Cell Death In Cortical Neurons Research Articles

Murine Neural Stem/Progenitor Cells Protect Neurons against Ischemia by HIF-1α–Regulated VEGF Signaling

Focal cerebral ischemia following middle cerebral artery occlusion (MCAO) stimulates a robust cytogenic response from the adult subventricular zone (SVZ) that includes massive proliferation of neural stem/progenitor cells (NSPCs) and cellular migration into the injury area. To begin to explore beneficial roles of NSPCs in this response, we investigated the ability of embryonic and postnatal NSPCs to promote neuronal survival under conditions of in vivo and in vitro ischemia. Intracerebral transplantation of NSPCs attenuated neuronal apoptosis in response to focal ischemia induced by transient MCAO, and prevented neuronal cell death of cortical neurons in response to oxygen-glucose deprivation (OGD) in culture. NSPC-mediated neuroprotection was blocked by the pharmacological inhibitors of vascular endothelial growth factor (VEGF), SU1498 and Flt-1Fc. Embryonic and postnatal NSPCs were both intrinsically resistant to brief OGD exposure, and constitutively expressed both hypoxia-inducible factor 1α (HIF-1α) transcription factor and its downstream target, VEGF. Genomic deletion of HIF-1α by Cre-mediated excision of exon 2 in NSPC cultures resulted in >50% reduction of VEGF production and ablation of NSPC-mediated neuroprotection. These findings indicate that NSPCs promote neuronal survival under ischemic conditions via HIF-1α-VEGF signaling pathways and support a role for NSPCs in promotion of neuronal survival following stroke.

Neurodegenerative, with expression ATF-2 by P38 in cortical neurons

DNA damage, as an important initiator of neuronal cell death, has been implicated in numerous neurodegenerative conditions. We previously delineated several pathways that control embryonic cortical neuronal cell death evoked by the DNA-damaging agent, camptothecin. The topisomerase-1 inhibitor, camptothecin, has been shown to induce cortical neuronal cell death in a reproducible and synchronistic manner. Primary embryonic neuronal cell culture cortical neurons were prepared. In the study, the survival % of neurons in camptothecin P38 group, after 6 hours (85%), 24 hours (64%) and 48 hours (50%), compared to camptothecin ATF-2 and P38 group after 4 hours (97 and 95%), have been significantly lower, and the expression % of neurons in camptothecin P38 group , after 6 hours (20%), 24 hours (40%) and 48 hours (55%), compared to camptothecin ATF-2 and P38 group after 4 hours (5 and 3%) have been significant lower (p<0·05). The expression % of neurons in camptothecin P38 group, after 24 hours (40%), compared to camptothecin ATF-2 group after 24hours (30%), have been significant lower (p<0·05). This study revealed that camptothecin induces P38 expression and P38 in embryonic cortical neurons to determine the importance of the P38 pathway in neuronal death following DNA damage, and P38 is induce phosphorylation of ATF-2 in embryonic cortical neurons following DNA damage.

Role of the sodium hydrogen exchanger in maitotoxin-induced cell death in cultured rat cortical neurons

Maitotoxin (MTX) is one of the most potent toxins known to date. It causes massive calcium (Ca 2+) influx and necrotic cell death in various tissues. However, the exact mechanism(s) underlying its cellular toxicity is not fully understood. In the present study, the role of the sodium hydrogen exchanger (NHE) in MTX-induced increases in intracellular Ca 2+ and subsequent cell death were investigated in cultured rat cortical neurons. Intracellular Ca 2+ concentrations ([Ca 2+] i) were measured fluorimetrically using FURA-2 as the fluorescence indicator. Cell death was measured with the alamarBlue™ cell viability assay and the vital dye ethidium bromide (EB) uptake assay. Results showed that MTX increased, in a concentration dependent manner, both [Ca 2+] i and cell death in cortical neurons. Decreasing the pH of the treatment medium from 7.5 to 6.0 diminished MTX-induced cell death. The protection offered by lowering extracellular pH was not due to MTX degradation, because it was still effective even if the cells were treated with MTX in normal pH and then switched to a lower pH. Pretreatment of cells with the specific NHE inhibitor, 5-( N-ethyl- N-isopropyl)-amiloride (EIPA), prevented MTX-induced increases in [Ca 2+] i, as well as cell death in a concentration dependent manner. Furthermore, knockdown of NHE1 by SiRNA transfection suppressed MTX-induced cell death in human embryonic kidney (HEK) cells. Together, these results suggest that NHE1 plays a major role in MTX-induced neurotoxicity.

