p62 protects SH-SY5Y neuroblastoma cells against H2O2-induced injury through the PDK1/Akt pathway

The p62 protein has been identified as a major component of the protein aggregations associated with neurodegenerative disease. Oxidative insult has also been identified as a principal cause of neurodegenerative disease. Thus, in the present study, we investigated the potential role of p62 in oxidative stress-induced cell death in SH-SY5Y human neuroblastoma cells. The results indicated that H₂O₂ treatment induced p62 expression in SH-SY5Y cells. In addition, p62 showed neuroprotective effects against H₂O₂-induced cell death in differentiated SH-SY5Y cells. p62 expression prolonged Akt phosphorylation during the later stages of H₂O₂-induced cell death. Furthermore, coexpression of p62 and wild-type PDK1, the upstream kinase of Akt, further increased Akt phosphorylation and cell viability, whereas the expression of kinase-defective PDK1 reversed the cytoprotective effects of p62 under oxidative stress. Overexpression of p62 led to the dissociation of PDK1 from the 14-3-3θ protein, which is thought to be a negative regulator of PDK1 kinase activity. These findings suggest a mechanism that involves the p62-mediated modulation of the interaction between signaling molecules and results in cell survival.     

Neuroglobin and cytoglobin overexpression protects human SH-SY5Y neuroblastoma cells against oxidative stress-induced cell death

Although reactive oxygen species (ROS) at physiological concentrations are required for normal cell function, excessive production of ROS is detrimental to cells. Neuroglobin and cytoglobin are two globins, whose functions are still a matter of debate. A potential role in the detoxification of ROS is suggested. The influence of neuroglobin and cytoglobin on cell death after oxidative stress in human neuroblastoma SH-SY5Y cells was evaluated. Exposure of SH-SY5Y cells to paraquat or H₂O₂ resulted in a concentration- and time-dependent induction of apoptotic and necrotic cell death. H₂O₂ was 16 times more potent to induce cell death as compared to paraquat. SH-SY5Y cells transfected with plasmid DNA containing the neuroglobin or cytoglobin sequence showed enhanced survival after exposure to 300 μM H₂O₂ for 24 h as compared to untransfected controls. This finding suggests that neuroglobin and cytoglobin protect SH-SY5Y cells against oxidative stress-induced cell death.     

Quercetin protects neuroblastoma SH-SY5Y cells against oxidative stress by inhibiting expression of Krüppel-like factor 4

Quercetin is one of flavonoids with cyto-protective activities. It has been demonstrated that quercetin inhibits oxidative stress in some animal models and specific cells, but the particular mechanism is known a little. In the present study, we found that quercetin could decrease the expression of Krüppel-like factor 4 (KLF4) induced by hydrogen peroxide (H₂O₂) in human neuroblastoma SH-SY5Y cells, further increase the expression ratio of bcl-2 to bax, which were apoptotic-related target genes of KLF4, thus alleviate the apoptotic rate and caspase-3 enzyme activity of SH-5YSY cells; the overexpression or inhibition of KLF4 demonstrated the mediated role of KLF4 for the protective effect of quercetin on cell damage induced by H₂O₂. All results suggest a potential molecular mechanism of quercetin protecting against the oxidative damage, which may be applied in the treatment of oxidative related diseases, such as neurodegeneration diseases.     

Phospholipids block nuclear factor-kappa B and tau phosphorylation and inhibit amyloid-beta secretion in human neuroblastoma cells

