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1.
Franco-Cea A Valencia A Sánchez-Armass S Domínguez G Morán J 《Neurochemical research》2004,29(1):227-238
Cultured cerebellar granule neurons (CGC) increase survival in a medium containing 25 mM KCl (K25), and they die apoptotically when cultures are treated with staurosporine (St) or are transferred to a 5-mM KCl containing medium (K5). Apoptotic CGC show nuclear condensation and caspase-3 activation. Cell death induced by these conditions was partially prevented when cultures were maintained under alkaline conditions, which also induced a marked reduction of the caspase-3 activation. The acidification of the medium further increased cell death induced by both stimuli. Cultures transferred to K5 suffered an immediate intracellular alkalinization that remained constant during the time K5 was present. In contrast, St did not modify cytosolic pH at any of the evaluated times. On the other hand, DIDS, furosemide, and bumetanide prevented CGC death induced by K5 and St. Other drugs such as amiloride, EIPA, tamoxifen, NEM, or NPPB did not modify cell death induced by these conditions. Both DIDS and bumetanide markedly inhibited the processing and activation of caspase-3, and DIDS prevented the nuclear condensation induced by K5 and St. These findings suggest that pH is a condition that could contribute to the modulation of cell death induced by some stimuli and that other ions, such as potassium, could have a role in the initial phase of apoptotic death of CGC. 相似文献
2.
Cultured cerebellar granule cells deprived of depolarizing concentrations of KCl and serum die by programmed cell death. Recently, it was shown that serum removal by itself can lead to oxidative stress and DNA fragmentation in these cells. We have modified the protocol which initiates cell death in such a way that only the effect of KCl withdrawal-induced cell death was observed. We have performed a series of experiments to correlate the structural and biochemical changes in this process of cell death. Significant morphological alterations occur in cell bodies and neurites during a 48-hour period of KCl removal. Cell viability dropped to 53%, 34% or 10% of control levels, respectively, as a result of 1-, 2-, or 3-day KCl removal. A series of experiments was conducted to determine the change of total protein level, protein synthesis rate, RNA synthesis rate, and mitochondrial activity during the first 48 hours of KCl removal. These studies not only provide a picture correlating the morphological and biochemical changes in the process of programmed cell death, but also serve as a reference for future studies of this complex phenomenon. 相似文献
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4.
Cerebellar granule neurons (CGN) cultured in a 25 mM KCl medium (K25) die apoptotically when they are transferred to a medium
containing 5 mM KCl (K5). It has been previously shown that apoptotic death of CGN induced by K5 is mediated by an increase
in the levels of reactive oxygen species (ROS). ROS may participate in the apoptotic program either as signaling molecules
or as effectors by causing oxidative damage to lipids, DNA and proteins. In this study we evaluated ROS production in CGN
treated with K5 for different periods of time and evaluated a possible correlation between ROS production and oxidation of
DNA proteins and two lipid peroxidation products, conjugated dienes and malondialdehyde. Under these conditions, we found
two episodes of ROS generation, one at an early time (4 h) and another at a later time point (18–24 h). We also identified
two peaks in the formation of conjugated dienes, the initial and transient by-products of lipoperoxidation. The first one
occurred after 4 h of K5 treatment and the other was observed after 18 h, both of them correlated with the formation of ROS.
In contrast, we found significant levels of the late product of lipidperoxidation, malondialdehyde only after 18 h of treatment.
Besides, we did not find significant levels of DNA and protein oxidation products that could be correlated with the observed
ROS production. These results support the idea that ROS produced early by K5 treatment could act primarily as a signal of
the apoptotic cell death and that ROS produced later could be mainly a product of the cell death that could contribute directly
to this process. 相似文献
5.