Brain-derived Neurotrophic Factor Activates ERK5 in Cortical Neurons via a Rap1-MEKK2 Signaling Cascade

The extracellular signal-regulated kinase 5 (ERK5) is activated in neurons of the central nervous system by neurotrophins including brain-derived neurotrophic factor (BDNF). Although MEK5 is known to mediate BDNF stimulation of ERK5 in central nervous system neurons, other upstream signaling components have not been identified. Here, we report that BDNF induces a sustained activation of ERK5 in rat cortical neurons and activates Rap1, a small GTPase, as well as MEKK2, a MEK5 kinase. Our data indicate that activation of Rap1 or MEKK2 is sufficient to stimulate ERK5, whereas inhibition of either Rap1 or MEKK2 attenuates BDNF activation of ERK5. Furthermore, BDNF stimulation of MEKK2 is regulated by Rap1. Our evidence also indicates that Ras and MEKK3, a MEK5 kinase in non-neuronal cells, do not play a significant role in BDNF activation of ERK5. This study identifies Rap1 and MEKK2 as critical upstream signaling molecules mediating BDNF stimulation of ERK5 in central nervous system neurons.

Neurotoxicity of Ecstasy Metabolites in Rat Cortical Neurons, and Influence of Hyperthermia

3,4-Methylenedioxymethamphetamine (MDMA or "Ecstasy") is a widely abused, psychoactive recreational drug. There is growing evidence that the MDMA neurotoxic profile may be highly dependent on both its hepatic metabolism and body temperature. Metabolism of MDMA involves N-demethylation to 3,4-methylenedioxyamphetamine (MDA), which is also a drug of abuse. MDMA and MDA are O-demethylenated to N-methyl-alpha-methyldopamine (N-Me-alpha-MeDA) and alpha-methyldopamine (alpha-MeDA), respectively, both of which are catechols that can undergo oxidation to the corresponding ortho-quinones. In the presence of glutathione (GSH), ortho-quinones may be conjugated with GSH to form glutathionyl adducts. In this study, we evaluated the neurotoxicity of MDMA and three of its metabolites obtained by synthesis, N-Me-alpha-MeDA, alpha-MeDA, and 5-(GSH)-alpha-MeDA [5-(glutathion-S-yl)-alpha-methyldopamine] in rat cortical neuronal serum-free cultures under normal (36.5 degrees C) and hyperthermic (40 degrees C) conditions. Cell viability was assessed, and the mechanism of cell death was also evaluated. Our study shows that these metabolites are more neurotoxic [5-(GSH)-alpha-MeDA being the most toxic] than the parent compound MDMA. The neurotoxicity of MDMA metabolites was partially prevented by the antioxidants N-acetylcystein and also, in a minor extent, by alpha-phenyl-N-tert-butyl nitrone. All the tested compounds induced apoptotic cell death in cortical neurons, and their neurotoxic effect was potentiated under hyperthermic conditions. These data suggest that MDMA metabolites, especially under hyperthermic conditions, contribute to MDMA-induced neurotoxicity.

Activation of protein kinase C-?? attenuates kainate-induced cell death of cortical neurons

We investigated the role of individual protein kinase C (PKC) isoforms during kainate toxicity in cortical neurons. Treatment with 50 microM kainate induced isoform-specific activation of PKC-delta according to the translocation from the soluble to the particulate fraction, while it caused remarkable decreases in PKC alpha, beta, epsilon and zeta in both fractions. Kainate-induced neuronal death was significantly increased by pharmacological inhibition of PKC-delta with rottlerin, suggesting a protective role of PKC-delta against kainate toxicity. A PKC activator phorbol 12-myristate 13-acetate remarkably attenuated the kainate-induced neuronal death. Although phorbol 12-myristate 13-acetate activates PKC-epsilon and PKC-delta, the protective effect of phorbol 12-myristate 13-acetate was almost completely abolished by rottlerin, but not by epsilonV1-2. These results suggest that activation of PKC-delta attenuates the kainate-induced cell death of cortical neurons.

The effects of early post-traumatic hyperthermia in female and ovariectomized rats.