Inflammation and oxidative stress have been shown to play a critical role in the pathophysiology that leads to neurodegeneration. Omega-6 phospholipids, e.g. dilinoleoylphosphatidylcholine (DLPC), have been shown to have anti-inflammatory properties and therefore experiments were undertaken to determine whether DLPC can prevent inflammatory neurodegenerative events in the model neuronal cell line, SH-SY5Y. Tumor necrosis factor (TNF-α) and H₂O₂ activate mitogen-activated protein kinase (MAPK) in SH-SY5Y cells within 5 min and this activation is completely blocked by DLPC (12 μM). DLPC blocks IκBα phosphorylation in the SH-SY5Y cells and prevents the phosphorylation and activation of nuclear factor-kappa B (NF-κB). The phospholipid inhibits induction of MAPK and NF-κB in similar fashion to the MEK1/2-inhibitor, U0126 (10 μM). DLPC completely abolishes TNF-α, H₂O₂ and lipopolysaccaride (LPS)-induced neuronal tau phosphorylation. Cellular amyloid precursor protein levels are reduced by DLPC and LPS-induced amyloid-β expression and secretion in SH-SY5Y cells are completely blocked by DLPC. Taken together, these data suggest that DLPC can act through MAPK to block neuronal inflammatory cascades and prevent potential pathological consequences in the neuronal metabolism of amyloid and tau proteins.     

Okadaic acid protects human neuroblastoma SH-SY5Y cells from 1-methyl-4-phenylpyridinium ion-induced apoptosis

1-Methyl-4-phenylpyridinium ion (MPP⁺) has been shown to selectively inhibit mitochondrial function and induce a parkinsonism-like syndrome. MPP⁺ stimulates the production of reactive oxygen species (ROS) and induces cell death in vitro. In this study, we investigated the protective effects of okadaic acid on MPP⁺-induced cell death in SH-SY5Y neuroblastoma cells. We found that MPP⁺-induced apoptosis and -ROS generation were blocked by okadaic acid. MPP⁺-mediated activation of AKT was also inhibited by okadaic acid. Taken together, these results demonstrate that okadaic acid protects against MPP⁺-induced apoptosis by blocking ROS stimulation and ROS-mediated signaling pathways in SH-SY5Y cells. These data indicated that okadaic acid could provide a therapeutic strategy for the treatment of neurodegenerative diseases including Parkinson's disease.     

Effect of Four Medicinal Plants on Amyloid-β Induced Neurotoxicity in SH-SY5Y Cells

Amyloid-beta peptide (Aβ) is implicated in the pathogenesis of Alzheimer''s disease (AD), a neurodegenerative disorder. This study was designed to determine the effect of four medicinal plants used to treat neurodegenerative diseases on Aβ-induced cell death. Cytotoxicity of the ethanol extracts of the plants was determined against SH-SY5Y (human neuroblastoma) cells which were untreated, as well as toxically induced with Aβ, using the MTT and neutral red uptake assays. Cell viability was reduced to 16% when exposed to 20 µM Aβ_25–35 for 72 h. The methanol extract of the roots of Ziziphus mucronata Willd., Lannea schweinfurthii (Engl.) Engl. and Terminalia sericea Burch. ex DC., were the least toxic to the SH-SY5Ycells at the highest concentration tested (100 µg/ml). All four plants tested were observed to reduce the effects of Aβ-induced neuronal cell death, indicating that they may contain compounds which may be relevant in the prevention of AD progression.     

Lithium and oxidative stress lessons from the MPTP model of Parkinson's disease

Lithium has been successfully employed therapeutically for treatment of bipolar depressive illness; however, its mechanism of action is poorly understood. Recently, it has been demonstrated by us that lithium can prevent 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) dopaminergic neurotoxicity in mice. From analyzing the pattern of protection in various parameters, we suggest that lithium protects against MPTP-induced depletion of striatal dopamine (DA) by preventing free radical-induced inactivation of tyrosine hydroxylase (TH), the rate limiting enzyme in dopamine synthesis. Possible neuroprotective effect of lithium against H₂O₂-induced cell death was assessed in human neuroblastoma; SH-SY5Y cell line. Pretreatment with LiCl (2 mM and 4 mM) for 7 days protected against H₂O₂ neurotoxicity in a dose-dependent manner. However, this protection could not be achieved through short-term incubation with LiCl. In agreement; we found that lithium lacks immediate antioxidant activity using the in vitro lipid peroxidation essay indicating that not acute but chronic treatment with lithium allows cells to deal better with oxidative stress.     