Nikolay K. Isaev Elena V. Stelmashook Sergey V. Lukin Dorette Freyer Philipp Mergenthaler Dmitry B. Zorov 《Cellular and molecular neurobiology》2010,30(6):877-883
Severe acidosis caused death of cultured cerebellar granule neurons (CGNs). Acidosis was accompanied by a progressive increase
of the intracellular zinc ions ([Zn2+]i) and decrease of [Ca2+]i. Zn2+ chelator, N,N,N′,N′-tetrakis(2-pyridylmethyl)ethylenediamine (TPEN), prevented the increase of [Zn2+]i and acidosis-induced neuronal death. However, neuronal death was insensitive to blockade of ASIC1 channels with amiloride,
as CGNs display considerably lower expression of ASIC1a than other neurons. The antioxidant trolox and menadione significantly
protected neurons from acidotic death. Earlier, we demonstrated that menadione rescues neurons from the deleterious effect
of inhibition of mitochondrial complex I (Isaev et al. Neuroreport 15:2227–2231, 2004). We speculate that excessive Zn2+-dependent production of reactive oxygen species by mitochondrial complex I may be a general motive for the induction of cell
death in CGNs under acidotic conditions. 相似文献
6.
Production of Reactive Oxygen Species and Release of l-Glutamate During Superoxide Anion-Induced Cell Death of Cerebellar Granule Neurons 总被引:3,自引:0,他引:3
Takumi Satoh Tadahiro Numakawa Yasuhiro Abiru Tomoko Yamagata Yasuyuki Ishikawa Yasushi Enokido Hiroshi Hatanaka 《Journal of neurochemistry》1998,70(1):316-324
Abstract: Enhanced production of superoxide anion (O2 − ) is considered to play a pivotal role in the pathogenesis of CNS neurons. Here, we report that O2 − generated by xanthine (XA) + xanthine oxidase (XO) triggered cell death associated with nuclear condensation and DNA fragmentation in cerebellar granule neuron. XA + XO induced significant increases in amounts of intracellular reactive oxygen species (ROS) before initiating loss of cell viability, as determined by measurement of 6-carboxy-2',7'-dichlorodihydrofluorescein diacetate, di(acetoxymethyl ester) (C-DCDHF-DA) for O2 − and other ROS and hydroethidine (HEt) specifically for O2 − by using fluorescence microscopy and flow cytometry. Catalase, but not superoxide dismutase (SOD), significantly protected granule neurons from the XA + XO-induced cell death. Catalase effectively reduced C-DCDHF-DA but not HEt fluorescence, whereas SOD reduced HEt but not C-DCDHF-DA fluorescence, indicating that HEt and C-DCDHF-DA fluorescence correlated with O2 − and hydrogen peroxide, respectively. The NMDA antagonist MK-801 prevented the death. XA + XO induced an increase in l -glutamate release from cerebellar granule neurons. These results indicate that elevation of O2 − induces cell death associated with increasing ROS production in cerebellar granule neurons and that XA + XO enhanced release of l -glutamate. 相似文献
7.
Abstract: The neuronal effects of the metabotropic glutamate receptor agonist (1 S ,3 R )-aminocyclopentane-1,3-dicarboxylic acid have been studied in cultured rat cerebellar granule cells, and compared with those of the endogenous excitotoxin glutamate, and the dietary excitotoxin β- N -methylamino- l -alanine. Glutamate, β- N -methylamino- l -alanine, and (1 S ,3 R )-aminocyclopentane-1,3-dicarboxylic acid all caused concentration-dependent cerebellar granule cell death over a 24-h exposure period. The metabotropic antagonist ( RS )-α-methyl-4-carboxyphenylglycine reduced glutamate-, β- N -methylamino- l -alanine-, and (1 S ,3 R )-aminocyclopentane-1,3-dicarboxylic acid-induced death by 50, 37, and 90%, respectively. (1 S ,3 R )-Aminocyclopentane-1,3-dicarboxylic acid-induced death was unaffected by the group I antagonist ( RS )-1-aminoindan-1,5-dicarboxylic acid, increased by the group II antagonist ethylglutamic acid, and markedly decreased by the group III antagonist ( RS )-α-methylserine- O -phosphate. Neither (1 S ,3 R )-aminocyclopentane-1,3-dicarboxylic acid nor the group I agonist ( RS )-3,5-dihydroxyphenylglycine caused an increase in intracellular free calcium levels. The group III agonist l -(+)-2-amino-4-phosphonobutyric acid also induced concentration-dependent cerebellar granule cell death, and so it was suggested that the group III metabotropic glutamate receptors were responsible for (1 S ,3 R )-aminocyclopentane-1,3-dicarboxylic acid-induced death. Blocking these receptors with ( RS )-α-methylserine- O -phosphate also prevented a proportion of glutamate- and β- N -methylamino- l -alanine-induced death. 相似文献
8.
