Induction of Neuronal Morphology in the 661W Cone Photoreceptor Cell Line with Staurosporine

Purpose

RGC-5 cells undergo differentiation into a neuronal phenotype with low concentrations of staurosporine. Although the RGC-5 cell line was initially thought to be of retinal ganglion cell origin, recent evidence suggests that the RGC-5 line could have been the result of contamination with 661W mouse cone photoreceptor cells. This raised the possibility that a cone photoreceptor cell line could be multipotent and could be differentiated to a neuronal phenotype.

Methods

661W and RGC-5 cells, non-neuronal retinal astrocytes, retinal endothelial cells, retinal pericytes, M21 melanoma cells, K562 chronic myelogenous leukemia cells, and Daudi Burkitt lymphoma cells, were differentiated with staurosporine. The resulting morphology was quantitated using NeuronJ with respect to neurite counts and topology.

Results

Treatment with staurosporine induced similar-appearing morphological differentiation in both 661W and RGC-5 cells. The following measures were not significantly different between 661W and RGC-5 cells: number of neurites per cell, total neurite field length, number of neurite branch points, and cell viability. Neuronal-like differentiation was not observed in the other cell lines tested.

Conclusions

661W and RGC-5 cells have virtually identical and distinctive morphology when differentiated with low concentrations of staurosporine. This result demonstrates that a retinal neuronal precursor cell with cone photoreceptor lineage can be differentiated to express a neuronal morphology.     

Neuroprotectin D1 Induces Dephosphorylation of Bcl-xL in a PP2A-dependent Manner during Oxidative Stress and Promotes Retinal Pigment Epithelial Cell Survival

Retinal pigment epithelial (RPE) cell integrity is critical for the survival of photoreceptor cells. Bcl-x_L is a major anti-apoptotic Bcl-2 protein required for RPE cell survival, and phosphorylation of Bcl-x_L at residue Ser-62 renders this protein pro-apoptotic. In this study, we identify serine/threonine protein phosphatase 2A (PP2A) as a key regulator of Bcl-x_L phosphorylation at residue Ser-62 in ARPE-19 cells, a spontaneously arising RPE cell line in which Bcl-x_L is highly expressed. We found that either PP2A inhibitor okadaic acid or depletion of catalytic subunit α of PP2A (PP2A/Cα) by small interfering RNA enhanced Bcl-x_L phosphorylation when activated with hydrogen peroxide and tumor necrosis factor α-induced oxidative stress. Disruption of PP2A/Cα exacerbated oxidative stress-induced apoptosis. PP2A/Cα colocalized and interacted with S62Bcl-x_L in cells stressed with H₂O₂/tumor necrosis factor α. By contrast, the omega-3 fatty acid docosahexaenoic acid derivative, neuroprotectin D1 (NPD1), a potent activator of survival signaling, down-regulated oxidative stress-induced phosphorylation of Bcl-x_L by increasing protein phosphatase activity. NPD1 also attenuated the oxidative stress-induced apoptosis by knockdown of PP2A/Cα and increased the association of PP2A/Cα with S62Bcl-x_L as well as total Bcl-x_L. NPD1 also enhanced the heterodimerization of Bcl-x_L with its counterpart, pro-apoptotic protein Bax. Thus, NPD1 modulates the activation of this Bcl-2 family protein by dephosphorylating in a PP2A-dependent manner, suggesting a coordinated, NPD1-mediated regulation of cell survival in response to oxidative stress.     

Geldanamycin Provides Posttreatment Protection Against Glutamate-Induced Oxidative Toxicity in a Mouse Hippocampal Cell Line

Abstract : The benzoquinoid ansamycin geldanamycin interferes with many cell signaling pathways and is currently being evaluated as an anticancer agent. The main intracellular target of geldanamycin is the 90-kDa heat shock protein, hsp90. In this report we demonstrate that geldanamycin is effective at preventing glutamate-induced oxidative toxicity in the HT22 mouse hippocampal cell line, even if given 4 h after glutamate treatment. Geldanamycin prevents glutamate-induced internucleosomal DNA cleavage in the HT22 cells but does not reverse the depletion of glutathione levels brought about by glutamate treatment. Both anabolic and catabolic effects are generated by geldanamycin treatment of HT22 cells, as evidenced by the induction of hsp70 expression and degradation of c-Raf-1 protein, respectively. Thus, geldanamycin may provide an effective strategy for manipulating signaling pathways in neuronal cells that use hsp90 as they proceed through a programmed cell death pathway in response to oxidative stress.     

