An early increase in the disialoganglioside GD3 contributes to the development of neuronal apoptosis in culture

We induced apoptosis in primary cultures of cerebellar granule neurons by switching the growing medium into a medium containing lower concentrations of K(+) (5 or 10 mM instead of 25 mM) or, alternatively, by addition of staurosporine. The apoptotic phenotype was always preceded by an early increase in the intracellular levels of the disialoganglioside GD3, which peaked at 2-6 h and returned back to normal at 12 h. GD3 synthase, the enzyme that forms GD3 from the monosialoganglioside GM3, was also induced at early times after the induction of apoptosis in granule cells. Immunofluorescent staining showed that GD3 increased in neuronal cell bodies and neurites, but was never localized in cell nuclei. In cultures switched into a low K(+)-containing medium, exogenously applied GD3, but not the disialoganglioside GD1a, accelerated the development of neuronal apoptosis. In contrast, the antisense-induced knock-down of GD3 synthase was protective against granule cell death induced by lowering extracellular K(+) from 25 to 10 - but not 5 - mM. These results demonstrate that an early and transient increase in GD3 synthesis is one of the factors that contribute to the induction of neuronal apoptosis in culture.     

Pyrrolidinium-type fullerene derivative-induced apoptosis by the generation of reactive oxygen species in HL-60 cells

The biological activities of C₆₀-bis(N,N-dimethylpyrrolidinium iodide), a water-soluble cationic fullerene derivative, on human promyeloleukaemia (HL-60) cells were investigated. The pyrrolidinium fullerene derivative showed cytotoxicity in HL-60 cells. The characteristics of apoptosis, such as DNA fragmentation and condensation of chromatin in HL-60 cells, were observed by exposure to the pyrrolidinium fullerene derivative. Caspase-3 and -8 were activated and cytochrome c was also released from mitochondria. The generation of reactive oxygen species (ROS) by the pyrrolidinium fullerene derivative was observed by DCFH-DA, a fluorescence probe for the detection of ROS. Pre-treatment with α-tocopherol suppressed cell death and intracellular oxidative stress caused by the pyrrolidinium fullerene derivative. The apoptotic cell death induced by the pyrrolidinium fullerene derivative was suggested to be mediated by ROS generated by the pyrrolidinium fullerene derivative.     

Different mechanisms of protection against apoptosis by valproate and Li+.

Acute treatment with valproate and Li+ was found to protect cultured cerebellar granule cells against apoptosis induced by low K+ (5 mM). Because the protection was unaffected by MK801 (N-methyl-D-aspartate receptor inhibitor), an increase in glutamate release cannot be responsible for the observed neuroprotection. Insulin also protects against low-K+-induced apoptosis of cerebellar granule cells. This protection is totally dependent on LY294002 (a phosphatidylinositol 3-kinase inhibitor). These results suggest a role for phosphatidylinositol 3-kinase in the neuroprotection induced by insulin. Likewise, and in contrast with the results observed with Li+, the protection induced by valproate is also dependent on insulin and LY294002. Moreover, valproate (a branched-chain fatty acid) does not change the plasma membrane microviscosity under physiological conditions. These results suggest that valproate protects against low-K+-induced apoptosis by acting in the phosphatidylinositol 3-kinase/protein kinase B pathway. The protection by Li+ is independent of this transduction pathway.     

Apoptosis-inducing and -preventing signal transduction pathways in cultured cerebellar granule neurons

