‐4 receptor α subunit (IL‐4Rα) is abundantly present in retina. Colocalization of IL‐4Rα and rhodopsin indicate a direct effect of this cytokine in rod photoreceptor cells. Moreover, IL‐4 increased the intracellular levels of cAMP in 7.4‐fold, indicating that the neuroprotective effect of this cytokine was completely blocked by RpcAMP, an inhibitor of protein kinase (PKA). Our data demonstrate, for the first time, the neuroprotective effect of IL‐4 through cAMP/PKA pathway in thapsigargin‐induced photoreceptor cell death. © 2009 Wiley‐Liss, Inc.     

Developmental neuronal death is not a universal phenomenon among cell types in the chick embryo retina

The goal of this study was to investigate whether all the cell types present in the chick embryo retina undergo developmental neuronal death. Apoptosis was investigated in retinal sections at different developmental stages, processed either with propidium iodide, which stains pyknotic nuclei intensely, or with terminal transferase-mediated deoxyuridine triphosphate (d-UTP)-biotin nick-end labeling (TUNEL). Internucleosomal DNA fragmentation was investigated in tissue extracts by agarose gel electrophoresis. TUNEL-positive (T+) cells and pyknotic nuclei were first detectable in the ganglion cell layer (GCL) around embryonic day (ED) 8 and peaked at ED 10. In the inner nuclear layer (INL), T+ and pyknotic cells first appeared on ED 8, reached maximum frequency on ED 11, and were largely absent after ED 14. DNA ladders were observed at all the stages, when T+ and pyknotic cells were abundant, but not on ED 4, when only scattered dead cells were observed histologically. Dying cells were virtually never detected in the outer nuclear layer (ONL) from ED 4 to postnatal day 2. After unilateral midbrain ablation on ED 5, there was a striking increase in the number of pyknotic and T+ cells in both the GCL and in the INL of the contralateral eye but not in the ONL. The absence of apoptotic cell death in the ONL during normal development and after tectal ablation shows that developmental death is not universal among the various cell populations present in the chick embryo retina and raises questions regarding mechanisms controlling both photoreceptor survival and the matching of pre- and postsynaptic elements in the outer plexiform layer of this species. J. Comp. Neurol. 396:12–19, 1998. © 1998 Wiley-Liss, Inc.     

Low sensitivity of retina to AMPA-induced calcification

Glutamate is involved in most CNS neurodegenerative diseases. In particular, retinal diseases such as retinal ischemia, retinitis pigmentosa, and diabetic retinopathy are associated with an excessive synaptic concentration of this neurotransmitter. To gain more insight into retinal excitotoxicity, we carried out a dose-response study in adult rats using alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA), a glutamate analogue. AMPA intraocular injections (between 0.27 and 10.8 nmol) caused no morphologic modification, but a 10.8 + 21 nmol double injection in a 10-day interval produced a lesion characterized by discrete neuronal loss, astroglial and microglial reactions, and calcium precipitation. Abundant calcium deposits similar to those present in rat and human brain excitotoxicity or hypoxia-ischemia neurodegeneration were detected by alizarin red staining within the retinal surface and the optic nerve. Glial reactivity, associated normally with astrocytes in the nerve fiber, was assessed in Müller cells. GABA immunoreactivity was detected not only in neuronal elements but also in Müller cells. In contrast to the high vulnerability of the brain to excitotoxin microinjection, AMPA-induced retinal neurodegeneration may provide a useful model of low central nervous system sensitivity to excitotoxicity.     

Selective sensitivity of early postmitotic retinal cells to apoptosis induced by inhibition of protein synthesis

In previous work we showed that apoptosis in retinal tissue from developing rats can be induced by inhibition of protein synthesis (Rehen et al. 1996, Development, 122, 1439-1448). Here we show that recent postmitotic cells are the cells sensitive to apoptosis triggered by blockade of protein synthesis. To label all proliferating cells in the retina, a series of injections of the nucleotide analogue, bromo-deoxy-uridine (BrdU, 60 mg/kg b.w.), was given in rat pups. Then, explants of the retina were incubated in vitro with the inhibitor of protein synthesis anisomycin (1.0-3.2 microg/mL) for 1 day to induce apoptosis. Detection of apoptotic bodies under differential interference contrast microscopy was combined with immunocytochemistry for BrdU, proliferating cell nuclear antigen (PCNA) or for various markers of retinal cell differentiation. Despite the large number of BrdU- and PCNA-labelled cells in the tissue, the vast majority of the cells that underwent apoptosis were postmitotic cells which have left the mitotic cycle 3-4 days before. However, these cells were not labelled with antibodies to calretinin, calbindin, rhodopsin or to a Muller glial cell marker, suggesting that these are early postmitotic neurons. We suggest that during migration and initial differentiation, the apoptotic machinery is blocked by suppressor proteins, thus allowing recent postmitotic cells to find their final positions and differentiate while protected from apoptosis.     

