Light regulation of the insulin receptor in the retina

The peptide hormone insulin binds its cognate cell-surface receptors to activate a coordinated biochemical-signaling network and to induce intracellular events. The retina is an integral part of the central nervous system and is known to contain insulin receptors, although their function is unknown. This article, describes recent studies that link the photobleaching of rhodopsin to tyrosine phosphorylation of the insulin receptor and subsequent activation of phosphoinositide 3-kinase (PI3K). We recently found a light-dependent increase in tyrosine phosphorylation of the insulin receptor-β-subunit (IRβ) and an increase in PI3K enzyme activity in isolated rod outer segments (ROS) and in anti-phosphotyrosine (PY) and anti-IRβ immunoprecipitates of retinal homogenates. The light effect, which was localized to photoreceptor neurons, is independent of insulin secretion. Our results suggest that light induces tyrosine phosphorylation of IRβ in outersegment membranes, which leads to the binding of p85 through its N-terminal SH2 domain and the generation of PI-3,4,5-P₃. We suggest that the physiological role of this process may be to provide neuroprotection of the retina against light damage by activating proteins that protect against stress-induced apoptosis. The studies linking PI3K activation through tyrosine phosphorylation of IRβ now provide physiological relevance for the presence of these receptors in the retina.     

Interaction between caspase-3 and caspase-5 in the stretch-induced programmed cell death in the human periodontal ligament cells

In our previous studies, programmed cell death (PCD) was induced in human periodontal ligament (PDL) cells, through activation of caspase-3 and upregulation of CASP5 gene (encoding caspase-5 protein), in response to mechanical stretch loading. The aim of this study is to explore the relationship between the inflammatory caspase, caspase-5, and the apoptotic executioner protein, caspase-3, in human PDL cells. Here, we found that cyclic stretching upregulated the activity and the protein expression level of caspase-3 and -5 and the addition of the caspase-3 inhibitor or caspase-5 inhibitor significantly inhibited the stretch-induced PCD. Meanwhile, the inhibition of caspase-5 inhibited the activation of caspase-3 and vice versa. The result of coimmunoprecipitation also demonstrated that the expression of caspase-3 was immunoprecipitated with caspase-5. Thus, our study revealed that the in vitro application of cyclic stretching induced PCD by activation of caspase-3 and -5 in human PDL cells, and these two caspases could interact with each other after mechanical stretch loading. The study may facilitate further studies on the mechanism of stretch-induced PCD and help us understand the force-related periodontal homeostasis and remodeling better.     

植物细胞程序化死亡的形态学和生理生化特征

植物细胞程序化死亡(PCD)是一种由基因控制的、主动的细胞死亡过程,它在植物正常生长发育过程中起着重要作用。发生程序化死亡的植物细胞在形态、生理生化方面表现出一些共性特点和个性特点,该文对这些特点进行了综述。     

Hyperglycemia-induced apoptotic cell death in the mouse blastocyst is dependent on expression of p53

Murine preimplantation embryos exposed to hyperglycemia experience decreased glucose transport, and overexpression of the proapoptotic protein BAX, leading to increased apoptosis. These changes may account for the increased rates of miscarriages and malformations seen in women with diabetes mellitus. To test whether p53 expression is necessary for hyperglycemia-induced apoptosis, p53+/+, +/-, -/- embryos were obtained by superovulation. Two-cell embryos were cultured to a blastocyst stage in 52 mM D- or L-glucose. Apoptosis was detected using terminal dUTP nick end labeling (TUNEL) assays. In vivo studies were performed in the same manner using blastocysts recovered from streptozotocin-induced diabetic mothers. Both in vitro and in vivo studies showed that wildtype embryos had a significantly higher percentage of TUNEL-positive nuclei than p53+/- and -/- embryos. To test whether p53 is upstream of BAX, immunofluorescent confocal microscopy and immunoprecipitation/ immunoblotting were performed on blastocysts cultured in high vs. control glucose conditions. Blastocysts from p53+/+ mice exhibited increased BAX staining vs. p53+/- and -/- embryos. Next, to determine whether a decrease in glucose transport was upstream or downstream of p53, deoxyglucose transport was measured in individual blastocysts from p53+/+ and +/- diabetic vs. nondiabetic mice. Embryos from diabetic p53+/- mice exhibit a 44% decrease in glucose transport, similar to the 38% decrease seen in embryos from diabetic p53+/+ mice. Taken together, these results strongly indicate that p53 plays a role in hyperglycemia-induced apoptosis, upstream of BAX overexpression and downstream of the decrease in glucose transport experienced by the mouse preimplantation embryo.     