1. Cultured cerebellar granule neurons maintained in medium containing 26 mM potassium (high K+ or HK+) undergo cell death when switched to medium with 5 mM potassium (low K+ or LK+). This low K(+)-induced cell death has typical features of apoptosis. The intracellular signaling pathway of low K(+)-induced apoptosis has been investigated. 2. Cerebellar granule neurons become committed to undergo apoptosis between 2 and 5 h after K+ deprivation, judging from the inability of high K+ to rescue them after this time. Although the levels of most mRNAs decrease markedly concomitant with commitment, expression of c-jun mRNA increases 2-3 h after K+ deprivation. Among the family of caspases, a caspase-3-like protease is activated within 4 h of lowering the K+ concentration. A caspase-1-like protease is also activated within 2 h of K+ deprivation. 3. Inhibition of phosphatidylinositol 3-kinase (PI3-K) activity by LY294002 or wortmannin also induces apoptosis in cerebellar granule neurons. The intracellular signaling pathway of LY294002-induced apoptosis has been investigated. The activity of c-Jun N-terminal kinase (JNK) increases 8 h after addition of LY294002 to high K+ medium or low K+ medium containing BDNF. Expression of c-Jun protein also increases almost simultaneously. 4. The low K(+)-induced apoptosis of cerebellar granule neurons is prevented by high K+ (membrane depolarization by high K+), BDNF, IGF-1, bFGF or cAMP. The intracellular signaling pathways by which these agents prevent low K(+)-induced apoptosis have been investigated. Agents other than cAMP prevent apoptosis through PI3-K and a Ser/Thr kinase, Akt/PKB. The survival-promoting effect of cAMP does not depend on the PI3-K-Akt pathway.     

Synthesis of beta-alanine C60 derivative and its protective effect on hydrogen peroxide-induced apoptosis in rat pheochromocytoma cells

Oxidative stress has been considered as a major cause of cellular injuries in a variety of clinical abnormalities, especially prominent in neural diseases. One of the effective ways to prevent the reactive oxygen species (ROS) mediated cellular injury is dietary or pharmaceutical augmentation of some free radical scavenger. Water-soluble amino-fullerene derivative is a novel compound that behaves as a free radical scavenger with excellent biocompatibility. In the present study, we synthesized a novel beta-alanine C(60) derivative. The product was characterized by FT-IR, (1)H NMR, (13)C NMR, LC-MS and elemental analysis. We investigated the protective effect on hydrogen peroxide-induced oxidative stress and apoptotic death in cultured rat pheochromocytoma (PC12) cells. PC12 cells treated with hydrogen peroxide underwent apoptotic death determined by MTT, flow cytometry analysis and PI/Hoechst 33342 staining. Moreover, the scavenging ability of beta-alanine C(60) derivative to reactive oxygen species both in vivo and in vitro of PC12 cells was measured. The results suggest that beta-alanine C(60) derivative has the potential to prevent oxidative stress-induced cell death without evident toxicity. Hence, on the basis of the above-mentioned studies, we can hypothesize that the protective effect of beta-alanine C(60) derivative on H(2)O(2) induced apoptosis is related to their known scavenger activity toward ROS.     

Roles of Thapsigargin-Sensitive Ca2+ Stores in the Survival of Developing Cultured Neurons

The roles of the intracellular calcium pool involved in regulating the Ca2+ profile and the neuronal survival rate during development were studied by using thapsigargin (TG), a specific inhibitor of endoplasmic reticulum (ER) Ca2+-ATPase in cultured cerebellar granule neurons. Measuring the neuronal [Ca2+]i directly in the culture medium, we found a bell-shaped curve for [Ca2+]i versus cultured days in cerebellar granule neurons maintained in medium containing serum and 25 mM K+. The progressive increase in [Ca2+]i of the immature granule neurons (1-4 days in vitro) was abolished by TG, which resulted in massive neuronal apoptosis. When the [K+] was lowered from 25 to 5 mM, neither the progressively increasing [Ca2+]i nor the survival of immature granule neurons was significantly changed over 24-h incubation. Similarly, TG caused a dramatic decrease in the [Ca2+]i and survival rate of these immature neurons when switched to 5 mM K+ medium. Following maturation, the granule neurons became less sensitive to TG for both [Ca2+]i and neuronal survival. However, TG can protect mature granule neurons from the detrimental effect of switching to a 5 mM K+ serum-free medium by decreasing [Ca2+]i to an even lower level than in the respective TG-free group. Based on these findings, we propose that during the immature stage, TG-sensitive ER Ca2+-ATPase plays a pivotal role in the progressive increase of [Ca2+]i, which is essential for the growth and maturation of cultured granule neurons.     

