Staurosporine-induced Neuronal Apoptosis Research Articles

Formaldehyde suppresses neuronal apoptosis via inhibition of outward K(+) currents in rat hippocampus.

Formaldehyde (FA) is widely present in the environment, and is also a mammalian metabolite. However, its biological role has not been well understood. Here, we show that FA plays an anti-apoptotic role in cultured hippocampal neurons: FA suppressed staurosporine-induced neuronal apoptosis and inhibited the activity of apoptosis-associated caspase-3/7 proteases in a concentration-dependent manner. Moreover, FA suppressed outward K(+) currents and attenuated the enhanced IK currents that are associated with neuronal apoptosis. As an increase in outward K(+) currents is critical for cell apoptosis, our results suggest that FA exerts its anti-apoptotic effects on neuronal cells, probably through its inhibitory effect on the outward K(+) currents.

Neuroprotective function of thymosin-β and its derivative peptides on the programmed cell death of chick and rat neurons

Thymosin-βs (Tβs) are small polypeptides with various biological functions, including cytoskeletal remodeling, angiogenesis, cellular migration, wound healing, and regulation of apoptosis. Recently, we found that Tβ is involved in the control of programmed cell death (PCD) of motoneurons (MNs) in chick embryo, and that the anti-apoptotic action of Tβ is independent of its actin-sequestering activity. In this study, we observed that a synthetic peptide derived from Tβ suppressed staurosporine-induced neuronal apoptosis in vitro, and PCD of chick or rat MNs in vivo. Furthermore, inhibition of Tβ4 in chick embryo by antibody significantly augmented the PCD of MNs, suggesting that secreted form of Tβ is physiological regulator of PCD. Based on these findings, we propose that extracellularly secreted Tβ is involved in the control of PCD of neurons during development, and Tβ-derived peptides could be useful for the anti-apoptotic therapy of neuropathologies related to neuronal apoptosis.

Meso-Dihydroguaiaretic acid attenuates the neurotoxic effect of staurosporine in primary rat cortical cultures

The effect of meso-dihydroguaiaretic acid (MDGA) on the staurosporine-induced neuronal apoptosis and its potential mechanism were investigated using primary cultures of rat cortical cells as an assay system. Treatment of MDGA at the concentrations of 0.1, 1.0 and 10 μM significantly protected neuronal cells against Staurosporine-induced apoptosis. The neuroprotective activity of MDGA was the most potent at the concentration of 1.0 μM and was not increased at higher concentration. MDGA reduced apoptotic characteristics induced by STS; MDGA reduced the condensed nuclei in staurosporine-injured rat cortical cells. MDGA diminished the calcium influx that accompanies the staurosporine-induced apoptosis, and inhibited the subsequent overproduction of reactive oxygen species and peroxide to the level of control cells. It also preserved cellular activity of superoxide dismutase, an antioxidative enzyme reduced by staurosporine insult. In addition, MDGA significantly inhibited caspase-3/7 activation and cytochrome c release. Taken together, these results suggested that MDGA protected neuronal cells against staurosporine-induced apoptosis through the inhibition of Ca 2+ influx, cellular oxidation, cytochrome c release and caspase-3/7 activation.

Wild-type PINK1 Prevents Basal and Induced Neuronal Apoptosis, a Protective Effect Abrogated by Parkinson Disease-related Mutations

