UV-induced apoptosis in SH-SY5Y cells: contribution to apoptosis by JNK signaling and cytochrome c

Activation of the c-Jun N-terminal kinase (JNK) pathway is suggested to be required for neuronal apoptosis. We investigated the role of JNK on phosphorylation of c-Jun, Bcl-2, and apoptotic translocation of cytochrome c (cyt c) in UV-induced apoptosis in human neuroblastoma SH-SY5Y cells. We confirm that UV irradiation induces both apoptosis and necrosis in SH-SY5Y cells and that phosphorylation of JNK at Thr183/Tyr185 in SH-SY5Y cells treated with UV is an early event preceding apoptosis. We also demonstrate that phosphorylation of c-Jun at Ser63 is an early event coinciding with JNK activation, and that the phosphorylation of c-Jun is partially prevented by the JNK inhibitor SP600125. Despite the use of SP600125, the amount of cyt c released into the cytoplasm is not diminished and SP600125 is also unable to decrease the extent of UV-induced apoptosis. These data support the hypothesis that in this system, UV-induced apoptosis is not dependent exclusively on JNK activation. Possible involvement of cyclin-dependent kinases (CDKs) in c-Jun phosphorylation at Ser63 was excluded by pretreating UV-irradiated SH-SY5Y cells with the CDK1/2/5 inhibitor roscovitine.     

Regulation of Bax translocation through phosphorylation at Ser-70 of Bcl-2 by MAP kinase in NO-induced neuronal apoptosis

The molecular mechanism of Bcl-2 phosphorylation and its relationship to Bax is largely unknown. Here we show that the phosphorylation of Bcl-2 is involved in the intracellular translocation of Bax from cytosol to mitochondria in NO-induced neuronal apoptosis. We examined how the phosphorylation of Bcl-2 is regulated during the apoptosis and found it to be mediated by the activation of p38 and ERK, members of the MAPK superfamily. Furthermore, we investigated whether Bcl-2 phosphorylation affected Bax translocation, using mutant Bcl-2 expression vectors. Cortical neuronal cells overexpressing the Bcl-2 mutant S70A (which cannot be phosphorylated) prevented the translocation of Bax. In contrast, transfection with Bcl-2 (S70D), a constitutively active Bcl-2 mutant, enhanced the translocation. Our results suggested that Bcl-2 phosphorylated at Ser-70 plays a critial role in the translocation of Bax from the cytosol to the mitochondria, and this may regulate NO-induced neuronal apoptosis.     

Neuroprotection against focal ischemic brain injury by inhibition of c-Jun N-terminal kinase and attenuation of the mitochondrial apoptosis-signaling pathway.

c-Jun N-terminal kinase (JNK) is an important stress-responsive kinase that is activated by various forms of brain insults. In this study, we have examined the role of JNK activation in neuronal cell death in a murine model of focal ischemia and reperfusion; furthermore, we investigated the mechanism of JNK in apoptosis signaling, focusing on the mitochondrial-signaling pathway. We show here that JNK activity was induced in the brain 0.5 to 24 h after ischemia. Systemic administration of SP600125, a small molecule JNK-specific inhibitor, diminished JNK activity after ischemia and dose-dependently reduced infarct volume. c-Jun N-terminal kinase inhibition also attenuated ischemia-induced expression of Bim, Hrk/DP5, and Fas, but not the expression of Bcl-2 or FasL. In strong support of a role for JNK in promoting the mitochondrial apoptosis-signaling pathway, JNK inhibition prevented ischemia-induced mitochondrial translocation of Bax and Bim, release of cytochrome c and Smac, and activation of caspase-9 and caspase-3. The potential mechanism by which JNK promoted Bax translocation after ischemia was further studied using coimmunoprecipitation, and the results revealed that JNK activation caused serine phosphorylation of 14-3-3, a cytoplasmic sequestration protein of Bax, leading to Bax disassociation from 14-3-3 and subsequent translocation to mitochondria. These results confirm the role of JNK as a critical cell death mediator in ischemic brain injury, and suggest that one of the mechanisms by which JNK triggers the mitochondrial apoptosis-signaling pathway is via promoting Bax and Bim translocation.     