Antiapoptotic effects of roscovitine in cerebellar granule cells deprived of serum and potassium: a cell cycle-related mechanism

Neuronal apoptosis may be partly due to inappropriate control of the cell cycle. We used serum deprivation as stimulus and reduced potassium from 25 to 5mM (S/K deprivation), which induces apoptosis in cerebellar granule neurons (CGNs), to evaluate the direct correlation between re-entry in the cell cycle and apoptosis. Roscovitine (10 microM), an antitumoral drug that inhibits cyclin-dependent kinase 1 (cdk1), cdk2 and cdk5, showed a significant neuroprotective effect on CGNs deprived of S/K. S/K deprivation induced the expression of cell cycle proteins such as cyclin E, cyclin A, cdk2, cdk4 and E2F-1. It also caused CGNs to enter the S phase of the cell cycle, measured by a significant incorporation of BrdU (30% increase over control cells), which was reduced in the presence of roscovitine (10 microM). On the other hand, roscovitine modified the expression of cytochrome c (Cyt c), Bcl-2 and Bax, which are involved in the apoptotic intrinsic pathway induced by S/K deprivation. We suggest that the antiapoptotic effects of roscovitine on CGNs are due to its anti-proliferative efficacy and to an action on the mitochondrial apoptotic mechanism.     

The role of induction and inhibition of nitric oxide synthesis in regulation of blood neutrophils cell death during oxidative imbalance

Modeling of oxidative stress in vitro with 5 mM H₂O₂ has demonstrated a protective role of nitric oxide in realization of constitutional blood neutrophil cell death. The NO synthase inductor, L-arginine, and the inhibitor of nitric oxide synthesis, L-NAME, influenced the amount of annexin-positive cells, the content of Bax protein, reactive oxygen species, cyclic nucleotides, and calcium homeostasis in neutrophils under conditions realizing programmed death during oxidative stress in vitro and under acute inflammation. During oxidative stress L-arginine normalized an increased intracellular Ca²⁺ level and the cAMP/cGMP ratio by increasing the cGìP level, stabilized metabolism and prolonged neutrophil lifetime. During acute inflammation NO induction was insufficient for limitation of Ca²⁺ release into cytosol and for onset of the apoptotic effect; blockade of NO synthesis deteriorated this situation by activating neutrophil apoptosis due to the sharp increase in the Ca²⁺ content and reduction of cytosolic cyclic nucleotides. The protective effect of NO on neutrophil cell death during oxidative imbalance was not associated with regulation of the proapoptotic protein Bax.     

Mitochondrial calcium uniporter protein MCU is involved in oxidative stress-induced cell death

Mitochondrial calcium uniporter (MCU) is a conserved Ca²⁺ transporter at mitochondrial in eukaryotic cells. However, the role of MCU protein in oxidative stressinduced cell death remains unclear. Here, we showed that ectopically expressed MCU is mitochondrial localized in both HeLa and primary cerebellar granule neurons (CGNs). Knockdown of endogenous MCU decreases mitochondrial Ca²⁺ uptake following histamine stimulation and attenuates cell death induced by oxidative stress in both HeLa cells and CGNs. We also found MCU interacts with VDAC1 and mediates VDAC1 overexpression-induced cell death in CGNs. This finding demonstrates that MCU-VDAC1 complex regulates mitochondrial Ca²⁺ uptake and oxidative stress-induced apoptosis, which might represent therapeutic targets for oxidative stress related diseases.     

Protective effect of a novel cystine C(60) derivative on hydrogen peroxide-induced apoptosis in rat pheochromocytoma PC12 cells