Episodes of post-traumatic hyperthermia commonly occur in the head-injured patient population. Although post-traumatic hyperthermia has been shown to worsen outcome in experimental studies using male rats, the consequences of secondary hyperthermia following traumatic brain injury (TBI) have not been investigated in female animals. Thus, the purpose of this study was to examine the effects of post-traumatic hyperthermia after fluid-percussion (F-P) brain injury in intact and ovariectomized female rats. Thirty-eight female Sprague-Dawley rats were used in these experiments. Intact female rats underwent TBI followed 30 min later by a 4-h period of normothermia (37 degrees C) or brain hyperthermia (40 degrees C). Female rats that had been ovariectomized 10 days prior to TBI were also traumatized and followed by a period of normothermia or hyperthermia. At 72 h after TBI, rats were perfusion-fixed for quantitative histopathological and immunocytochemical evaluation. Following normothermic TBI, intact female rats demonstrated significantly smaller contusion volumes, decreased frequency of axonal beta-amyloid precursor protein (beta-APP) profiles, and greater numbers of NeuN-positive cortical neurons compared to traumatized ovariectomized females. Although post-traumatic hyperthermia increased contusion volume, cortical neuronal cell death and axonal damage in both intact and ovariectomized female groups, the effects of the induced hyperthermic period were more pronounced in ovariectomized animals. These findings demonstrate for the first time that post-traumatic hyperthermia worsens histopathological outcome in female rats, and that neural hormones, including estrogen and progesterone, may protect against secondary hyperthermic insults.

Oxidative stress inhibits ionomycin-mediated cell death in cortical neurons.

Thiol-proteases play important roles in many cellular processes, including maintenance of protein homeostasis and execution of cell death. Therefore, determining how this family of enzymes is regulated is critical for our understanding of both physiological and pathological conditions. Because these proteases require a reduced cysteine residue for activity, the cellular redox state plays a crucial role in regulating the activity of thiol proteases. Importantly, increased oxidative stress can result in the direct modification of the active site cysteine, leading to enzyme inactivation. This would suggest that oxidative stress that occurs during pathological insults could prolong cell survival by preventing the execution of thiol-protease-dependent cell death pathways. To test this hypothesis, cultured rat cortical neurons were treated with the oxidizing agent diamide or doxorubicin in the presence or absence of the calcium ionophore ionomycin. Under normoxic conditions, ionomycin treatment resulted in approximately 70% cell death, which was prevented by addition of the calpain-selective inhibitor benyzloxycarbonyl-Leu-Leu-Tyr fluoromethylketone. Similarly, pretreatment of neurons with either oxidant was also protective. Protection resulting from oxidative stress was not due to new protein synthesis, insofar as cycloheximide did not affect oxidant-mediated protection. Interestingly, treatment with the antioxidant Trolox to reverse or prevent oxidative stress blocked the protective effects of both oxidants against ionomycin-induced cell death. We interpret these findings to suggest that, in diseases or conditions in which oxidative stress is increased, the ability of thiol-proteases to execute cell death pathways fully is decreased and may prolong cell survival.

Increased oxidative stress is associated with chronic intermittent hypoxia-mediated brain cortical neuronal cell apoptosis in a mouse model of sleep apnea

Chronic intermittent hypoxia (CIH), as occurs in obstructive sleep apnea (SA), is associated with substantial cortico-hippocampal damage leading to impairments of neurocognitive, respiratory and cardiovascular functions. Previous studies in a rat model have shown that CIH increases brain cortical neuronal cell death. However, the molecular events leading to CIH-mediated neuronal cell death remain largely undefined. The oscillation of O 2 concentrations during CIH remarkably mimics the processes of ischemia/re-oxygenation and could therefore increase cellular production of reactive oxygen species (ROS). We extended the CIH paradigm to a mouse model of SA to identify the molecular mechanisms underlying cortical neuronal cell death. A significant increase of ROS production in mouse brain cortex and cortical neuronal cells was detected by fluorescent oxidation assays upon exposure of mice to CIH, followed by increased expression of oxidative stress response markers, c-Fos, c-Jun and NF-κB in mouse brain cortex, as revealed by immunohistochemical and LacZ reporter assays respectively. Long-term exposure of mice to CIH increased the levels of protein oxidation, lipid peroxidation and nucleic acid oxidation in mouse brain cortex. Furthermore, exposure of mice to CIH induced caspase-3 activation and increased some cortical neuronal cell apoptosis. On the other hand, transgenic mice overexpressing Cu,Zn-superoxide dismutase exposed to CIH conditions had a lower level of steady-state ROS production and reduced neuronal apoptosis in brain cortex compared with that of normal control mice. Taken together, these findings suggest that the increased ROS production and oxidative stress propagation contribute, at least partially, to CIH-mediated cortical neuronal apoptosis and neurocognitive dysfunction.