HYPERTROPHY OF SPINAL ROOTS IN CHRONIC INFLAMMATORY DEMYELINATING NEUROPATHY

We have demonstrated in the SH-SY5Y human neuroblastoma cell line that the antineoplastic drug paclitaxel induces apoptosis associated with the phosphorylation of c-raf and Bcl-2, thus causing Bcl-2 inactivation. In addition, we have observed the cleavage of caspase 7 and PARP, and the involvement of JNK/SAPK cascade.
In this study, we investigated the possible anti-apoptotic effect of resveratrol on paclitaxel-induced apoptosis. Resveratrol is a natural antioxidant occurring in grapes and wine that as been shown to have anticancer and anti-inflammatory effects. Studies on the effect(s) of resveratrol on nervous cells are very few and limited to testing its ability in preventing oxidative stress in rat pheocromocytoma PC12 cells. However, the biological and pharmacological properties of resveratrol suggest that this natural compound might act on neuronal cells with a mode of action that is different from the antioxidant ones. To confirm this hypothesis, we studied the possible antagonist effect(s) of resveratrol on paclitaxel-induced apoptosis pathways.
Our results indicate that the simultaneous treatment of 50M resveratrol and 1M paclitaxel induces a significant reduction of the percentage of apoptotic cells in comparison with 1M paclitaxel alone. Furthermore, we have demonstrated that resveratrol treatment determines a significant reduction of the phosphorylation of c-raf and Bcl-2 and a partial reduction of cleavage of caspase 7 and PARP. Finally, we observed an evident reduction of activation of JNK/SAPK in the presence of resveratrol. On the contrary, resveratrol does not affect the tubulin polymerization induced by paclitaxel.
In conclusion, our results suggest that resveratrol is able to partially antagonize a nonoxidative stress apoptotic stimulus by influencing the typical signal pathways involved in apoptosis.     

Dynorphin up-regulation in the dentate granule cell mossy fiber pathway following chronic inhibition of GluN2B-containing NMDAR is associated with increased CREB (Ser 133) phosphorylation,but is independent of BDNF/TrkB signaling pathways

Emerging evidence suggests that neuronal responses to N-methyl-d-aspartate (NMDAR) activation/inactivation are influenced by subunit composition. For example, activation of synaptic NMDAR (comprised of GluN2A > GluN2B) phosphorylates cAMP-response-element-binding protein (CREB) at Ser 133, induces BDNF expression and promotes neuronal survival. Activation of extrasynaptic NMDAR (comprised of GluN2B > GluN2) dephosphorylates CREB (Ser 133), reduces BDNF expression and triggers neuronal death. These results led us to hypothesize that chronic inhibition of GluN2B-containing NMDAR would increase CREB (Ser 133) phosphorylation, increase BDNF levels and subsequently alter downstream dynorphin (DYN) and neuropeptide Y (NPY) expression. We focused on DYN and NPY because these neuropeptides can decrease excitatory neurotransmission and seizure occurrence and we reported previously that seizure-like events are reduced following chronic treatment with GluN2B antagonists. Consistent with our hypothesis, chronic treatment (17–21 days) of hippocampal slice cultures with the GluN2B-selective antagonists ifenprodil or Ro25,6981 increased both CREB (Ser 133) phosphorylation and granule cell mossy fiber pathway DYN expression. Similar treatment with the non-subtype-selective NMDAR antagonists d-APV or memantine had no significant effect on either CREB (Ser 133) phosphorylation or DYN expression. In contrast to our hypothesis, BDNF levels were decreased following chronic treatment with Ro25,6981, but not ifenprodil, d-APV or memantine. Blockade of BDNF actions and TrkB activation did not significantly augment hilar DYN expression in vehicle-treated cultures and had no effect in Ro25,6981 treated cultures. These findings suggest that chronic exposure to GluN2B-selective NMDAR antagonists increased DYN expression through a putatively pCREB-dependent, but BDNF/TrkB-independent mechanism.     