Oxidative stress has been considered as a major cause of cellular injuries in a variety of clinical abnormalities, especially neural diseases. One of the effective ways to prevent the reactive oxygen species (ROS) mediated cellular injury is dietary or pharmaceutical augmentation of free radical scavengers. In the present study, we describe the synthesis and characterization of a novel cystine C(60) derivative (CFD). The compound was analyzed by FT-IR, (1)H NMR, (13)C NMR, LC-MS and elemental analysis. It contains five cystine moieties per C(60) molecule. This water-soluble amino-fullerene derivative was able to scavenge both superoxide and hydroxyl radical with biocompatibility. We investigated its potential protective effects on hydrogen peroxide-induced oxidative stress and apoptotic death in cultured rat pheochromocytoma (PC12) cells. Cells treated with hydrogen peroxide underwent cytotoxicity and apoptotic death determined by MTT assay, flow cytometry analysis, PI/Hoechst 33342 staining and glutathione peroxidase assay. The CFD was able to reduce the accumulation of reactive oxygen species and cellular damage caused by hydrogen peroxide in PC12 cells. RF assay demonstrated that CFD could penetrate through the cell membrane and it has played its distinguished role in protecting PC12 cells against hydrogen peroxide-induced cytotoxicity. The results suggest that CFD has the potential to prevent oxidative stress-induced cell death without evident toxicity. Hence, we can hypothesize that the protective effect of CFD on hydrogen peroxide-induced apoptosis is related to its scavenger activity.     

The octadecaneuropeptide ODN prevents 6‐hydroxydopamine‐induced apoptosis of cerebellar granule neurons through a PKC‐MAPK‐dependent pathway

Oxidative stress, induced by various neurodegenerative diseases, initiates a cascade of events leading to apoptosis, and thus plays a critical role in neuronal injury. In this study, we have investigated the potential neuroprotective effect of the octadecaneuropeptide (ODN) on 6‐hydroxydopamine (6‐OHDA)‐induced oxidative stress and apoptosis in cerebellar granule neurons (CGN). ODN, which is produced by astrocytes, is an endogenous ligand for both central‐type benzodiazepine receptors (CBR) and a metabotropic receptor. Incubation of neurons with subnanomolar concentrations of ODN (10^?18 to 10^?12 M) inhibited 6‐OHDA‐evoked cell death in a concentration‐dependent manner. The effect of ODN on neuronal survival was abrogated by the metabotropic receptor antagonist, cyclo_1–8[DLeu⁵]OP, but not by a CBR antagonist. ODN stimulated polyphosphoinositide turnover and ERK phosphorylation in CGN. The protective effect of ODN against 6‐OHDA toxicity involved the phospholipase C/ERK MAPK transduction cascade. 6‐OHDA treatment induced an accumulation of reactive oxygen species, an increase of the expression of the pro‐apoptotic gene Bax, a drop of the mitochondrial membrane potential and a stimulation of caspase‐3 activity. Exposure of 6‐OHDA‐treated cells to ODN blocked all the deleterious effects of the toxin. Taken together, these data demonstrate for the first time that ODN is a neuroprotective agent that prevents 6‐OHDA‐induced oxidative stress and apoptotic cell death.     

N-t-Butyl hydroxylamine regulates heat shock-induced apoptosis in U937 cells

Heat shock may increase oxidative stress due to increased production of reactive oxygen species and/or the promotion of cellular oxidation events. Therefore, compounds that scavenge reactive oxygen species may regulate heat shock-induced cell death. Recently, it has been shown that the decomposition product of the spin-trapping agent alpha-phenyl-N-t-butylnitrone, N-t-butyl hydroxylamine (NtBHA), mimics alpha-phenyl-N-t-butylnitrone and is much more potent in delaying reactive oxygen species-associated senescence. We investigated the protective role of NtBHA against heat shock-induced apoptosis in U937 cells. Upon exposure to heat shock, there was a distinct difference between the untreated cells and the cells pre-treated with 0.1 mM NtBHA for 2 h in regard to apoptotic parameters, cellular redox status, and mitochondrial function. Upon exposure to heat shock, NtBHA pre-treated cells showed significant inhibition of apoptotic features such as activation of caspase-3, up-regulation of Bax, and down-regulation of Bcl-2 compared to untreated cells. This study indicates that NtBHA may play an important role in regulating the apoptosis induced by heat shock, presumably through scavenging of reactive oxygen species.     

p38 mitogen-activated protein kinase regulates low potassium-induced c-Jun phosphorylation and apoptosis in cultured cerebellar granule neurons