Mauro Sparapani Marco Virgili Giuseppe Bardi Manuela Tregnago Barbara Monti Monia Bentivogli Antonio Contestabile 《Journal of neurochemistry》1998,71(5):1898-1904
Abstract: Ornithine decarboxylase (ODC), the key enzyme for polyamine biosynthesis, dramatically decreases in activity during normal cerebellar development, in parallel with the progressive differentiation of granule neurons. We have studied whether a similar pattern is displayed by cerebellar granule neurons during survival and differentiation in culture. We report that when granule cells were kept in vitro under trophic conditions (high K+ concentration), ODC activity progressively decreased in parallel with neuronal differentiation. Under nontrophic conditions (cultures kept in low K+ concentration), the enzymatic activity dropped quickly in parallel with an increased apoptotic elimination of cells. Cultures kept in high K+ but chronically exposed to 10 m M lithium showed both an increased rate of apoptotic cell death at 2 and 4 days in vitro and a quicker drop of ODC activity and immunocytochemical staining. A short chronic treatment of rat pups with lithium also resulted in transient decrease of cerebellar ODC activity and increased programmed cell death, as revealed by in situ detection of apoptotic granule neurons. The present data indicate that a sustained ODC activity is associated with the phase of survival and differentiation of granule neurons and that, conversely, conditions that favor their apoptotic elimination are accompanied by a down-regulation of the enzymatic activity. 相似文献
9.
Activation of G Proteins Bidirectionally Affects Apoptosis of Cultured Cerebellar Granule Neurons 总被引:17,自引:0,他引:17
§Guang-Mei Yan Sui-Zhen Lin †Robert P. Irwin ‡§ Steven M. Paul 《Journal of neurochemistry》1995,65(6):2425-2431
Abstract: Cultured cerebellar granule neurons maintained in depolarizing concentrations of K+ (25 m M ) and then switched to physiological concentrations of K+ (5 m M ) undergo apoptosis. We now report that activation of specific G proteins robustly and bidirectionally affects apoptosis of cultured rat cerebellar granule neurons. Stimulation of Gs with cholera toxin completely blocks apoptosis induced by nondepolarizing concentrations of K+ , whereas stimulation of Go /Gi with the wasp venom peptide mastoparan induces apoptosis of cerebellar granule neurons even in high (depolarizing) concentrations of K+ . Moreover, pretreatment of cerebellar granule neurons with cholera toxin attenuates neuronal death induced by mastoparan. By contrast, pertussis toxin, cell-permeable analogues of cyclic AMP, and activators of protein kinase A do not affect apoptosis of cultured cerebellar granule neurons. These data suggest that G proteins may function as key switches for controlling the programmed death of mammalian neurons, especially in the developing CNS. 相似文献
10.
Stephen D. Skaper Maura Floreani Alessandro Negro Laura Facci Pietro Giusti 《Journal of neurochemistry》1998,70(5):1859-1868
Abstract: Cerebellar granule neurons maintained in medium containing serum and 25 mM K+ reliably undergo an apoptotic death when switched to serum-free medium with 5 mM K+. New mRNA and protein synthesis and formation of reactive oxygen intermediates are required steps in K+ deprivation-induced apoptosis of these neurons. Here we show that neurotrophins, members of the nerve growth factor gene family, protect from K+/serum deprivation-induced apoptotic death of cerebellar granule neurons in a temporally distinct manner. Switching granule neurons, on day in vitro (DIV) 4, 10, 20, 30, or 40, from high-K+ to low-K+/serum-free medium decreased viability by >50% when measured after 30 h. Treatment of low-K+ granule neurons at DIV 4 with nerve growth factor, brain-derived neurotrophic factor (BDNF), neurotrophin-3, or neurotrophin-4/5 (NT-4/5) demonstrated concentration-dependent (1–100 ng/ml) protective effects only for BDNF and NT-4/5. Between DIV 10 and 20, K+-deprived granule neurons showed decreasing sensitivity to BDNF and no response to NT-4/5. Cerebellar granule neuron death induced by K+ withdrawal at DIV 30 and 40 was blocked only by neurotrophin-3. BDNF and NT-4/5 also circumvented glutamate-induced oxidative death in DIV 1–2 granule neurons. Granule neuron death caused by K+ withdrawal or glutamate-triggered oxidative stress was, moreover, limited by free radical scavengers like melatonin. Neurotrophin-protective effects, but not those of antioxidants, were blocked by selective inhibitors of phosphatidylinositol 3-kinase or the mitogen-activated protein kinase pathway, depending on the nature of the oxidant stress. These observations indicate that the survival-promoting effects of neurotrophins for central neurons, whose cellular antioxidant defenses are challenged, require activation of distinct signal transduction pathways. 相似文献
11.