Interfering effect and mechanism of neuregulin on experimental dementia model in rats

Objective: To investigate the effect of neuregulin 1β (NRG1β) on the neuronal apoptosis and the expressions of Bcl-2 and Bax proteins in experimental dementia model rats. Methods: Thirty adult healthy male Wistar rats were randomly divided into control group, model group and treated group consisting of 10 rats, respectively. The experimental dementia models were established by injecting beta-amyloid protein 1-40 (Aβ1-40) stereotactically into the left lateral ventricle, and treated by injecting NRG1β into right lateral ventricle. The cognitive capacity of rats was evaluated with Y-electric maze. The neuronal apoptosis was counted by TUNEL assay. The expressions of Bcl-2 and Bax were determined with immunohistochemistry assay and double immunofluorescence labeling. Results: The cognitive ability in model group rats decreased, along with the number of neuronal apoptosis and the expressions of Bcl-2 and Bax increased significantly than those in control group (P < 0.05). After treatment with NRG1β, the cognitive ability of rats improved, the number of neuronal apoptosis reduced and the expression of Bcl-2 increased while Bax decreased significantly than those in model group (P < 0.05). Conclusion: NRG1β could inhibit neuronal apoptosis by regulating the expressions of Bcl-2/Bax to improve the capacity of learning and memory in experimental dementia rats.     

TRPV1 receptors modulate retinal development

We investigated the possible participation of TRPV1 channels in retinal apoptosis and overall development. Retinas from newborn, male albino rats were treated in vitro with capsazepine, a TRPV1 antagonist. The expression of cell cycle markers was not changed after TRPV1 blockade, whereas capsazepine reduced the number of apoptotic cells throughout the retina,increased ERK1/2 and p38 phosphorylation and slightly reduced JNK phosphorylation. The expression of BAD, Bcl-2, as well as integral and cleaved capsase-3 were similar in all experimental conditions. Newborn rats were kept for 2 months after receiving high doses of capsazepine. In their retinas, calbindin and parvalbumin protein levels were upregulated, but only the number of amacrine-like, parvalbumin-positive cells was increased. The numbers of calretinin, calbindin, ChAT, vimentin, PKC-alpha and GABA-positive cells were similar in both conditions. Protein expression of synapsin Ib was also increased in the retinas of capsazepine-treated rats. Calretinin, vimentin, GFAP, synapsin Ia, synaptophysin and light neurofilament protein levels were not changed when compared to control values.Our results indicate that TRPV1 channels play a role in the control of the early apoptosis that occur during retinal development, which might be dependent on MAPK signaling. Moreover, it seems that TRPV1 function might be important for neuronal and synaptic maturation in the retina.     

Effects of blockade of voltage-sensitive Ca/Na channels by a novel phenylpyrimidine derivative, NS-7, on CREB phosphorylation in focal cerebral ischemia in the rat

NS-7 is a novel blocker of voltage-sensitive Ca²⁺ and Na⁺ channels, and it significantly reduces infarct size after occlusion of the middle cerebral artery. Persistent activation of cyclic AMP response element binding protein (CREB), which can be induced by increase in intracellular Ca²⁺ concentrations or other second messengers, has recently been found to be closely associated with neuronal survival in cerebral ischemia. The present study was therefore undertaken to evaluate the neuroprotective effects of NS-7 by analyzing changes in CREB phosphorylation in a focal cerebral ischemia model. CREB phosphorylation in the brain of rats was investigated immunohistochemically at 3.5–48-h recirculation after 1.5-h occlusion of the middle cerebral artery. NS-7 (1 mg/kg; NS-7 group) or saline (saline group) was intravenously injected 5 min after the start of recirculation. The NS-7 group showed significantly milder activation of CREB phosphorylation in various cortical regions after 3.5 h of recirculation than the saline group. The inner border zone of ischemia in the NS-7 group subsequently exhibited a moderate, but persistent, increase in number of phosphorylated CREB-positive neurons with no apparent histological damage. By contrast, the saline group displayed a marked, but only transient, increase in number of immunopositive neurons in this border zone after 3.5 h of recirculation, and this was followed by clear suppression of CREB phosphorylation and subsequent loss of normal neurons. These findings suggest that: (1) the marked enhancement of CREB phosphorylation in the acute post-ischemic phase may be triggered largely by an influx of calcium ions as a result of activation of the voltage-sensitive Ca²⁺ and Na⁺ channels; and that (2) the neuroprotective effects of NS-7 may be accompanied by persistent activation of CREB phosphorylation in the inner border zone of ischemia.     