Cultured rat cerebellar granule neurons are widely used as a model system for studying neuronal apoptosis. After maturation by culturing in medium containing 26 mm potassium (high K(+)), changing to medium containing 5 mm potassium (low K(+); LK) rapidly induces neuronal apoptosis. Then over 50% of granule cells die within 24 h. However, the molecular mechanisms by which the LK-induced apoptosis occurs in cultured cerebellar granule cells remain unclear. In the present study, we found that p38 MAP kinase (p38) was an important factor for LK-induced apoptosis. Three hours after changing to LK medium, p38 was markedly activated. In addition, SB203580, a specific inhibitor of p38, strongly inhibited the phosphorylation and expression of c-Jun in LK-induced apoptosis of cultured cerebellar granule cells. In vitro kinase assay using glutathione S-transferase-c-Jun as a substrate showed that p38 directly phosphorylated c-Jun. Furthermore, in the presence of SB203580, about 80% of neurons survived. These results indicate that p38 regulates LK-induced apoptosis of cerebellar granule neurons.     

A New Ibuprofen Derivative Inhibits Platelet Aggregation and ROS Mediated Platelet Apoptosis

Thrombocytopenia is a serious issue connected with the pathogenesis of several human diseases including chronic inflammation, arthritis, Alzheimer''s disease, cardiovascular diseases (CVDs) and other oxidative stress-associated pathologies. The indiscriminate use of antibiotics and other biological drugs are reported to result in thrombocytopenia, which is often neglected during the treatment regime. In addition, augmented oxidative stress induced by drugs and pathological conditions has also been shown to induce thrombocytopenia, which seems to be the most obvious consequence of elevated rate of platelet apoptosis. Thus, blocking oxidative stress-induced platelet apoptosis would be of prime importance in order to negotiate thrombocytopenia and associated human pathologies. The current study presents the synthesis and platelet protective nature of novel ibuprofen derivatives. The potent anti-oxidant ibuprofen derivative 4f was selected for the study and the platelet protective efficacy and platelet aggregation inhibitory property has been demonstrated. The compound 4f dose dependently mitigates the oxidative stress-induced platelet apoptosis in both platelet rich plasma and washed platelets. The platelet protective nature of compound 4f was determined by assessing various apoptotic markers such as ROS generation, cytosolic Ca²⁺ levels, PS externalization, cytochrome C translocation, Caspase activation, mitochondrial membrane depolarization, cytotoxicity, LDH leakage and tyrosine phosphorylation of cytosolic proteins. Furthermore, compound 4f dose dependently ameliorated agonist induced platelet aggregation. Therefore, compound 4f can be estimated as a potential candidate in the treatment regime of pathological disorders associated with platelet activation and apoptosis. In addition, compound 4f can be used as an auxiliary therapeutic agent in pathologies associated with thrombocytopenia.     

Oxalomalate, a competitive inhibitor of NADP+-dependent isocitrate dehydrogenase, regulates heat shock-induced apoptosis

Heat shock may increase oxidative stress due to increased production of reactive oxygen species and/or the promotion of cellular oxidation events. Recently, we demonstrated that the control of cytosolic and mitochondrial redox balance and the cellular defense against oxidative damage is one of the primary functions of NADP(+)-dependent isocitrate dehydrogenase (ICDH) by supplying NADPH for antioxidant systems. The protective role of ICDH against heat shock-induced apoptosis in U937 cells was investigated in the control and the cells pre-treated with oxalomalate, a competitive inhibitor of ICDH. Upon exposure to heat shock, there was a distinct difference between the control cells and the cells pre-treated with 3mM oxalomalate for 3h in regard to apoptotic parameters, cellular redox status, and mitochondrial function. The oxalomalate pre-treated cells showed significant enhancement of apoptotic features such as activation of caspase-3, up-regulation of Bax, and down-regulation of Bcl-2 compared to the control cells upon exposure to heat shock. This study indicates that ICDH may play an important role in regulating the apoptosis induced by heat shock presumably through maintaining the cellular redox status.     