CEP-1347/KT7515 prevents motor neuronal programmed cell death and injury-induced dedifferentiation in vivo

CEP-1347, also known as KT7515, a derivative of a natural product indolocarbazole, inhibited motor neuronal death in vitro, inhibited activation of the stress-activated kinase JNK1 (c-jun NH terminal kinase) in cultured spinal motor neurons, but had no effect on the mitogen-activated protein kinase ERK1 in these cells. Results reported here profile the functional activity of CEP-1347/KT7515 in vivo in models of motor neuronal death or dedifferentiation. Application of CEP-1347/KT7515 to the chorioallantoic membrane of embryonic chicks rescued 40% of the lumbar motor neurons that normally die during the developmental period assessed. Peripheral administration of low doses (0.5 and 1 mg/kg daily) of CEP-1347/KT7515 reduced death of motor neurons of the spinal nucleus of the bulbocavernosus in postnatal female rats, with efficacy comparable to testosterone. Strikingly, daily administration of CEP-1347/KT7515 during the 4-day postnatal window of motor neuronal death resulted in persistent long-term motor neuronal survival in adult animals that received no additional CEP-1347/KT7515. In a model of adult motor neuronal dedifferentiation following axotomy, local application of CEP-1347/KT7515 to the transected hypoglossal nerve substantially reduced the loss of choline acetyl transferase immunoreactivity observed 7 days postaxotomy compared to untreated animals. Results from these experiments demonstrate that a small organic molecule that inhibits a signaling pathway associated with stress and injury also reduces neuronal death and degeneration in vivo. © 1998 John Wiley & Sons, Inc. J Neurobiol 35: 361–370, 1998     

Zinc induces cell death in immortalized embryonic hippocampal cells via activation of Akt-GSK-3beta signaling

Zinc is an essential catalytic and structural element of many proteins and a signaling messenger that is released by neuronal activity at many central excitatory synapses. Excessive synaptic release of zinc followed by entry into vulnerable neurons contributes severe neuronal cell death. We have previously observed that zinc-induced neuronal cell death is accompanied by Akt activation in embryonic hippocampal progenitor (H19-7) cells. In the present study, we examined the role of Akt activation and its downstream signaling events during extracellular zinc-induced neuronal cell death. Treatment of H19-7 cells with 10 microM of zinc plus zinc ionophore, pyrithione, led to increased phosphorylation of Akt at Ser-473/Thr-308 and increased Akt kinase activity. Zinc-induced Akt activation was accompanied by increased Tyr-phosphorylated GSK-3beta as well as increased GSK-3beta kinase activity. Transient overexpression of a kinase-deficient Akt mutant remarkably suppressed GSK-3beta activation and cell death. Furthermore, tau phosphorylation, but not the degradation of beta-catenin, was dependent upon zinc-induced GSK-3beta activation and contributed to cell death. The current data suggest that, following exposure to zinc, the sequential activation of Akt and GSK-3beta plays an important role directing hippocampal neural precursor cell death.     