Ceramide in rafts (detergent-insoluble fraction) mediates cell death in neurotumor cell lines

Detergent-resistant lipid microdomains (Rafts) were isolated from human oligodendroglioma (HOG), human neuroblastoma (LA-N-5), and immortalized dorsal root ganglion (F-11) cell lines by sucrose-density gradient ultracentrifugation and shown to be enriched in cholesterol, sphingomyelin, and ceramide. [(3)H]palmitate labeling allowed the Raft fraction to be easily identified as a sharp peak of (3)H radioactivity in the 5-30% sucrose interphase. Treatment of [(3)H]palmitate-labeled cells with staurosporine (to activate caspase 8 and induce apoptosis) or exogenous sphingomyelinase specifically increased the [(3)H]ceramide content of the Raft fraction. Depletion of cholesterol with beta-methylcyclodextran decreased Raft formation and partially blocked staurosporine-induced apoptosis. Similarly, treatment of cells with Fumonisin B1 to inhibit de novo sphingolipid synthesis by 50% reduced the labeling of the Raft fraction and partially blocked staurosporine-induced apoptosis. Staurosporine treatment activated neutral sphingomyelinase but had no effect on acid sphingomyelinase activity or on other lysosomal hydrolases, such as alpha-L-fucosidase. Most of the neutral sphingomyelinase activity is in the Raft fraction, suggesting that the conversion of sphingomyelin to ceramide in Rafts is an important event in neural cell apoptosis.     

1,25-Dihydroxyvitamin D3 induces programmed cell death in a rat glioma cell line

1,25-Dihydroxyvitamin D3 (1,25(OH)₂D₃), a seco-steroid hormone with potential antitumoral activities, has been recently reported to exert cytotoxic effects on C6 glioma cells. However, the molecular mechanisms which trigger this cell death remain unknown. We show here that this 1,25(OH)₂D₃-induced cell death is dependent upon protein synthesis and is accompanied by the expression of c-myc, p53, and gadd45 genes. Two other genes, coding for interleukin-6 and vaso-endothelial growth factor, are also up-regulated after addition of 1,25(OH)₂D₃. This programmed cell death can be suppressed when cells are treated with forskolin, a drug which increases intracellular cAMP concentration, or with genistein, an inhibitor of tyrosine protein kinases. However, in spite of the demonstration of fragmented DNA in 1,25(OH)₂D₃-treated cells, the C6.9 cells used in this study do not show the classical morphological features of apoptosis. These results provide the first evidence for the existence of a programmed cell death triggered by 1,25(OH)₂D₃ in glioma cells and may provide a basis for the development of new therapeutic strategies. In addition, these data also suggest that the treatment of C6.9 cells with 1,25(OH)₂D₃ may be a useful model to study the molecular mechanisms involved in the programmed cell death of a cell of glial origin. © 1996 Wiley-Liss, Inc.     

Microglial response to light‐induced photoreceptor degeneration in the mouse retina

The microglial response elicited by degeneration of retinal photoreceptor cells was characterized in BALB/c mice exposed to bright light for 7 hours and then kept in complete darkness for survival times ranging from 0 hours to 10 days. Photodegeneration resulted in extensive cell death in the retina, mainly in the outer nuclear layer (ONL), where the photoreceptor nuclei are located. Specific immunolabeling of microglial cells with anti‐CD11b, anti‐CD45, anti‐F4/80, anti‐SRA, and anti‐CD68 antibodies revealed that microglial cells were activated in light‐exposed retinas. They migrated to the ONL, changed their morphology, becoming rounded cells with short and thick processes, and, finally, showed immunophenotypic changes. Specifically, retinal microglia began to strongly express antigens recognized by anti‐CD11b, anti‐CD45, and anti‐F4/80, coincident with cell degeneration. In contrast, upregulation of the antigen recognized by anti‐SRA was not detected by immunocytochemistry until 6 hours after light exposure. Differences were also observed at 10 days after light exposure: CD11b, CD45, and F4/80 continued to be strongly expressed in retinal microglia, whereas the expression of CD68 and SRA had decreased to near‐normal values. Therefore, microglia did not return to their original state after photodegeneration and continued to show a degree of activation. The accumulation of activated microglial cells in affected regions simultaneously with photoreceptor degeneration suggests that they play some role in photodegeneration. J. Comp. Neurol. 518:477–492, 2010. © 2009 Wiley‐Liss, Inc.     