ERK-Mediated Activation of Fas Apoptotic Inhibitory Molecule 2 (Faim2) Prevents Apoptosis of 661W Cells in a Model of Detachment-Induced Photoreceptor Cell Death

In this study, we examined the role of Fas apoptotic inhibitory molecule 2 (Faim2), an inhibitor of the Fas signaling pathway, and its regulation by stress kinase signaling during Fas-mediated apoptosis of 661W cells, an immortalized photoreceptor-like cell line Treatment of 661W cells with a Fas-activating antibody led to increased levels of Faim2. Both ERK and JNK stress kinase pathways were activated in Fas-treated 661W cells, but only the inhibition of the ERK pathway reduced the levels of Faim2. Blocking the ERK pathway using a pharmacological inhibitor increased the susceptibility of 661W cells to Fas-induced caspase activation and apoptosis. When the levels of Faim2 were reduced in 661W cells by siRNA knockdown, Fas activating antibody treatment resulted in earlier and more robust caspase activation, and increased cell death. These results demonstrate that Faim2 acts as a neuroprotectant during Fas-mediated apoptosis of 661W cells. The expression of Faim2 is triggered, at least in part, by Fas-receptor activation and subsequent ERK signaling. Our findings identify a novel protective pathway that auto-regulates Fas-induced photoreceptor apoptosis in vitro. Modulation of this pathway to increase Faim2 expression may be a potential therapeutic option to prevent photoreceptor death.     

Hippocampal Neuro-Networks and Dendritic Spine Perturbations in Epileptogenesis Are Attenuated by Neuroprotectin D1

Purpose

Limbic epileptogenesis triggers molecular and cellular events that foster the establishment of aberrant neuronal networks that, in turn, contribute to temporal lobe epilepsy (TLE). Here we have examined hippocampal neuronal network activities in the pilocarpine post-status epilepticus model of limbic epileptogenesis and asked whether or not the docosahexaenoic acid (DHA)-derived lipid mediator, neuroprotectin D1 (NPD1), modulates epileptogenesis.

Methods

Status epilepticus (SE) was induced by intraperitoneal administration of pilocarpine in adult male C57BL/6 mice. To evaluate simultaneous hippocampal neuronal networks, local field potentials were recorded from multi-microelectrode arrays (silicon probe) chronically implanted in the dorsal hippocampus. NPD1 (570 μg/kg) or vehicle was administered intraperitoneally daily for five consecutive days 24 hours after termination of SE. Seizures and epileptiform activity were analyzed in freely-moving control and treated mice during epileptogenesis and epileptic periods. Then hippocampal dendritic spines were evaluated using Golgi-staining.

Results

We found brief spontaneous microepileptiform activity with high amplitudes in the CA1 pyramidal and stratum radiatum in epileptogenesis. These aberrant activities were attenuated following systemic NPD1 administration, with concomitant hippocampal dendritic spine protection. Moreover, NPD1 treatment led to a reduction in spontaneous recurrent seizures.

Conclusions

Our results indicate that NPD1 displays neuroprotective bioactivity on the hippocampal neuronal network ensemble that mediates aberrant circuit activity during epileptogenesis. Insight into the molecular signaling mediated by neuroprotective bioactivity of NPD1 on neuronal network dysfunction may contribute to the development of anti-epileptogenic therapeutic strategies.     

Association of Shank 1A Scaffolding Protein with Cone Photoreceptor Terminals in the Mammalian Retina

Photoreceptor terminals contain post-synaptic density (PSD) proteins e.g., PSD-95/PSD-93, but their role at photoreceptor synapses is not known. PSDs are generally restricted to post-synaptic boutons in central neurons and form scaffolding with multiple proteins that have structural and functional roles in neuronal signaling. The Shank family of proteins (Shank 1–3) functions as putative anchoring proteins for PSDs and is involved in the organization of cytoskeletal/signaling complexes in neurons. Specifically, Shank 1 is restricted to neurons and interacts with both receptors and signaling molecules at central neurons to regulate plasticity. However, it is not known whether Shank 1 is expressed at photoreceptor terminals. In this study we have investigated Shank 1A localization in the outer retina at photoreceptor terminals. We find that Shank 1A is expressed presynaptically in cone pedicles, but not rod spherules, and it is absent from mice in which the Shank 1 gene is deleted. Shank 1A co-localizes with PSD-95, peanut agglutinin, a marker of cone terminals, and glycogen phosphorylase, a cone specific marker. These findings provide convincing evidence for Shank 1A expression in both the inner and outer plexiform layers, and indicate a potential role for PSD-95/Shank 1 complexes at cone synapses in the outer retina.     