Neurogenesis and cell death in olfactory epithelium

The olfactory epithelium (OE) of the mammal is uniquely suited as a model system for studying how neurogenesis and cell death interact to regulate neuron number during development and regeneration. To identify factors regulating neurogenesis and neuronal death in the OE, and to determine the mechanisms by which these factors act, investigators studied OE using two major experimental paradigms: tissue culture of OE; and ablation of the olfactory bulb or severing the olfactory nerve in adult animals, procedures that induce cell death and a subsequent surge of neurogenesis in the OE in vivo. These studies characterized the cellular stages in the olfactory receptor neuron (ORN) lineage, leading to the realization that at least three distinct stages of proliferating neuronal precursor cells are employed in generating ORNs. The identification of a number of factors that act to regulate proliferation and survival of ORNs and their precursors suggests that these multiple developmental stages may serve as control points at which cell number is regulated by extrinsic factors. In vivo surgical studies, which have shown that all cell types in the neuronal lineage of the OE undergo apoptotic cell death, support this idea. These studies, and the possible coregulation of neuronal birth and apoptosis in the OE, are discussed. © 1996 John Wiley & Sons, Inc.     

Dopamine-induced programmed cell death is associated with cytochrome c release and caspase-3 activation in snail salivary gland cells

Background information. PCD (programmed cell death) is a common mechanism to remove unwanted and excessive cells from organisms. In several exocrine cell types, PCD mode of release of secretory products has been reported. The molecular mechanism of the release, however, is largely unknown. Our aim was to study the molecular mechanism of saliva release from cystic cells, the specific cell type of snail SGs (salivary glands). Results. SG cells in active feeding animals revealed multiple morphological changes characteristic of PCD. Nerve stimulation and DA (dopamine) increased the number of TUNEL (terminal deoxynucleotidyl transferase‐mediated dUTP nick‐end labelling)‐positive cells both in inactive and feeding animals. The DA‐induced PCD was prevented by TEA (tetraethylammonium chloride) and eticlopride, emphasizing the role of K channels and D2 receptors in the PCD of cystic cells. DA enhanced cyto‐c (cytochrome c) translocation into the cytosol and methyl‐β‐cyclodextrin prevented it, suggesting apoptosome formation and ceramide involvement in the PCD linking of the surface DA receptor to mitochondria. Western blot analysis revealed that the release of cyto‐c was under the control of Bcl‐2 and Bad. DA also increased the active caspase‐3 in gland cells while D2 receptor antagonists and TEA attenuated it. Conclusion. Our results provide evidence for a type of transmitter‐mediated pathway that regulates the PCD of secretory cells in a mitochondrial‐caspase‐dependent manner. The activation of specific molecules, such as K channels, DA receptors, cyto‐c, ceramide, Bcl‐2 proteins and caspase‐3, but not caspase‐8, was demonstrated in cells involved in the DA‐induced PCD, suggesting that PCD is a physiological method for the release of saliva from SG cells.     

Target‐derived and locally derived neurotrophins support retinal ganglion cell survival in the neonatal rat retina

Brain‐derived neurotrophic factor (BDNF) and neurotrophin‐4/5 (NT‐4/5) protein and mRNA are found in the neonatal rat retina and also in target sites such as the superficial layers of the superior colliculus. Both neurotrophins support neonatal retinal ganglion cell survival in vitro. In vivo, injections of recombinant BDNF and NT‐4/5 reduce naturally occurring cell death as well as death induced by removal of the contralateral superior colliculus. In the latter case, the peak of retinal ganglion cell death occurs about 24 h postlesion. We wished to determine: whether a similar time‐course of degeneration occurs after selective removal of target cells or depletion of target‐derived trophic factors, and whether ganglion cell viability also depends on intraretinally derived neurotrophins. Retinal ganglion cell death was measured 24 and 48 h following injections of kainic acid or a mixture of BDNF and NT‐4/5 blocking antibodies into the superior colliculus and 24 h after intraocular injection of the same antibodies. Retinotectally projecting ganglion cells were identified by retrograde labeling with the nucleophilic dye diamidino yellow. We show that collicular injections of either kainic acid or BDNF and NT‐4/5 blocking antibodies significantly increased retinal ganglion cell death in the neonatal rat 24 h postinjection, death rates returning to normal by 48 h. This increase in death was greatest following collicular injections; however, death was also significantly increased 24 h following intravitreal antibody injection. Thus retinal ganglion cell survival during postnatal development is not only dependent upon trophic factors produced by central targets but may also be influenced by local intraretinal neurotrophin release. © 2004 Wiley Periodicals, Inc. J Neurobiol 60: 319–327, 2004     