Adenovirus-Mediated Gene Transfer of Inhibitors of Apoptosis Proteins
Delays Apoptosis in Cerebellar Granule Neurons 总被引:1,自引:0,他引:1
M. Simons S. Beinroth M. Gleichmann P. Liston R. G. Korneluk A. E. MacKenzie M. Bähr T. Klockgether G. S. Robertson† M. Weller & J. B. Schulz 《Journal of neurochemistry》1999,72(1):292-301
Abstract : The inhibitor of apoptosis (IAP) family of anti-apoptotic genes, originally discovered in baculovirus, exists in animals ranging from insects to humans. Here, we investigated the ability of IAPs to suppress cell death in both a neuronal model of apoptosis and excitotoxicity. Cerebellar granule neurons undergo apoptosis when switched from 25 to 5 m M potassium, and excitotoxic cell death in response to glutamate. We examined the endogenous expression of four members of the IAP family, X chromosome-linked IAP (XIAP), rat IAP1 (RIAP1), RIAP2, and neuronal apoptosis inhibitory protein (NAIP), by semiquantitative reverse PCR and immunoblot analysis in cultured cerebellar granule neurons. Cerebellar granule neurons express significant levels of RIAP2 mRNA and protein, but expression of RIAP1, NAIP, and XIAP was not detected. RIAP2 mRNA content and protein levels did not change when cells were switched from 25 to 5 m M potassium. To determine whether ectopic expression of IAP influenced neuronal survival after potassium withdrawal or glutamate exposure, we used recombinant adenoviral vectors to target XIAP, human IAP1 (HIAP1), HIAP2, and NAIP into cerebellar granule neurons. We demonstrate that forced expression of IAPs efficiently blocked potassium withdrawal-induced N -acetly-Asp-Glu-Val-Asp-specific caspase activity and reduced DNA fragmentation. However, neurons were only protected from apoptosis up to 24 h after potassium withdrawal, not at later time points suggesting that IAPS delay but do not block apoptosis in cerebellar granule neurons. In contrast, treatment with 100 μ M or 1 m M glutamate did not induce caspase activity and adenoviral-mediated expression of IAPs had no influence on subsequent excitotoxic cell death. 相似文献
12.
Jeffery D. Fritz Andrea L. Utz Nannette M. Hale †Lan Wu †‡§Alvin C. Powers Todd A. Verdoorn ‡ David Robertson 《Journal of neurochemistry》1997,68(1):204-212
Abstract: The human glutamic acid decarboxylase (GAD) gene was transferred into rat cerebellar granule neurons. Following adenoviral-mediated gene transfer, nearly 100% of the neurons had transgene expression that persisted for the duration of their survival in culture. GABA levels were elevated both in the growth media and in lysates of GAD-modified granule neurons. In GAD-modified neurons, extracellular GABA levels steadily increased with time, whereas intracellular GABA levels peaked 10 days after gene transfer. GAD-modified neurons released both glutamate and GABA into the surrounding media before and after potassium-induced stimulation, but only the release of glutamate was sensitive to potassium stimulation. These data suggest that glutamatergic neurons, which initially contained no detectable GABA, can be genetically modified to release GABA constitutively. 相似文献
13.