Mutations in the PTEN-induced kinase 1 (PINK1) gene have recently been implicated in autosomal recessive early onset Parkinson Disease (1, 2). To investigate the role of PINK1 in neurodegeneration, we designed human and murine neuronal cell lines expressing either wild-type PINK1 or PINK1 bearing a mutation associated with Parkinson Disease. We show that under basal and staurosporine-induced conditions, the number of terminal deoxynucleotidyltransferase-mediated dUTP nick end labeling (TUNEL)-positive cells was lower in wild-type PINK1 expressing SH-SY5Y cells than in mock-transfected cells. This phenotype was due to a PINK1-mediated reduction in cytochrome c release from mitochondria, which prevents subsequent caspase-3 activation. We show that overexpression of wild-type PINK1 strongly reduced both basal and staurosporine-induced caspase 3 activity. Overexpression of wild-type PINK1 also reduced the levels of cleaved caspase-9, caspase-3, caspase-7, and activated poly(ADP-ribose) polymerase under both basal and staurosporine-induced conditions. In contrast, Parkinson disease-related mutations and a kinase-inactive mutation in PINK1 abrogated the protective effect of PINK1. Together, these results suggest that PINK1 reduces the basal neuronal pro-apoptotic activity and protects neurons from staurosporine-induced apoptosis. Loss of this protective function may therefore underlie the degeneration of nigral dopaminergic neurons in patients with PINK1 mutations.

Linking Alterations in Tau Phosphorylation and Cleavage during Neuronal Apoptosis

Neurofibrillary tangles (NFTs) are classic lesions of Alzheimer's disease. NFTs are bundles of abnormally phosphorylated tau, the paired helical filaments. The initiating mechanisms of NFTs and their role in neuronal loss are still unknown. Accumulating evidence supports a role for the activation of proteolytic enzymes, caspases, in neuronal death observed in brains of patients with Alzheimer's disease. Alterations in tau phosphorylation and tau cleavage by caspases have been previously reported in neuronal apoptosis. However, the links between the alterations in tau phosphorylation and its proteolytic cleavage have not yet been documented. Here, we show that, during staurosporine-induced neuronal apoptosis, tau first undergoes transient hyperphosphorylation, which is followed by dephosphorylation and cleavage. This cleavage generated a 10-kDa fragment in addition to the 17- and 50-kDa tau fragments previously reported. Prior tau dephosphorylation by a glycogen synthase kinase-3beta inhibitor, lithium, enhanced tau cleavage and sensitized neurons to staurosporine-induced apoptosis. Caspase inhibition prevented tau cleavage without reversing changes in tau phosphorylation linked to apoptosis. Furthermore, the microtubule depolymerizing agent, colchicine, induced tau dephosphorylation and caspase-independent tau cleavage and degradation. Both phenomena were blocked by inhibiting protein phosphatase 2A (PP2A) by okadaic acid. These experiments indicate that tau dephosphorylation precedes and is required for its cleavage and degradation. We propose that the absence of cleavage and degradation of hyperphosphorylated tau (due to PP2A inhibition) may lead to its accumulation in degenerating neurons. This mechanism may contribute to the aggregation of hyperphosphorylated tau into paired helical filaments in Alzheimer's disease where reduced PP2A activity has been reported.

Homer–PIKE complex: a novel link between mGluRI and PI 3-kinase

Metabotropic glutamate (mGlu) receptors, a family of G-protein-coupled receptors, are thought to signal through the phospholipase and inositol (1,4,5)-trisphosphate receptor system, or through the adenylyl cyclase and protein kinase C system. Rong et al. have recently identified a new phosphoinositide (PI) 3-kinase enhancer (PIKE-L) that links group I mGlu receptors (mGluRI) to PI 3-kinase through Homer proteins, adaptors that bind mGluRI. mGluRI agonists enhanced mGluRI-Homer-PIKE-L complex formation, leading to activation of PI 3-kinase and inhibition of staurosporine-induced neuronal apoptosis. These results reveal a novel anti-apoptotic signaling mechanism that involves formation of an mGluRI signaling complex.

Huperzine A attenuates the neurotoxic effect of staurosporine in primary rat cortical neurons

The effects of huperzine A (HupA) on staurosporine-induced neuronal apoptosis and potential mechanisms were investigated in primary cultured rat cortical neurons. Pretreatment of the cells with HupA (0.1–100 μM) 2 h prior to 0.5 μM staurosporine exposure for 24 h, markedly elevated cell survival. Incubation with HupA at higher dose (1 μM) also reduced the DNA fragmentation caused by staurosporine. This dose of HupA also reduced the upregulation of the pro-apoptotic gene, bax, downregulation of the anti-apoptotic gene, bcl-2, and antagonized the decrease in immunoreactive caspase-3 proenzyme. These results demonstrated that HupA protect neurons against staurosporine-induced apoptosis via regulating the apoptotic related genes and caspase-3 proenzyme.