Developmental mercury exposure elicits acute hippocampal cell death, reductions in neurogenesis, and severe learning deficits during puberty

Normal brain development requires coordinated regulation of several processes including proliferation, differentiation, and cell death. Multiple factors from endogenous and exogenous sources interact to elicit positive as well as negative regulation of these processes. In particular, the perinatal rat brain is highly vulnerable to specific developmental insults that produce later cognitive abnormalities. We used this model to examine the developmental effects of an exogenous factor of great concern, methylmercury (MeHg). Seven-day-old rats received a single injection of MeHg (5 μg/gbw). MeHg inhibited DNA synthesis by 44% and reduced levels of cyclins D1, D3, and E at 24 h in the hippocampus, but not the cerebellum. Toxicity was associated acutely with caspase-dependent programmed cell death. MeHg exposure led to reductions in hippocampal size (21%) and cell numbers 2 weeks later, especially in the granule cell layer (16%) and hilus (50%) of the dentate gyrus defined stereologically, suggesting that neurons might be particularly vulnerable. Consistent with this, perinatal exposure led to profound deficits in juvenile hippocampal-dependent learning during training on a spatial navigation task. In aggregate, these studies indicate that exposure to one dose of MeHg during the perinatal period acutely induces apoptotic cell death, which results in later deficits in hippocampal structure and function.     

Cell death in the oligodendrocyte lineage

We have recently found that about 50% of newly formed oligodendrocytes normally die in the developing rat optic nerve. When purified oligodendrocytes or their precursors are cultured in the absence of serum or added signalling molecules, they die rapidly with the characteristics of programmed cell death. This death is prevented either by the addition of medium conditioned by cultures of their normal neighboring cells in the developing optic nerve, or by the addition of platelet-derived growth factor (PDGF) or insulin-like growth factors (IGFs). Increasing PDGF in the developing optic nerve decreases normal oligodendrocyte death by up to 90% and doubles the number of oligodendrocytes, suggesting that this normally occurring glial cell death might result from a competition for limiting amounts of survival signals. These results suggest that competition for limiting amounts of survival factors is not confined to developing neurons, and raise the possibility that a similar mechanism may be responsible for some naturally occurring cell deaths in nonneural tissues. © 1992 John Wiley & Sons, Inc.     

Akt activation prevents Apop-1-induced death of cells

Apop-1 is a novel protein identified in cultured atherosclerotic smooth muscle cells of ApoE-deficient mice, and the expression of the Apop-1 protein induces the death of cultured cells. Insulin-like growth factor-1 (IGF-1) is a well-characterized survival factor for VSMC; however, the interaction between Apop-1 and survival factor IGF-1 in the mediation of cell death is poorly understood. In this report, we show that the IGF-1 signaling cascade protects VSMC against Apop-1-induced death. Furthermore, our data indicate that the inhibition of Apop-1-induced death by IGF-1 is mediated by the activation of the PI3K/Akt signaling pathway.     

Heat-induced programmed cell death in Leishmania infantum is reverted by Bcl-XL expression

An increasing number of reports indicate that single-celled organisms are able to die following what seems to be an ordered program of cell death with strong similarities to apoptosis from higher eukaryotes. DNA degradation and several other apoptotic-like processes have also been described in the parasitic protozoa Leishmania. However, the existence of an apoptotic death in this parasite is still a matter of controversy. Our results indicate that most of the processes of macromolecular degradation and organelle dysfunction observed in mammalian cells during apoptosis can also be reproduced in promastigotes of the genus Leishmania when incubated at temperatures above 38°C. These processes can be partially reversed by the expression of the anti-apoptotic mammalian gene Bcl-X_L, which suggests that this family of apoptosis-regulating proteins was present very early in the evolution of eukaryotic cells.     