Koshimizu H Suzuki S Araki T Yamada M Kojima M Hatanaka H 《Cellular and molecular neurobiology》2011,31(7):1027-1032
Brain immunoglobulin-like molecule with tyrosine-based activation motifs/SHP substrate 1 (BIT/SHPS-1) is a neuronal adhesion
molecule that is highly expressed in cerebellar granule neurons (CGNs); however its function in CGNs remains unclear. Our
previous studies indicated that BIT/SHPS-1 is able to modulate the antiapoptotic effect of brain-derived neurotrophic factor
(BDNF) on CNS neurons by cell type-specific mechanisms. In this article, we have studied the role of BIT/SHPS-1 in the antiapoptotic
function of BDNF on low potassium (LK)-induced cell death of cultured CGNs which is an in vitro model system of neuronal apoptosis
during brain development. Cultured rat CGNs were transduced with wild-type rat BIT/SHPS-1 (BIT/SHPS-1WT), its 4F-mutant (BIT/SHPS-14F, in which all cytoplasmic tyrosine residues were substituted with phenylalanine), or nuclear localization signal-attached
beta-galactosidase (NLS-LacZ, as control)-expressing adenoviruses. Expression of BIT/SHPS-1WT and BIT/SHPS-14F alone did not affect steady-state cell viability. Tyrosine phosphorylation of BIT/SHPS-1 was only detected in BIT/SHPS-1WT-expressing cultures in the presence and the absence of BDNF. When subjected to LK in the presence of BDNF, BIT/SHPS-1WT- and BIT/SHPS-14F-expressing cultures showed a significant resistance to cell death, while the control virus-transfected culture did not. In
addition, a phosphatidylinositol 3-kinase (PI3-K) inhibitor, LY294002, attenuated the antiapoptotic effect of BDNF on BIT/SHPS-1WT-, and BIT/SHPS-14F-expressing cultures. These results demonstrated that in both tyrosine phosphorylation-independent and PI3-K-dependent manners,
BIT/SHPS-1 promotes the antiapoptotic effect of BDNF on the LK-induced cell death of CGNs. 相似文献
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15.
Respiration in Primary Cultured Cerebellar Granule Neurons and Cerebral Cortical Neurons 总被引:1,自引:1,他引:0
Respiration was measured polarographically in primary cultures enriched with cerebellar granule neurons or cerebral cortical neurons. The basal respiratory rate, measured on the sixth day after culturing, was 12.00 natom equiv. O/mg protein/min for the cortical neurons and 12.70 natom equiv. O/mg protein/min for the granule neurons. Maximal stimulation by 2,4-dinitrophenol produced a 20-40% increase over the basal rate for both neuronal types. Oligomycin inhibited neuronal basal respiration by 45%. These respiratory rates in neurons from primary culture are markedly lower than those measured in astrocytes grown under similar conditions. 相似文献
16.
Bax Deletion Further Orders the Cell Death Pathway in Cerebellar Granule Cells and Suggests a Caspase-independent Pathway to Cell Death 总被引:21,自引:2,他引:19 下载免费PDF全文
Timothy M. Miller Krista L. Moulder C. Michael Knudson Douglas J. Creedon Mohanish Deshmukh Stanley J. Korsmeyer Eugene M. Johnson Jr. 《The Journal of cell biology》1997,139(1):205-217
Dissociated cerebellar granule cells maintained in medium containing 25 mM potassium undergo an apoptotic death when switched to medium with 5 mM potassium. Granule cells from mice in which Bax, a proapoptotic Bcl-2 family member, had been deleted, did not undergo apoptosis in 5 mM potassium, yet did undergo an excitotoxic cell death in response to stimulation with 30 or 100 μM NMDA. Within 2 h after switching to 5 mM K+, both wild-type and Bax-deficient granule cells decreased glucose uptake to <20% of control. Protein synthesis also decreased rapidly in both wild-type and Bax-deficient granule cells to 50% of control within 12 h after switching to 5 mM potassium. Both wild-type and Bax −/− neurons increased mRNA levels of c-jun, and caspase 3 (CPP32) and increased phosphorylation of the transactivation domain of c-Jun after K+ deprivation. Wild-type granule cells in 5 mM K+ increased cleavage of DEVD–aminomethylcoumarin (DEVD-AMC), a fluorogenic substrate for caspases 2, 3, and 7; in contrast, Bax-deficient granule cells did not cleave DEVD-AMC. These results place BAX downstream of metabolic changes, changes in mRNA levels, and increased phosphorylation of c-Jun, yet upstream of the activation of caspases and indicate that BAX is required for apoptotic, but not excitotoxic, cell death. In wild-type cells, Boc-Asp-FMK and ZVAD-FMK, general inhibitors of caspases, blocked cleavage of DEVD-AMC and blocked the increase in TdT-mediated dUTP nick end labeling (TUNEL) positivity. However, these inhibitors had only a marginal effect on preventing cell death, suggesting a caspase-independent death pathway downstream of BAX in cerebellar granule cells. 相似文献
17.