Patterns of cell death in the ganglion cell layer of the human fetal retina

The distribution of dying cells in the ganglion cell layer (GCL) of retinae from human fetuses has been analysed. Both whole-mounted and sectioned retinae have been studied. Results suggest that cells are lost from the GCL between weeks 14 and 30 of the gestation period, approximately. This period corresponds to the period during which axons are lost from the developing optic nerve. Cell loss is greatest between weeks 16 and 21 of the gestation period. The pattern of cell loss is nonuniform, and between weeks 16 and 24, the relative frequency of pyknotic cells (pyknotic cells:viable cells) in peripheral retina is considerably higher than in central retina. This pattern of cell loss predominates during the period in which a distinct centroperipheral gradient of cell densities emerges in the GCL of the human fetal retina (between 18 and 23 weeks gestation). It is suggested that the regional loss of ganglion cells may contribute to the formation of the cell density gradient.     

Differential effects of cyclin-dependent kinase blockers upon cell death in the developing retina

Pharmacological blockers of cyclin-dependent kinases (CDKs) can inhibit cell cycle progression. Deferoxamine (DFO) and mimosine (MIMO) arrest cells reversibly at the G1/S transition and olomoucine (OLO) inhibits the cell cycle at both G1/S and G2/M. We investigated the effect of these drugs upon cell death in histotypical explants taken from the retina of neonatal rats. Degeneration of retinal ganglions cells (RGC) induced by axotomy was inhibited by OLO (100 microM) but not by DFO (up to 2 mM) or MIMO (up to 1 mM). On the other hand, after 1 day in vitro, all cell cycle inhibitors induced cell death in the neuroblastic layer (NBL) of the explants. DFO and MIMO induced cell death only of proliferating cells, identified either by their incorporation of bromodeoxyuridine or by immunolabeling the proliferating cell nuclear antigen. In turn, OLO induced cell death of both proliferating and post-mitotic cells. However, the post-mitotic cells were unlabeled with markers of retinal differentiation. Our results indicate that cyclin-dependent kinases are involved in the control of sensitivity to cell death in the retina, and that retinal cells present differentiation-dependent responses to modulation of CDK activity.     

Analysis of the mechanisms underlying increased histogenetic cell death in developing cerebellum of the hypothyroid rat: determination of the time required for granule cell death

The connection between the date of formation of granule cells and their final position in the internal granular layer of the cerebellum has been described previously. In rats made hypothyroid since the end of gestation, the distribution of the pyknotic cells in the internal granular layer of the cerebullar cortex was also previously found to be age-related. In 14-day-old hypothyroid rats, it was compared with that of the granule cells which were labeled after a pulse of [³H]thymidine at various stages of development. It appeared that the localization of the dying cells corresponded roughly to that of granule cells labeled on day 9. Therefore the maximum time required for granule cell death was about 5 days. Since the migratory phase through the molecular layer lasted about two days, the granule cells died after a maximum time of 3 days following their deposition in the internal granular layer. Information concerning the time of survival of the dying granule cells was important for subsequent investigation of the mechanisms underlying increased granule cell death in the hypothyroid cerebellum and the corrective effects of thyroid hormone.     

Postnatal changes in retinal ganglion cell and optic axon populations in the pigmented rat

The number of ganglion cells in the retina of the postnatal rat has been examined. We estimated both the number of axons in the optic nerve and the number of cells which can be retrogradely labelled with horseradish peroxidase from injections into the brain. In the retina of the newborn rat there are at least twice as many ganglion cells as in the adult rat. By retrograde labelling of the ganglion cells and following transection of their axons 24-48 hrs later we can find no evidence that ganglion cells withdraw their axon without degeneration of the patent cell body. We have found that the excess ganglion cells are lost over the first ten postnatal days and during this period we observe pyknotic nuclei in the ganglion cell layer. From our estimates of the total number of neurones in the ganglion cell layer and the number of ganglion cells found at different ages we conclude that the migration of amacrine cells into the ganglion cell layer occurs in the first five postnatal days.     