Protective Impact of Edaravone Against ZnO NPs-induced Oxidative Stress in the Human Neuroblastoma SH-SY5Y Cell Line

Cellular and Molecular Neurobiology - Extensive applications of ZnO NPs (zinc oxide nanoparticles) in daily life have created concern about their biotoxicity. Zinc oxide nanoparticles induce...     

Distinct and Atypical Intrinsic and Extrinsic Cell Death Pathways between Photoreceptor Cell Types upon Specific Ablation of Ranbp2 in Cone Photoreceptors

Non-autonomous cell-death is a cardinal feature of the disintegration of neural networks in neurodegenerative diseases, but the molecular bases of this process are poorly understood. The neural retina comprises a mosaic of rod and cone photoreceptors. Cone and rod photoreceptors degenerate upon rod-specific expression of heterogeneous mutations in functionally distinct genes, whereas cone-specific mutations are thought to cause only cone demise. Here we show that conditional ablation in cone photoreceptors of Ran-binding protein-2 (Ranbp2), a cell context-dependent pleiotropic protein linked to neuroprotection, familial necrotic encephalopathies, acute transverse myelitis and tumor-suppression, promotes early electrophysiological deficits, subcellular erosive destruction and non-apoptotic death of cones, whereas rod photoreceptors undergo cone-dependent non-autonomous apoptosis. Cone-specific Ranbp2 ablation causes the temporal activation of a cone-intrinsic molecular cascade highlighted by the early activation of metalloproteinase 11/stromelysin-3 and up-regulation of Crx and CoREST, followed by the down-modulation of cone-specific phototransduction genes, transient up-regulation of regulatory/survival genes and activation of caspase-7 without apoptosis. Conversely, PARP1⁺-apoptotic rods develop upon sequential activation of caspase-9 and caspase-3 and loss of membrane permeability. Rod photoreceptor demise ceases upon cone degeneration. These findings reveal novel roles of Ranbp2 in the modulation of intrinsic and extrinsic cell death mechanisms and pathways. They also unveil a novel spatiotemporal paradigm of progression of neurodegeneration upon cell-specific genetic damage whereby a cone to rod non-autonomous death pathway with intrinsically distinct cell-type death manifestations is triggered by cell-specific loss of Ranbp2. Finally, this study casts new light onto cell-death mechanisms that may be shared by human dystrophies with distinct retinal spatial signatures as well as with other etiologically distinct neurodegenerative disorders.     

Oxidative Stress and Mechanisms of Protection Against It in Bacteria

In the review contemporary data on the effects of oxidative stresses of various kinds in bacteria are summarized. A general theory of oxidative stress, peculiarities of oxidative stress in eukaryotes and prokaryotes, and natural and induced oxidative stresses are described. Data on the mechanisms of protection against oxidative stress are given, including prevention of the generation of oxidative stress, prevention of propagation of free radical chain reactions, and the mechanisms of repair of damaged DNA. The regulation of effector genes via redox-sensitive iron-containing proteins is analyzed. Special attention is given to the expression of so-called antioxidant and associated enzymes as protection mechanisms and to the space–time organization of the response of bacteria to oxidative stress.     

Human Surfactant Protein D Alters Oxidative Stress and HMGA1 Expression to Induce p53 Apoptotic Pathway in Eosinophil Leukemic Cell Line

Surfactant protein D (SP-D), an innate immune molecule, has an indispensable role in host defense and regulation of inflammation. Immune related functions regulated by SP-D include agglutination of pathogens, phagocytosis, oxidative burst, antigen presentation, T lymphocyte proliferation, cytokine secretion, induction of apoptosis and clearance of apoptotic cells. The present study unravels a novel ability of SP-D to reduce the viability of leukemic cells (eosinophilic leukemic cell line, AML14.3D10; acute myeloid leukemia cell line, THP-1; acute lymphoid leukemia cell lines, Jurkat, Raji; and human breast epithelial cell line, MCF-7), and explains the underlying mechanisms. SP-D and a recombinant fragment of human SP-D (rhSP-D) induced G2/M phase cell cycle arrest, and dose and time-dependent apoptosis in the AML14.3D10 eosinophilic leukemia cell line. Levels of various apoptotic markers viz. activated p53, cleaved caspase-9 and PARP, along with G2/M checkpoints (p21 and Tyr15 phosphorylation of cdc2) showed significant increase in these cells. We further attempted to elucidate the underlying mechanisms of rhSP-D induced apoptosis using proteomic analysis. This approach identified large scale molecular changes initiated by SP-D in a human cell for the first time. Among others, the proteomics analysis highlighted a decreased expression of survival related proteins such as HMGA1, overexpression of proteins to protect the cells from oxidative burst, while a drastic decrease in mitochondrial antioxidant defense system. rhSP-D mediated enhanced oxidative burst in AML14.3D10 cells was confirmed, while antioxidant, N-acetyl-L-cysteine, abrogated the rhSP-D induced apoptosis. The rhSP-D mediated reduced viability was specific to the cancer cell lines and viability of human PBMCs from healthy controls was not affected. The study suggests involvement of SP-D in host’s immunosurveillance and therapeutic potential of rhSP-D in the eosinophilic leukemia and cancers of other origins.     