Programmed cell death 4: A novel player in the pathogenesis of polycystic ovary syndrome

Polycystic ovary syndrome (PCOS) is a pathological condition recognized by menstrual cycle irregularities, androgen excess, and polycystic ovarian morphology, affecting a significant proportion of women of childbearing age and accounting for the most prevalent cause of anovulatory sterility. In addition, PCOS is frequently accompanied by metabolic and endocrine disturbances such as obesity, dyslipidemia, insulin resistance, and hyperinsulinemia, indicating the multiplicity of mechanisms implicated in the progression of PCOS. However, the exact pathogenesis of PCOS is yet to be elucidated. Programmed cell death 4 (PDCD4) is a ubiquitously expressed protein that contributes to the regulation of various cellular processes, including gene expression, cell cycle progression, proliferation, and apoptosis. Despite some disparities concerning its exact cellular effects, PDCD4 is generally characterized as a protein that inhibits cell cycle progression and proliferation and instead drives the cell into apoptosis. The apoptosis of granulosa cells (GCs) is speculated to take a major part in the occurrence and progression of PCOS by ceasing antral follicle development and compromising oocyte competence. Given the possible involvement of GC apoptosis in the progression of PCOS, as well as the contribution of PDCD4 to the regulation of cell apoptosis and the development of metabolic diseases, the current review aimed to discuss whether or how PDCD4 can play a role in the pathogenesis of PCOS by affecting GC apoptosis.     

Ultrastructural characterization of alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid-induced cell death in embryonic dopaminergic neurons

Developing neuronal populations undergo significant attrition by natural cell death. Dopaminergic neurons in the substantia nigra pars compacta undergo apoptosis during synaptogenesis. Following this time window, destruction of the anatomic target of dopaminergic neurons results in dopaminergic cell death but the morphology is no longer apoptotic. We describe ultrastructural changes that appear unique to dying embryonic dopaminergic neurons. In primary cultures of mesencephalon, death of dopaminergic neurons is triggered by activation of glutamate receptors sensitive to alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA), and differs ultrastructurally from both neuronal apoptosis or typical excitotoxicity. AMPA causes morphological changes selectively in dopaminergic neurons, without affecting other neurons in the same culture dishes. Two hours after the onset of treatment swelling of Golgi complexes is apparent. At 3 h, dopaminergic neurons display loss of membrane asymmetry (coinciding with commitment to die), as well as nuclear membrane invagination, irregular aggregation of chromatin, and mitochondrial swelling. Nuclear changes continue to worsen until loss of cytoplasmic structures and cell death begins to occur after 12 h. These changes are different from those described in neurons undergoing either apoptosis or excitotoxic death, but are similar to ultrastructural changes observed in spontaneous death of dopaminergic neurons in the natural mutant weaver mouse.     

Formic acid induces Yca1p-independent apoptosis-like cell death in the yeast Saccharomyces cerevisiae

Formic acid disrupts mitochondrial electron transport and sequentially causes cell death in mammalian ocular cells by an unidentified molecular mechanism. Here, we show that a low concentration of formic acid induces apoptosis-like cell death in the budding yeast Saccharomyces cerevisiae, with several morphological and biochemical changes that are typical of apoptosis, including chromatin condensation, DNA fragmentation, externalization of phosphatidylserine, reactive oxygen species (ROS) production, loss of mitochondrial membrane potential and mitochondrion destruction. This process may not be dependent on the activation of Yca1p, the yeast caspase counterpart. In addition, the cell death induced by formic acid is associated with ROS burst,while intracellular ROS accumulate more rapidly and to a higher level in the YCA1 disruptant than in the wild-type strain during the progression of cell death. Our data indicate that formic acid induces yeast apoptosis via an Yca1p-independent pathway and it could be used as an extrinsic inducer for identifying the regulators downstream of ROS production in yeast.     