Increased expression of AGS3 in rat brain cortex after traumatic brain injury

Receptor‐independent activators of G protein signaling (AGS) offer alternative modes of signal processing for the G protein signaling system that has broad mechanistic and functional significance. Previous studies have demonstrated that AGS3, which belongs to the AGS family, is involved in a number of different cellular activities. However, the distribution and function of AGS3 in the central nervous system (CNS) remain unclear. To investigate whether AGS3 is involved in CNS injury and repair, we used an acute traumatic brain injury (TBI) model in adult rats. Western blot analysis and immunohistochemistry showed a significant upregulation of AGS3 in ipsilateral peritrauma cortex. Double immunofluorescence staining showed that AGS3 was coexpressed with NeuN but rarely with glial fibrillary acidic protein. In addition, we detected that active caspase‐3 had colocalization with NeuN and AGS3, suggesting that AGS3 might be involved in the neuron apoptosis after TBI. To investigate the potential function of AGS3 further, a neuronal cell line, PC12, was employed to establish a cell apoptosis model. Western blot analysis indicated that AGS3 shared a similar dynamic variation in animal experiments, and phosphorylated cyclic AMP response element‐binding protein (CREB) increased in parallel. Additionally, knocking down AGS3 with siRNA partially attenuated the protein level of phosphorylated CREB in PC12 stimulated by H₂O₂, while reinforcing active caspase‐3 expression, demonstrating a probable antiapoptotic role through CREB played by AGS3 in neuronal apoptosis. We hypothesize that AGS3 might play an important antiapoptotic role through enhancing phosphorylation of CREB. © 2013 Wiley Periodicals, Inc.     

Acupuncture at Baihui and Dazhui reduces brain cell apoptosis in heroin readdicts简

Acupuncture at Baihui （GV20） and Dazhui （GV14） reduces neuronal loss and attenuates ultra- structural damage in cerebral ischemic rats. However, whether acupuncture can treat addiction and prevent readdiction through changes to brain cell ultrastructure remains unknown. In this study, cell apoptosis was observed in the hippocampus and frontal lobe of heroin readdicted rats by electron microscopy. Immunohistochemical staining displayed a reduction in Bcl-2 ex- pression and an increase in Bax expression in the hippocampus and frontal lobe. After rats were given acupuncture at Baihui and Dazhui, the pathological damage in the hippocampus and frontal lobe was significantly reduced, Bcl-2 expression was upregulated and Bax expression was downregulated. Acupuncture exerted a similar effect with methadone, a commonly used drug for clinical treatment of drug addiction. Experimental findings suggest that acupuncture at Dazhui and Baihui can prevent brain cell apoptosis in heroin readdicted rats.     

Differential regulation of AKT, MAPK and GSK3β during C2-ceramide-induced neuronal death

Evidence has implicated apoptosis as a mechanism underlying cell demise in diverse neurodegenerative diseases including Parkinson's disease (PD). Endogenous toxins and other stress signals activate the sphingomyelin pathway increasing the levels of ceramide, an important regulator of cell death.In the present paper we have analysed the contribution of PI3K/AKT-GSK3β and MAPK (ERK and JNK) pathways to cell death in a catecholaminergic cell line following exposure to C₂-ceramide. We also explored the potential neuroprotective action of insulin-like growth factor-1 (IGF-1) and neurotrophin-3 (NT3).We demonstrated that C₂-ceramide-induced cell death is associated to an early decrease in phosphorylation (inhibition) of PI3K/AKT and ERK, followed by phosphorylation (activation) of JNK and de-phosphorylation (activation) of glycogen synthase kinase-3 beta (GSK3β). NT3 and IGF-1 increased survival at early time points, but only IGF-1 is capable to attenuate C₂-ceramide-mediated neuronal death, and this neuroprotection is associated to strong and permanent activation of AKT and inhibition of GSK3β.In conclusion, C₂-ceramide initiates a series of events including an early inactivation of PI3K/AKT and ERK pathways followed by activation of JNK and activation of GSK3β and neuronal death, changes that are counteracted by IGF-1.     