The Motor Neuron-Like Cell Line NSC-34 and Its Parent Cell Line N18TG2 Have Glycogen that is Degraded Under Cellular Stress

Brain glycogen has a long and versatile history: Primarily regarded as an evolutionary remnant, it was then thought of as an unspecific emergency fuel store. A dynamic role for glycogen in normal brain function has been proposed later but exclusively attributed to astrocytes, its main storage site. Neuronal glycogen had long been neglected, but came into focus when sensitive technical methods allowed quantification of glycogen at low concentration range and the detection of glycogen metabolizing enzymes in cells and cell lysates. Recently, an active role of neuronal glycogen and even its contribution to neuronal survival could be demonstrated. We used the neuronal cell lines NSC-34 and N18TG2 and could demonstrate that they express the key-enzymes of glycogen metabolism, glycogen phosphorylase and glycogen synthase and contain glycogen which is mobilized on glucose deprivation and elevated potassium concentrations, but not by hormones stimulating cAMP formation. Conditions of metabolic stress, namely hypoxia, oxidative stress and pH lowering, induce glycogen degradation. Our studies revealed that glycogen can contribute to the energy supply of neuronal cell lines in situations of metabolic stress. These findings shed new light on the so far neglected role of neuronal glycogen. The key-enzyme in glycogen degradation is glycogen phosphorylase. Neurons express only the brain isoform of the enzyme that is supposed to be activated primarily by the allosteric activator AMP and less by covalent phosphorylation via the cAMP cascade. Our results indicate that neuronal glycogen is not degraded upon hormone action but by factors lowering the energy charge of the cells directly.

     

干旱胁迫下大豆与玉米叶片光破坏的防御

随干旱强度的增加,大豆叶片光呼吸速率（Pr）降低的幅度小于Pn,使Pr／Pn比率升高。在轻度及中度干旱下,PSII光化学效率（Fv／Fm）能在暗置后较快地恢复。随干旱强度的增加,叶片光化学猝灭系数下降,非光化学猝死系数升高。干旱使叶片β－胡萝卜素及紫黄质含量下降,而玉米黄质含量（Z）与叶黄素库增加。与大豆相比,玉米在干旱条件下具有较高的Z含量和较大的叶黄素库。     

Lrit1, a Retinal Transmembrane Protein,Regulates Selective Synapse Formation in Cone Photoreceptor Cells and Visual Acuity

1. Download high-res image (191KB)
2. Download full-size image

     

NCAM1 is the Target of miRNA-572: Validation in the Human Oligodendroglial Cell Line

The neural cell adhesion molecule 1 (NCAM1) is a fundamental protein in cell–cell interaction and in cellular developmental processes, and its dysregulation is involved in a number of diseases including multiple sclerosis. Studies in rats suggest that the modulation of NCAM1 expression is regulated by miRNA-572, but no data are available confirming such interaction in the human system. We analyzed whether this is the case using a human oligodendroglial cell line (MO3.13). MO3.13 cells were transfected with miRNA-572 mimic and inhibitor separately; NCAM1 mRNA and protein expression levels were analyzed at different time points after transfection. Results indicated that NCAM1 expression is increased after transfection with miRNA-572 inhibitor, whereas it is decreased after transfection with the mimic (p < 0.005). The interaction between NCAM1 and miRNA-572 was subsequently confirmed in a Vero cell line that does not express NCAM1, by luciferase assay after transfection with NCAM1. These results confirm that miRNA-572 regulates NCAM1 and for the first time demonstrate that this interaction regulates NCAM1 expression in human cells. Data herein also support the hypothesis that miRNA-572 is involved in diseases associated with NCAM1 deregulation, suggesting its possible use as a biomarker in these diseases.     