Nitric oxide induces oxidative stress and apoptosis in neuronal cells

Within the central nervous system and under normal conditions, nitric oxide (NO) is an important physiological signaling molecule. When produced in large excess, NO also displays neurotoxicity. In our previous report, we have demonstrated that the exposure of neuronal cells to NO donors induced apoptotic cell death, while pretreatment with free radical scavengers L-ascorbic acid 2-[3, 4-dihydro-2,5,7,8-tetramethyl-2-(4,8, 12-trimethyltridecyl)-2H-1-benzopyran-6-yl-hydrogen phosphate] potassium salt (EPC-K1) or superoxide dismutase attenuated apoptosis effectively, suggesting that reactive oxygen species (ROS) may be involved in the cascade of events leading to apoptosis. In the present investigation, we directly studied the kinetic generation of ROS in NO-treated neuronal cells by flow cytometry using 2', 7'-dichloro-fluorescein diacetate and dihydrorhodamine 123 as redox-sensitive fluorescence probes. The results indicated that exposure of cerebellar granule cells to the NO donor S-nitroso-N-acetylpenicillamine (SNAP) induced oxidative stress, which was characterized by the accumulation of cytosolic and mitochondrial ROS, the increase in the extracellular hydrogen peroxide level, and the formation of lipid peroxidation products. SNAP treatment also induced apoptotic cell death as confirmed by the formation of cytosolic mono- and oligonucleosomes. Pretreating cells with the novel antioxidant EPC-K1 effectively prevented oxidative stress induced by SNAP, and attenuated cells from apoptosis.     

Reactive oxygen species are related to ionic fluxes and volume decrease in apoptotic cerebellar granule neurons: role of NOX enzymes

Reactive oxygen species (ROS) are produced early during apoptosis of cerebellar granule neurons induced by low potassium (K5) and staurosporine (Sts). In addition, K5 and Sts activate NADPH oxidases (NOX). Recently, we described that K5 and Sts induce apoptotic volume decrease (AVD) at a time when ROS generation and NOX activity occur. In the present study, we evaluated the relationship between ROS generation and ionic fluxes during AVD. Here, we showed that K5- and Sts-induced AVD was inhibited by antioxidants and that direct ROS production induced AVD. Moreover, NOX inhibitors eliminated AVD induced by both K5 and Sts. Sts, but not K5, failed to induce AVD in cerebellar granule neurons from NOX2 knockout mice. These findings suggest that K5- and Sts-induced AVD is largely mediated by ROS produced by NOX. On the other hand, we also found that the blockage of ionic fluxes involved in AVD inhibited both ROS generation and NOX activity. These findings suggest that ROS generation and NOX activity are involved in ionic fluxes activation, which in turn could maintain ROS generation by activating NOX, leading to a self-amplifying cycle.     

Oxidative stress-induced apoptosis in neurons correlates with mitochondrial DNA base excision repair pathway imbalance

Neurodegeneration can occur as a result of endogenous oxidative stress. Primary cerebellar granule cells were used in this study to determine if mitochondrial DNA (mtDNA) repair deficiencies correlate with oxidative stress-induced apoptosis in neuronal cells. Granule cells exhibited a significantly higher intracellular oxidative state compared with primary astrocytes as well as increases in reductants, such as glutathione, and redox sensitive signaling molecules, such as AP endonuclease/redox effector factor-1. Cerebellar granule cultures also exhibited an increased susceptibility to exogenous oxidative stress. Menadione (50 μM) produced twice as many lesions in granule cell mtDNA compared with astrocytes, and granule cell mtDNA repair was significantly less efficient. A decreased capacity to repair oxidative mtDNA damage correlates strongly with mitochondrial initiated apoptosis in these neuronal cultures. Interestingly, the mitochondrial activities of initiators for base excision repair (BER), the bifunctional glycosylase/AP lyases as well as AP endonuclease, were significantly higher in cerebellar granule cells compared with astrocytes. The increased mitochondrial AP endonuclease activity in combination with decreased polymerase γ activity may cause an imbalance in oxidative BER leading to an increased production and persistence of mtDNA damage in neurons when treated with menadione. This study provides evidence linking neuronal mtDNA repair capacity with oxidative stress-related neurodegeneration.