Dephosphorylation-induced decrease of anti-apoptotic function of Bcl-2 in neuronally differentiated P19 cells following ischemic insults

It is known that Bcl-2 has a protective effect against neuronal ischemia. Some reports speculate anti-apoptotic function of Bcl-2 depends not on the expression level but on the phosphorylation state. We found induction of apoptosis and CPP32 activation by energy impairment (3-nitropropionic acid (3-NP)-treatment or glucose-deprivation) in the neuronally differentiated P19 cells. Time course study of cell viability following ischemic insults showed that the number of viable cells decreased along with the increase in the amount of dephosphorylated Bcl-2 without obvious quantitative alteration of the protein. Then, we generated differentiated P19 cells overexpressing wild-type Bcl-2 (P19/wt. Bcl-2) or phosphorylation-negative Bcl-2 mutant (P19/mut.Bcl-2), in which alanine was substituted for serine 70. When the cell viability was examined within 24 h, P19/mut.Bcl-2 was more vulnerable to energy impairment as compared with P19/wt.Bcl-2. In addition, overexpression of wild-type Bcl-2 inhibited DNA laddering and CPP32 activation induced by the insults, while that of mutant Bcl-2 did not. These findings suggest that the phosphorylation state, as well as the expression level, of Bcl-2 plays an important role to modulate its protective effect against ischemic insults.     

Neuronal NR2B-containing NMDA receptor mediates spinal astrocytic c-Jun N-terminal kinase activation in a rat model of neuropathic pain

Spinal N-methyl d-aspartate receptor (NMDAR) plays a pivotal role in nerve injury-induced central sensitization. Recent studies suggest that NMDAR also contributes to neuron-astrocyte signaling. c-Jun N-terminal kinase (JNK) is persistently and specifically activated (indicated by phosphorylation) in spinal cord astrocytes after nerve injury and thus it is considered as a dependable indicator of pain-related astrocytic activation. NMDAR-mediated JNK activation in spinal dorsal horn might be an important form of neuron-astrocyte signaling in neuropathic pain. In the present study, we observed that intrathecal injection of MK-801, a noncompetitive NMDA receptor antagonist, or Ro25-6981 and ifenprodil, which are selective antagonists of NR2B-containing NMDAR each significantly reduced nerve injury-induced JNK activation. Double immunostaining showed that NR2B was highly expressed in neurons, indicating the effect of NMDAR antagonists on JNK activation was indirect. We further observed that intrathecal injection of NMDA (twice a day for 3 days) significantly increased spinal JNK phosphorylation. Besides, NMDAR-related JNK activation could be blocked by a neuronal nitric oxide synthase (nNOS) selective inhibitor (7-nitroindazole sodium salt) but not by a nNOS sensitive guanylyl cyclase inhibitor (1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one). Finally, real-time RT-PCR and immunostaining showed that nerve injury-induced interleukin-1beta expression was dependent on astrocytic JNK activation. Treatments targeting NMDAR-nNOS pathway also influenced interleukin-1beta expression, which further confirmed our hypothesis. Taken together, our results suggest that neuronal NMDAR-nNOS pathway could activate astrocytic JNK pathway. Excitatory neuronal transmission initiates astrocytic activation-induced neuroinflammation in this way, which contributes to nerve injury-induced neuropathic pain.     