P2X7 Mediates Superoxide Production in Primary Microglia and Is Up-regulated in a Transgenic Mouse Model of Alzheimer's Disease

Primary rat microglia stimulated with either ATP or 2'- and 3'-O-(4-benzoylbenzoyl)-ATP (BzATP) release copious amounts of superoxide (O(2)(-)*). ATP and BzATP stimulate O(2)(-)* production through purinergic receptors, primarily the P2X(7) receptor. O(2)(-)* is produced through the activation of the NADPH oxidase. Although both p42/44 MAPK and p38 MAPK were activated rapidly in cells stimulated with BzATP, only pharmacological inhibition of p38 MAPK attenuated O(2)(-)* production. Furthermore, an inhibitor of phosphatidylinositol 3-kinase attenuated O(2)(-)* production to a greater extent than an inhibitor of p38 MAPK. Both ATP and BzATP stimulated microglia-induced cortical cell death indicating this pathway may contribute to neurodegeneration. Consistent with this hypothesis, P2X(7) receptor was specifically up-regulated around beta-amyloid plaques in a mouse model of Alzheimer's disease (Tg2576).

A distinct death mechanism is induced by 1-methyl-4-phenylpyridinium or by 6-hydroxydopamine in cultured rat cortical neurons: degradation and dephosphorylation of tau

We examined whether the well-known neurotoxins 6-hydroxydopamine (6-OHDA) and 1-methyl-4-phenylpyridinium ion (MPP +) recruit distinct cell death mechanisms using primary cultured neurons derived from day 16 embryonic rat cortices. Electron microscopy revealed that cell death induced by both 6-OHDA and MPP + was typified by a condensation of chromatin while prominent mitochondrial swelling was observed only in those cells treated with MPP +. Co-treatment of cells with a pan-caspase inhibitor, Z-VAD-fmk, attenuated 6-OHDA-induced chromatin condensation and neuronal death. Co-treatment with such antioxidants as N-acetylcysteine or Mn-TBAP also suppressed 6-OHDA-induced cell death. None of these treatments attenuated MPP +-induced cell death although caspase inhibition abolished MPP +-induced chromatin condensation. Interestingly, in these paradigms of cell death, the N-terminus of tau was specifically cleaved and the levels of phosphorylated tau were markedly decreased following 6-OHDA treatment. By contrast, the C-terminus of tau was cleaved in MPP +-induced cell death while the levels of phosphorylated tau remained largely unaltered. Taken together, our results indicate that distinct cellular mechanisms appear to underlie neurotoxin-induced cortical neuronal cell death.

A narrow window for rescuing cells by the inhibition of calcium influx and the importance of influx route in rat cortical neuronal cell death induced by glutamate

A rescue window for glutamate-insulted cells with regard to Ca 2+ influx was first investigated. The addition of EGTA, an impermeable calcium chelator to the culture within 5 min after the beginning of glutamate stimulation potently suppressed the neuronal cell death examined at 22 h. The effect of EGTA on rescuing cells decreased with the time delay of its addition to the system. MK-801, an antagonist of N-methyl- d-asparate (NMDA) receptor channels also inhibited the neuronal cell death in a similar manner to EGTA, suggesting Ca 2+ influx up to 5 min after the insult determined the fate of cells. But we also demonstrated that the elevated intracellular Ca 2+ did not always induce neurotoxicity. High concentration of potassium chloride plus FPL64176, an agonist of L-type Ca 2+ channels did not induce neuronal cell death, even though their combination elicited equal elevation of intracellular Ca 2+ to that by toxic concentration of glutamate, demonstrating that locally elevated intracellular Ca 2+ around NMDA receptors is important in the induction of neuronal cell death.

(-)1-(Benzofuran-2-yl)-2-propylaminopentane shows survival effect on cortical neurons under serum-free condition through sigma receptors.

1. The rapid cell death of cortical neurons in serum-free culture was rescued by the condition medium from the high-density culture, but not by brain-derived neurotrophic factor or basic fibroblast growth factor. 2. Similar rescue was observed by the addition of (-)BPAP, an impulse enhancer, and (+)-pentazocine, a sigma receptor agonist. These actions were blocked by BD1063, a sigma receptor antagonist. 3. (-)BPAP showed a weak displacement activity in the [3H]pentazocine binding to synaptic membranes from rat cerebral cortex. 4. These findings suggest that (-)BPAP and (+)-pentazocine have unique survival activity on cortical neurons through sigma receptors.