Implication of Bcl-2 and Caspase-3 in age-related Purkinje cell death in murine organotypic culture: an in vitro model to study apoptosis

Neuronal cell death is an essential feature of nervous system development and neurodegenerative diseases. Most Purkinje cells in murine cerebellar organotypic culture die when taken from 1-5-day-old mice (P1-P5), whereas they survive when taken before or after these ages. Using DNA gel electrophoresis, terminal deoxynucleotidyl transferase-mediated dUTP nick-end labelling (TUNEL) and electron microscopic analyses, we were able to show that this massive Purkinje cell death is apoptotic in nature and reaches a peak at P3. From the several endogenous genes known to be involved in the apoptotic process, we have focused on two: the bcl-2 and the caspase-3 that encode for anti-apoptotic and pro-apoptotic proteins, respectively. Immunostaining for activated Caspase-3 correlated with Purkinje cell death. A better survival of Purkinje cells was observed in P3 slices taken from hu-bcl-2 transgenic mice, and in slices treated with z-DEVD.fmk (an inhibitor of numerous caspases). Thus, these two genes are implicated in the age-related Purkinje cell apoptosis in organotypic culture. As Purkinje cell death in vitro takes place at the same age as Purkinje cells engaged in intense synaptogenesis and dendritic remodeling in vivo, we propose that this apoptosis reflects a naturally occurring Purkinje cell death during this critical period.     

Changing sensitivity to cell death during development of retinal photoreceptors

Photoreceptor cell death occurs during both normal and pathological retinal development. We tested for selective induction and blockade of cell death in either retinal photoreceptors or their precursors. Organotypical retinal explants from rats at postnatal days 3-11 were treated in vitro for 24 hr with thapsigargin, okadaic acid, etoposide, anisomycin, or forskolin. Explant sections were examined for cell death, and identification of either photoreceptors or proliferating/immediate postmitotic cells followed imunohistochemistry for either rhodopsin or bromodeoxyuridine and proliferating cell nuclear antigen, respectively. Photoreceptor cell death was selectively induced by either thapsigargin or okadaic acid, whereas death of proliferating/immediate postmitotic cells was induced by etoposide. Prelabeling of proliferating precursors allowed direct demonstration of changing sensitivity of photoreceptors to various chemicals. Degeneration of both photoreceptors and proliferating/immediate postmitotic cells depended on protein synthesis. Increase of intracellular cyclic AMP blocked degeneration of postmitotic, but not of proliferating, photoreceptor precursors. The selective induction and blockade of cell death show that developing photoreceptors undergo progressive changes in mechanisms of programmed cell death associated with phenotypic differentiation.     

Cell death in the normal developing brain, and following ionizing radiation, methyl-azoxymethanol acetate, and hypoxia-ischaemia in the rat

Naturally occurring (programmed) cell death in the developing brain has morphological characteristics of apoptosis and is associated with internucleosomal DNA fragmentation. Apoptosis also plays a role in cell death following hypoxia-ischaemia in the developing rat brain. Ionizing radiation-induced cell death in the brain of the young rat has morphological characteristics of apoptosis. is mediated by protein synthesis and is associated with internucleosomal UNA fragmentation. Methylazoxymethanol (MAM) acetate injection in the young rat produces apoptotic cell death in the external granule cell layer of the cerebellum. In addition, strong c-Jun immunore-activity is observed in apoptotic cells during normal development and following experimentally induced cell death. Moreover, c-Jun mRNA induction and de novo c-Jun protein synthesis, together with activation of c-Jun/AP-1, as revealed with gel mobility shift assay, occurs in irradiated animals. Western blotting of total brain homogenates shows a c-Jun-immunoreactive band at p39, which corresponds to the molecular weight of c-Jun. in control rats. However, a thick c-Jun-immunoreactive band at about p62, accompanied by a decrease of the p39 band, occurs in irradiated and MAM-treated rats. A thin band immediately above the thick p62 band, suggestive of c-Jun phosphorylation, is also observed in treated rats. Taken together, these observations indicate that c-Jun expression is associated with apoptotic cell death in the developing central nervous system.     

Apparent apoptotic cell death in the olfactory epithelium of adult rodents: Death occurs at different developmental stages

In the olfactory epithelium of adult rodent, receptor neurons are generated continually. Despite the ongoing generation of new neurons, no corresponding increase occurs in the thickness of the mature olfactory epithelium. Thus, epithelial cell death must occur to offset the continual generation of new cells. In the present study, a sensitive method to label nicked DNA in dying cells was combined with immunocytochemistry to determine the identity of dying olfactory cells. In addition, the positions of putative apoptotic cells were mapped to provide additional information about the identity of dying cells. Double labeling experiments revealed that each of the olfactory cell types, i.e., basal cells (keratin-positive), immature neurons (GAP43-positive) and mature receptor neurons (olfactory marker protein (OMP)-positive), were positive for fragmented DNA, suggesting that they undergo apoptotic cell death. The results of the mapping study suggest that apoptotic cell death occurs primarily among GAP43-positive neurons. © 1996 Wiley-Liss, Inc.