Selective Survival Rescue in 15-Lipoxygenase-1-deficient Retinal Pigment Epithelial Cells by the Novel Docosahexaenoic Acid-derived Mediator, Neuroprotectin D1

The integrity of the retinal pigment epithelial (RPE) cell is essential for the survival of rod and cone photoreceptor cells. Several stressors, including reactive oxygen species, trigger apoptotic damage in RPE cells preceded by an anti-inflammatory, pro-survival response, the formation of neuroprotectin D1 (NPD1), an oxygenation product derived from the essential omega-3 fatty acid family member docosahexaenoic acid. To define the ability of NPD1 and other endogenous novel lipid mediators in cell survival, we generated a stable knockdown human RPE (ARPE-19) cell line using short hairpin RNA to target 15-lipoxygenase-1. The 15-lipoxygenase-1-deficient cells exhibited 30% of the protein expression, and 15-lipoxygenase-2 remained unchanged, as compared with an ARPE-19 cell line control established using nonspecific short hairpin RNA transfected cells. NPD1 synthesis was stimulated by tumor necrosis factor α/H₂O₂-mediated oxidative stress in nonspecific cells (controls), whereas in silenced cells, negligible amounts of NPD1, 12(S)- and 15(S)-hydroxyeicosatetraenoic acid, and lipoxin A₄ were found under these conditions. Neither control nor the deficient cells showed an increase in 15-lipoxygenase-1 protein content after 16 h of oxidative stress, suggesting that the increased activity of 15-lipoxygenase-1 is due to activation of pre-existing proteins. 15-Lipoxygenase-silenced cells also displayed an exacerbated sensitivity to oxidative stress-induced apoptosis when compared with the control cells. NPD1 selectively and potently rescued 15-lipoxygenase-silenced cells from oxidative stress-induced apoptosis. These results demonstrate that 15-lipoxygenase-1 is activated by oxidative stress in ARPE-19 cells and that NPD1 is part of an early survival signaling in RPE cells.     

Evidence that Plasmalogen is Protective Against Oxidative Stress in the Rat Brain

The antioxidant capabilities of phosphatidylethanolamine plasmalogen (PlsEtn), in vivo, against lipid peroxidation were investigated via acute phosphine (PH₃) administration in rats. Oxidative stress was assessed from measures of malondialdehyde and various enzyme activities, while NMR analyses of lipid and aqueous tissue extracts provided metabolic information in cerebellum, brainstem, and cortex. Brainstem had the highest basal [PlsEtn], and showed only moderate PH₃-induced oxidative damage with no loss of ATP. The lowest basal [PlsEtn] was observed in cortex, where PH₃ caused a 51% decrease in [ATP]. The largest oxidative effect occurred in cerebellum, but [ATP] was unaffected. Myo-inositol+ethanolamine pretreatment attenuated all PH₃ effects. Specifically, the pretreatment attenuated the ATP decrease in cortex, and elevated brain [PlsEtn] in the cerebellum, nearly abolishing the cerebellar oxidative effects. Our data suggest a high basal [PlsEtn], or the capacity to synthesize new ethanolamine lipids (particularly PlsEtn) may protect against PH₃ toxicity.     

Oxidative Stress and Antioxidant Cell Protection Systems in the Microaerophilic Bacterium Spirillum winogradskii

The influence of oxygen availability during cultivation on the biosynthetic processes and enzymatic activities in the microaerophilic bacterium Spirillum winogradskii D-427 was studied, and the roles played by different systems of the defense against oxidation stress were determined. The metabolic adjustments caused by transition from microaerobic (2% O₂) aerobic conditions (21% O₂ of the gas phase) were found to slow down constructive metabolism and increase synthesis of exopolysaccharides as a means of external protection of cells from excess oxygen. This resulted in a twofold decline of the growth yield coefficient. Even though the low activity of catalase is compensated for by a multifold increase in the activities of other cytoplasmic enzymes that defend against toxic forms of O₂—peroxidase and enzymes of the redox system of glutathione (glutathione peroxidase and glutathione reductase)—massive lysis of cells starts in the midexponential phase and leads to culture death in the stationary phase because of H₂O₂ accumulation in the periplasm (up to 10 g/mg protein). The absence in cells of cytochrome-c-peroxidase, a periplasmic enzyme eliminating H₂O₂, was shown. It follows that the major cause of oxidative stress in cells is that active antioxidant defenses are located in the cytoplasm, whereas H₂O₂ accumulates in the periplasm due to the lack of cytochrome-c-peroxidase. The addition to the medium of thiosulfate promotes elimination of H₂O₂, stops cell lysis under aerobic conditions, lends stability to cultures, and results in a threefold increase in the growth yield.