Selective protection by phosphatidic acid against staurosporine-induced neuronal apoptosis.

Phosphatidic acid, the main product of lipid breakdown through phospholipase D activation, has been implicated in important signal transduction pathways able to influence cell fate in many ways. The purpose of this work was to determine possible effects of phosphatidic acid on neuronal cell death pathways. Here we used cerebellar granular cell cultures and cell death was triggered with either staurosporine or H(2)O(2). Cell viability was quantified by spectrophotometry, using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT) test. Staurosporine (1-3 microM) or H(2)O(2) (50-800 microM) induced cell death in a dose-dependent manner. Using fluorescent staining (propidium iodide or annexin V-Cy3/6-carboxyfluorescein) we showed that cell death was mostly apoptotic in staurosporine treated cells and mostly non-apoptotic (necrotic) in H(2)O(2) treated cells. Phosphatidic acid was able to increase cell viability in staurosporine-, but not in H(2)O(2) - treated cells. We therefore conclude that phosphatidic acid has neuroprotective potential in neurons exposed to stimuli that trigger apoptosis.

Preconditioning-induced neuroprotection is mediated by reactive oxygen species and activation of the transcription factor nuclear factor-kappaB.

Preconditioning by a sublethal stimulus induces tolerance to a subsequent, otherwise lethal insult and it has been suggested that reactive oxygen species (ROS) are involved in this phenomenon. In the present study, we determined whether preconditioning activates the transcription factor nuclear factor-kappaB (NF-kappaB) and how this activation contributes to preconditioning-induced inhibition of neuronal apoptosis. Preconditioning was performed by incubating mixed cultures of neurons and astrocytes from neonatal rat hippocampus with xanthine/xanthine oxidase or FeSO4 for 15 min followed by 24 h of recovery which protected the neurons against subsequent staurosporine-induced (200 nM, 24 h) apoptosis. The cellular ROS content increased during preconditioning, but returned to basal levels after removal of xanthine/xanthine oxidase or FeSO4. We detected a transient activation of NF-kappaB 4 h after preconditioning as shown by immunocytochemistry, by a decrease in the protein level of IkappaBalpha as well as by electrophoretic mobility shift assay. Preconditioning-mediated neuroprotection was abolished by antioxidants, inhibitors of NF-kappaB activation and cycloheximide suggesting the involvement of ROS, an activation of NF-kappaB and de novo protein synthesis in preconditioning-mediated rescue pathways. Furthermore, preconditioning increased the protein level of Mn-superoxide dismutase which could be blocked by antioxidants, cycloheximide and kappaB decoy DNA. Our data suggest that inhibition of staurosporine-induced neuronal apoptosis by preconditioning with xanthine/xanthine oxidase or FeSO4 involves an activation of NF-kappaB and an increase in the protein level of Mn-superoxide dismutase.

Enalapril and moexipril protect from free radical-induced neuronal damage in vitro and reduce ischemic brain injury in mice and rats

Angiotensin-converting enzyme inhibitors have been demonstrated to protect spontaneously hypertensive rats from cerebral ischemia. The present study investigated the protective effect of enalapril and moexipril in models of permanent focal cerebral ischemia in normotensive mice and rats. To elucidate the mechanism of neuroprotection the influence of these angiotensin-converting enzyme inhibitors on glutamate-, staurosporine- or Fe 2+/3+-induced generation of reactive oxygen species and neuronal cell death in primary cultures from chick embryo telencephalons was studied. Treatment with moexipril or enalapril dose-dependently reduced the percentage of damaged neurons, as well as mitochondrial reactive oxygen species generation induced by glutamate, staurosporine or Fe 2+/3+. Furthermore, moexipril and enalapril attenuated staurosporine-induced neuronal apoptosis as determined by nuclear staining with Hoechst 33258. In mice, 1 h pretreatment with enalapril (0.03 mg/kg) or moexipril (0.3 mg/kg) significantly reduced brain damage after focal ischemia as compared to control animals. Additionally, moexipril (0.01 mg/kg) was able to reduce the infarct volume in the rat model after focal cerebral ischemia. The results of the present study indicate that the angiotensin-converting enzyme inhibitors enalapril and moexipril promote neuronal survival due to radical scavenging properties.