TAXOL NEUROTOXICITY: CELLULAR MECHANISMS INVOLVED IN APOPTOSIS

The antineoplastic drug paclitaxel (Taxol™) is neurotoxic on the peripheral nervous system. Both activity and neurotoxicity of paclitaxel are due to enhancement in tubulin polymerization. In addition, it has been demonstrated that paclitaxel induces gene expression. To elucidate the signal transduction pathways involved in paclitaxel neurotoxicity we studied in vitro the effect(s) of paclitaxel on the human neuroblastoma (HN) SH-SY5Y cells, which express genes associated with neuronal differentiation and may be considered neuroblasts at various stages of maturation.
The exposure of SH-SY5Y cells to paclitaxel produces apoptosis, as demonstrated with Annexin V assay and orange acridin staining. Electron microscopy showed typical condensation of nuclear chromatin with normal nuclear membrane. DNA electrophoresis confirmed the occurrence of apoptosis by demonstrating the typical laddering.
We studied regulator proteins as Bcl-2 and c-raf, and investigated the classes of caspases activated in this process by immunoblotting. We observed that 1μM paclitaxel induces a simultaneous phosphorylation of c-raf and Bcl-2, thus causing Bcl-2 inactivation. Caspase 3, which is commonly present and activated in the nervous cells, was not activated, while paclitaxel-induced apoptotic process in SH-SY5Y cells is induced by activation of caspase 7. The involvement of caspase 7 is also confirmed by the same pattern of cleavage of PARP. Finally, we also studied MAPKs, a family of serine/threonine kinases that represent key signaling proteins known to be involved in many cellular events, including apoptosis.
Our results indicate that paclitaxel does not activate ERK cascade, whereas it induces JNK/SAPK and p38 phosphorylation, suggesting that these specific pathways are involved in the cell-death induced by paclitaxel.     

Scutellaria baicalensis stem-leaf total flavonoid reduces neuronal apoptosis induced by amyloid beta-peptide (25-35)

Scutellaria baicalensis stem-leaf total flavonoid might attenuate learning/memory impairment and neuronal loss in rats induced by amyloid beta-peptide. This study aimed to explore the effects of Scutellaria baicalensis stem-leaf total flavonoid on amyloid beta-peptide-induced neuronal apoptosis and the expression of apoptosis-related proteins in the rat hippocampus. Male Wistar rats were given intragastric administration of Scutellaria baicalensis stem-leaf total flavonoid, 50 or 100 mg/kg, once per day. On day 8 after administration, 10 μg amyloid beta-peptide (25-35) was injected into the bilateral hippocampus of rats to induce neuronal apoptosis. On day 20, hippocampal tissue was harvested and probed with the terminal deoxyribonucleotidyl transferase-mediated biotin-16-dUTP nick-end labeling assay. Scutellaria baicalensis stem-leaf total flavonoid at 50 and 100 mg/kg reduced neuronal apoptosis induced by amyloid beta-peptide (25-35 in the rat hippocampus. Immunohistochemistry and western blot assay revealed that expression of the pro-apoptotic protein Bax, cytochrome c and caspase-3 was significantly diminished by 50 and 100 mg/kg Scutellaria baicalensis stem-leaf total flavonoid, while expression of the anti-apoptotic protein Bcl-2 was increased. Moreover, 100 mg/kg Scutellaria baicalensis stem-leaf total flavonoid had a more dramatic effect than the lower dosage. These experimental findings indicate that Scutellaria baicalensis stem-leaf total flavonoid dose-dependently attenuates neuronal apoptosis induced by amyloid beta-peptide in the hippocampus, and it might mediate this by regulating the expression of Bax, cytochrome c, caspase-3 and Bcl-2.     

Nuclear translocation and calpain-dependent reduction of Bcl-2 after neonatal cerebral hypoxia–ischemia

Apoptosis-related mechanisms are important in the pathophysiology of hypoxic–ischemic injury in the neonatal brain. Caspases are the major executioners of apoptosis, but there are a number of upstream players that influence the cell death pathways. The Bcl-2 family proteins are important modulators of mitochondrial permeability, working either to promote or prevent apoptosis. In this study we focused on the anti-apoptotic Bcl-2 protein after neonatal cerebral hypoxia–ischemia (HI) in 8-day-old rats. Bcl-2 translocated to nuclei and accumulated there over the first 24 h of reperfusion after HI, as judged by immunohistochemistry and immuno-electron microscopy. We also found that the total level of Bcl-2 decreased after HI in vivo and after ionophore challenge in cultured human neuroblastoma (IMR-32) cells in vitro. Furthermore, the Bcl-2 reduction was calpain-dependent, because it could be prevented by the calpain inhibitor CX295 both in vivo and in vitro, suggesting cross-talk between excitotoxic and apoptotic mechanisms.