Increase in p53 protein expression following cortical infarction in the spontaneously hypertensive rat

Using stroke-prone spontaneously hypertensive (SH-SP) rats with permanent occlusion of the middle cerebral artery (MCA), we investigated the expression of wild type p53 (wt-p53) protein and the occurrence of DNA fragmentation in cerebral neurons after ischemia. Three days following MCA occlusion, terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end labeling (TUNEL staining) revealed a distinct pattern of nuclear staining in many neurons around the ischemic core. On the lesioned side of the cerebral cortex one day after MCA occlusion, wt-p53 immunoreactivity was observed specifically in the cortical neurons, in the same regions as the TUNEL staining. Mutant type p53 (mt-p53) immunoreactivity was not observed at any time following MCA occlusion. These findings suggest that wt-p53 dependent cell death of cortical neurons occurred in the ischemic periphery following cerebral ischemia and that this pathway for the induction of cell death may play an important role in the exaggeration of cerebral ischemic injury.

Attenuation of Oxidative Neuronal Necrosis by a Dopamine D1 Agonist in Mouse Cortical Cell Cultures

Events which lead to an increase in intracellular free radicals induce necrotic cell death of cultured cortical neurons. In the present study, we report that treatment with 1 μM(±)-SKF-38393 hydrochloride, a selective D1 agonist, as well as 100 μMtrolox, a lipophilic vitamin E analogue, significantly prevented oxidative-related necrotic cell death following exposure to 10 μMFe2+or 1 mMbuthionine sulfoximine, an inhibitor of γ-glutamylcysteine synthetase. The neuroprotective effect of (±)-SKF-38393 hydrochloride was partially reversed by addition of (±)-SKF-83566 hydrochloride, a selective D1 antagonist. Quinelorane dihydrochloride, a selective D2 agonist, did not influence free radical neurotoxicity. Interestingly, inclusion of (±)-SKF-38393 hydrochloride or quinelorane dihydrochloride did not attenuate apoptotic cell death of cortical neurons deprived of serum. The present study provides evidence that (±)-SKF-38393 hydrochloride attenuates oxidative neuronal necrosis, which has unique therapeutic potential for the treatment of various neurodegenerative diseases linked to oxidative stress.

Caspase Inhibition Selectively Reduces the Apoptotic Component of Oxygen-Glucose Deprivation-Induced Cortical Neuronal Cell Death

Cultured mouse cortical neurons undergo apoptosis when exposed to staurosporine. The cell-permeable caspase inhibitor Z-Val-Ala-Asp fluoromethylketone (Z-VAD.FMK) attenuated this death, without altering overall protein synthesis. Z-VAD.FMK also attenuated cortical neuronal apoptosis induced by removal of serum. However, Z-VAD.FMK did not attenuate the excitotoxic necrosis induced by 5-min exposure to 100 μMNMDA, 24-h exposure to 100 μMkainate, or 90-min exposure to oxygen-glucose deprivation. We have previously shown that blockade of the excitotoxic component of oxygen-glucose deprivation-induced neuronal death with glutamate antagonists unmasks an apoptotic death. Treatment with Z-VAD.FMK, but not the cathepsin-B protease inhibitor Z-Phe-Ala fluoromethylketone (Z-FA.FMK), also attenuated this oxygen-glucose deprivation-induced neuronal apoptosis. These data support the idea that brain caspases mediate the apoptotic component of oxygen-glucose deprivation-induced neuronal death and raise the possibility that combining caspase inhibitors with glutamate antagonists might attenuate brain damage induced by hypoxic-ischemic insultsin vivo.

Receptorpharmacological models for the therapy of labyrinthine vertigo.

In the mammalian labyrinth, GABAA receptor subtypes are involved in the excitatory neurotransmission between the vestibular type II hair cells and the afferent neurons. Additional afferent ionophoric receptor channels, sensitive to further transmitter candidates, are discussed for both types I and II hair cells. GABA accelerates excitotoxic cell death in cortical neurons. This GABA-ergic neurotoxic action forms an appropriate pathophysiological model explaining peripheral labyrinthine disorders characterized by the spontaneous labyrinthine nystagmus and vertigo in man. A calculated GABAA receptor antagonism was envisaged in order to attenuate the pathological vestibular imbalance following one-sided GABA-accelerated vestibular neurotoxicity. Moderate allosteric blockers of the GABAA receptor channel and weak inverse agonists of the benzodiazepine binding site meet some requirements for potentially successful clinical application. The suppressing action of the suitable drugs picrotoxin and flumazenil on labyrinthine nystagmus and vertigo, tested in clinical trials, supports the hypothesis that GABAA receptors are involved in vestibular neurotransmission, even in humans. The test results promise the development of a successful vestibular receptorpharmacology in the near future.