Saito Mariko Saito Mitsuo Berg Martin J. Guidotti Alessandro Marks Neville 《Neurochemical research》1999,24(9):1107-1115
Ethanol significantly enhances cell death of differentiated rat cerebellar granule neurons on culture in a serum-free medium containing a depolarizing concentration of KCl (25 mM), 5 M MK-801 (an NMDA receptor antagonist), and 20–200 mM ethanol for 1–4 days. Cell death augmented by ethanol was concentration- and time-dependent with neurons displaying hallmark apoptotic morphology and DNA fragmentation that correlated with the activation of cytosolic caspase-3. Inclusion of 5 M MK-801 or 100 M glycine in culture media did not alter rates of cell death indicating ethanol toxicity is mediated via an NMDA receptor-independent pathway. Preincubation with 50 M gangliosides GM1, GD1a, GD1b or GT1b for 2 h, or preincubation with 10 M LIGA20 (a semisynthetic GM1 with N-dichloroacetylsphingosine) for 10 min, attenuated caspase-3 activity and ethanol-induced cell death. Data show native gangliosides and a synthetic derivative are potently neuroprotective in this model of ethanol toxicity, and potentially serve as useful probes to further unravel the mechanisms relevant to neuronal apoptosis. 相似文献
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Lead (Pb2+) is a common pollutant and potent central neurotoxin. We have studied its pathways of permeation by two-photon fluorescence
microscopy in rat cerebellar granule neurons loaded with the fluorescent dye indo-1. Pb2+ binds indo-1 with high affinity acting as a quencher. Its permeation through the neuronal membrane was indicated by a decrease
of the fluorescence emission, which occurred even in resting condition. In the presence of 20 μM Pb2+, uptake reached a plateau level (≈45% of initial fluorescence) in 4 min and was partially antagonized by 25 μM lanthanum.
Subsequent addition of a membrane permeant ionophore caused a further (>70%) quenching of the dye, suggesting that previous
saturation was due to inactivation of the transport system. Intracellular Pb2+ concentrations were evaluated from the fluorescence intensity and this estimate indicated that the concentration of free
Pb2+ sufficient to inactivate the transport system is close to 50 pM. 相似文献
20.
Migration Behavior of Granule Cell Neurons in Cerebellar Cultures. II. An Electron Microscopic Study
We examined the fine structure of migrating granule cell neurons in cerebellar microexplant cultures. Radially migrating bipolar cells extended microspikes or small filopodia from their soma and processes and frequently made contact with neighboring cells. These microspikes contained microfilaments but no microtubules. At the later phase of the migration, in which they had symmetrical bipolar long processes, filopodia extending from perikarial region of cells contained microtubules, suggesting that they are precursors of the future thick perpendicular processes. When cell bodies changed orientation from radial to perpendicular, microtubules that were nucleated from perinuclear centrioles frequently extended into both thick radial and perpendicular processes from the perikarial region. Bundles of 10nm intermediate filaments also appeared in these processes. During migration by the perpendicular contact guidance, many filopodia extending from both the thick leading processes and thin trailing processes made close contacts with the radial parallel neurite. These findings suggest that; 1) The direct contact of the filopodia from both the growth cones and their processes of the granule cells to the neurite bundle plays roles in both the parallel and perpendicular contact guidances. 2) The spacial and temporal changes of cytoskeletons and the association of microtubules with perinuclear centrioles are important for the formation of perpendicular processes and initiation of the perpendicular contact guidance. 相似文献