Overexpression of regucalcin suppresses cell death and apoptosis in cloned rat hepatoma H4-II-E cells induced by insulin or insulin-like growth factor-I

The role of regucalcin, a regulatory protein in intracellular signaling pathway, in cell death was investigated by using the cloned rat hepatoma H4-II-E cells overexpressing regucalcin. The hepatoma cells (wild-type) and stable regucalcin/pCXN2 transfectants were cultured for 72 h in a medium containing 10% fetal bovine serum (FBS) to obtain subconfluent monolayers. After culture for 72 h, cells were further cultured for 24-72 h in a medium containing either vehicle, insulin (10(-8) or 10(-7) M) or insulin-like growth factor-I (IGF-I; 10(-9) or 10(-8) M) in the absence of FBS. The number of wild-type cells was significantly decreased by culture for 24, 48, or 72 h in the presence of insulin (10(-8) or 10(-7) M) or IGF-I (10(-9) or 10(-8) M). Agarose gel electrophoresis showed the presence of low-molecular-weight deoxyribonucleic acid (DNA) fragments of adherent wild-type cells cultured with insulin or IGF-I. The effect of insulin or IGF-I in stimulating cell death and DNA fragmentation in hepatoma cells (wild-type) was significantly prevented in transfectants overexpressing regucalcin. Meanwhile, epinephrine (10(-6) or 10(-5) M) or transforming growth factor-beta1 (10(-13) or 10(-12) M) did not cause cell death of hepatoma cells. Insulin-induced decrease in the number of wild-type cells was significantly prevented by culture with caspase-3 inhibitor (10(-8) M), although the effect of IGF-I was not inhibited. The effect of insulin or IGF-I in inducing the death of hepatoma cells (wild-type) was significantly prevented in the presence of N omega-nitro-L-arginine methylester (NAME), an inhibitor of nitric oxide synthase. Genistein (10(-6) M), an inhibitor of protein tyrosine kinase, or vanadate (10(-5) M), an inhibitor of protein tyrosine phosphatase, caused a significant decrease in the number of hepatoma cells (wild-type). The effect of insulin in inducing the death of wild-type cells was not seen in the presence of genistein or vanadate. The effect of IGF-I on the death of wild-type cells was observed in the presence of genistein or vanadate. The effect of genistein on cell death was significantly prevented in transfectants. Such effect was not seen with vanadate. This study demonstrates that insulin or IGF-I stimulates cell death and apoptosis in the hepatoma cells, and that overexpression of regucalcin has a suppressive effect on cell death induced by insulin or IGF-I that is mediated through different signaling pathway.     

Programmed cell death in neurotumour cells involves the generation of ceramide

Ceramide has been typically thought of as the membrane anchor for the carbohydrate in glycosphingolipids but many studies have suggested that it may cause apoptosis. Apoptosis or programmed cell death (PCD) is thought to be responsible for the death of one-half of neurons surviving the development of the nervous system. The potential involvement of the sphingomyelin-ceramide signaling process as an integral part of PCD was therefore examined in several neurotumour cell lines. We show that synthetic C2-ceramide (N-acetylsphingosine), a soluble ceramide analogue, can rapidly trigger PCD in these cells, characterized by: 1) classic DNA laddering on agarose gels; 2) DNA fragmentation as determined by Hoechst Dye; and 3) cell viability (mitochondrial function and intact nuclei) assays. We report that staurosporine can both activate PCD (by all three criteria above) in neurotumour cells and increase both the formation of ceramide and ceramide mass. Both ceramide formation and the induction of PCD were further enhanced by the co-addition of a ceramidase inhibitor oleoylethanolamine (25 µM). Staurosporine and oleoylethanolamine were similarly effective in inducing ceramide formation and PCD in immortalized hippocampal neurons (HN-2) and immortalized dorsal root ganglion cells (F-11). Our data suggests that formation of ceramide is a key event in the induction of PCD in neuronally derived neurotumour cells.Abbreviations PCD programmed cell death - PKC protein kinase C - HPTLC high-performance thin-layer chromatography - DETAPAC diethylenetriaminepentaacetic acid - DMEM Dubelco's modified Eagle's medium - FCS fetal calf serum - PBS phosphate-buffered saline - DAG diacylglycerol - DDI distilled-deionized - Cer ceramide - SM sphingomyelin Dedicated to Dr Sen-itiroh Hakomori in celebration of his 65th birthday.