Inhibition of serum deprivation- and staurosporine-induced neuronal apoptosis by Ginkgo biloba extract and some of its constituents

Previous studies have already demonstrated that some constituents of an extract of Ginkgo biloba (EGb), such as ginkgolide B and bilobalide, protect cultured neurons from hypoxia- and glutamate-induced damage. This prompted us to investigate whether they were also able to inhibit neuronal apoptosis. We induced apoptosis in cultured chick embryonic neurons as well as in mixed cultures of neurons and astrocytes from neonatal rat hippocampus by serum deprivation and staurosporine. The increase in the percentage of apoptotic chick neurons from 12% in controls to 30% after 24 h of serum deprivation was reduced to control level by EGb (10 mg/l), ginkgolide B (10 μM), ginkgolide J (100 μM) and bilobalide (1 μM). After treatment with staurosporine (200 nM) for 24 h we observed 74% apoptotic chick neurons. This percentage of apoptotic neurons was reduced to 24%, 62% and 31% in the presence of EGb (100 mg/l), ginkgolide J (100 μM) and ginkgolide B (10 μM), respectively. Bilobalide (10 μM) decreased apoptotic damage induced by staurosporine treatment for 12 h nearly to the control level. In mixed neuronal/glial cultures, the extract of EGb (100 mg/l) and bilobalide (100 μM) rescued rat neurons from apoptosis caused by serum deprivation, whereas, bilobalide (100 μM) and ginkgolide B (100 μM) reduced staurosporine-induced apoptotic damage. Ginkgolide A revealed no anti-apoptotic effect in either serum-deprived or staurosporine-treated neurons. Our results suggest that EGb and some of its constituents possess anti-apoptotic capacity and that bilobalide is the most potent constituent.

Inhibition of neuronal apoptosis by a metalloporphyrin superoxide dismutase mimic.

The objective of this study was to determine whether free radicals play a pathogenic role in neuronal apoptosis. The ability of Mn(III) tetrakis(benzoic acid) porphyrin (MnTBAP), a superoxide dismutase mimic, to inhibit staurosporine-induced neuronal apoptosis was tested in mixed cerebrocortical cultures. Staurosporine produced concentration-dependent cell death that was markedly inhibited by MnTBAP. Immunocytochemical analyses of cultures for neuron- and astrocyte-specific markers revealed that high concentrations of staurosporine induced the death of both neurons and astrocytes; both cell types were protected by MnTBAP. A less active congener of MnTBAP failed to protect cells against staurosporine-induced apoptosis. MnTBAP also protected cortical cultures against ceramide-induced apoptosis. These results support a role for oxidative stress in neuronal apoptosis.

Ca2+ and reactive oxygen species in staurosporine-induced neuronal apoptosis.

Staurosporine (0.03-0.5 microM) induced a dose-dependent, apoptotic degeneration in cultured rat hippocampal neurons that was sensitive to 24-h pretreatments with the protein synthesis inhibitor cycloheximide (1 microM) or the cell cycle inhibitor mimosine (100 microM). To investigate the role of Ca2+ and reactive oxygen species in staurosporine-induced neuronal apoptosis, we overexpressed calbindin D28K, a Ca2+ binding protein, and Cu/ Zn superoxide dismutase, an antioxidative enzyme, in the hippocampal neurons using adenovirus-mediated gene transfer. Infection of the cultures with the recombinant adenoviruses (100 multiplicity of infection) resulted in a stable expression of the respective proteins assessed 48 h later. Overexpression of both calbindin D28K and Cu/Zn superoxide dismutase significantly reduced staurosporine neurotoxicity compared with control cultures infected with a beta-galactosidase overexpressing adenovirus. Staurosporine-induced neuronal apoptosis was also significantly reduced when the culture medium was supplemented with 10 or 30 mM K+, suggesting that Ca2+ influx via voltage-sensitive Ca2+ channels reduces this apoptotic cell death. In contrast, neither the glutamate receptor agonist NMDA (1-10 microM) nor the NMDA receptor antagonist dizocilpine (MK-801; 1 microM) was able to reduce staurosporine neurotoxicity. Cultures treated with the antioxidants U-74500A (1-10 microM) and N-acetylcysteine (100 microM) also demonstrated reduced staurosporine neurotoxicity. These results suggest a fundamental role for both Ca2+ and reactive oxygen species in staurosprine-induced neuronal apoptosis.

Apoptosis induced in neuronal cultures by either the phosphatase inhibitor okadaic acid or the kinase inhibitor staurosporine is attenuated by isoquinolinesulfonamides H-7, H-8, and H-9.

Protein phosphorylation is kept in balance by an orchestrated action of kinases and phosphatases; when this balance is lost, neuronal apoptosis may occur. Okadaic acid (OKA), a marine toxin that inhibits specifically protein phosphatases 1 and 2A (EC 3.1.3.16), and staurosporine, an inhibitor of protein kinase C (PKC; EC 2.7.1.37), induced apoptosis in primary cultures of rat cerebellar granule neurons. We assayed apoptosis by the DNA gel electrophoresis, by the in situ TUNEL assay, and by morphological appearance following propidium iodide staining. Cell viability was assessed by the Trypan blue assay. Both OKA- and staurosporine-induced neuronal apoptosis were prevented by a macromolecular synthesis inhibitor actinomycin D and by a group of isoquinolinesulfonamide kinase inhibitors (H-7, 1-[5-isoquinolinesulfonyl]-2-methylpiperazine; H-8, N-¿2-[methylamino]ethyl¿-5-isoquinolinesulfonamide; H-9, N-(2-aminoethyl)-5-isoquinolinesulfonamide, but not by inhibitors of PKC, cyclic-GMP- and cyclic-AMP-dependent kinases, calcium/calmodulin-dependent kinases, tyrosine kinases, or by antioxidants. We postulate that a common mechanism, possibly an increased protein phosphorylation, is responsible for apoptosis triggered by an inhibition of phosphatases 1 and 2A and PKC. Elucidating the isoquinolinesulfonamide-sensitive mechanism may help us find new therapies for neurodegenerative diseases that involve apoptosis.

Staurosporine-Induced Neuronal Apoptosis

Staurosporine, a nonselective protein kinase inhibitor, has been shown to induce apoptosis in several different nonneuronal cell types. We tested the hypothesis that staurosporine would also induce apoptosis in central neurons. Exposure of murine cortical cell cultures to 30-100 nM staurosporine induced concentration-dependent selective neuronal degeneration over the following day; at higher concentrations, staurosporine damaged glial cells as well. Staurosporine-induced neuronal death was accompanied by cell body shrinkage, chromatin condensation, and DNA laddering. In contrast, NMDA-induced neuronal death was accompanied by acute cell body swelling without DNA laddering. Staurosporine-induced neuronal death, unlike excitotoxic death, was markedly attenuated by the protein synthesis inhibitor cycloheximide; this protective effect was not reversed by a glutathione synthesis inhibitor, buthionine sulfoximine. Interestingly, the glial cell death induced by 1 μM staurosporine was markedly potentiated by cycloheximide. Staurosporine-induced neuronal death was not accompanied by an increase in intracellular free Ca2+ and was attenuated by 30 mM K+; this protective effect of high K+ was blocked by nimodipine or Co2+. Present data suggest that staurosporine can induce apoptosis in cultured cortical neurons and that this apoptosis can be blocked by raising intracellular Ca2+ or by blocking protein synthesis. Staurosporine exposure may be useful as a model for studying central neuronal apoptosis in vitro.