Protective effects of the citrus flavanones to PC12 cells against cytotoxicity induced by hydrogen peroxide

Oxidative stress has been considered as a major cause of cellular injuries in a variety of clinical abnormalities. One of the plausible ways to prevent the reactive oxygen species (ROS)-mediated cellular injury is dietary or pharmaceutical augmentation of endogenous antioxidant defense capacity. In this study, we investigated the protective actions of citrus flavanones naringin and nobiletin against the cytotoxicity induced by exposure to hydrogen peroxide (H₂O₂) (150 μM, 3 h) in PC12 cells. The results showed that naringin and nobiletin inhibited the decrease of cell viability (MTT reduction), prevented membrane damage (LDH release), scavenged ROS formation, reduced caspase-3 activity, and attenuated the decrease of mitochondrial membrane potential (MMP), respectively, in H₂O₂-induced PC12 cells. Meanwhile, naringin and nobiletin increased superoxide dismutase (SOD) and glutathione (GSH) activity, while decreased malondialdehyde (MDA), the production of lipid peroxidation, in H₂O₂-induced PC12 cells. In addition, the percentage of cells undergoing H₂O₂-induced apoptosis was decreased in the presence of naringin and nobiletin. These results first demonstrate that naringin and nobiletin, even at physiological concentrations, have neuroprotective effects against H₂O₂-induced cytotoxicity in PC12 cells. All the above results suggest that these dietary antioxidants are potential candidates for use in the intervention for neurodegenerative diseases.     

Hydrogen sulfide protects neurons against hypoxic injury via stimulation of ATP-sensitive potassium channel/protein kinase C/extracellular signal-regulated kinase/heat shock protein90 pathway

Cerebral hypoxia is one of the main causes of cerebral injury. This study was conducted to investigate the potential protective effect of H₂S in in vitro hypoxic models by subjecting SH-SY5Y cells to either oxygen–glucose deprivation or Na₂S₂O₄ (an oxygen scavenger) treatment. We found that treatment with NaHS (an H₂S donor, 10–100 μM) 15 min prior to hypoxia increased cell viability in a concentration-dependent manner. Time-course study showed that NaHS was able to exert its protective effect even when added 8 h before or less than 4 h after hypoxia induction. Interestingly, endogenous H₂S level was markedly reduced by hypoxia induction. Over-expression of cystathionine-β-synthase prevented hypoxia induced cell apoptosis. Blockade of ATP-sensitive K⁺ (K_ATP) channels with glibenclamide and HMR-1098, protein kinase C (PKC) with its three specific inhibitors (chelerythrine, bisindolylmaleide I and calphostin C), extracellular signal-regulated kinase 1/2 (ERK1/2) with PD98059 and heat shock protein 90 (Hsp90) with geldanamycin and radicicol significantly attenuated the protective effects of NaHS. Western blots showed that NaHS significantly stimulated ERK1/2 activation and Hsp90 expression. In conclusion, H₂S exerts a protective effect against cerebral hypoxia induced neuronal cell death via K_ATP/PKC/ERK1/2/Hsp90 pathway. Our findings emphasize the important neuroprotective role of H₂S in the brain during cerebral hypoxia.     

Early induction of c-Myc is associated with neuronal cell death

Neuronal cell cycle activation has been implicated in neurodegenerative diseases such as Alzheimer's disease, while the initiating mechanism of cell cycle activation remains to be determined. Interestingly, our previous studies have shown that cell cycle activation by c-Myc (Myc) leads to neuronal cell death which suggests Myc might be a key regulator of cell cycle re-entry mediated neuronal cell death. However, the pattern of Myc expression in the process of neuronal cell death has not been addressed. To this end, we examined Myc induction by the neurotoxic agents camptothecin and amyloid-β peptide in a differentiated SH-SY5Y neuronal cell culture model. Myc expression was found to be significantly increased following either treatment and importantly, the induction of Myc preceded neuronal cell death suggesting it is an early event of neuronal cell death. Since ectopic expression of Myc in neurons causes the cell cycle activation and neurodegeneration in vivo, the current data suggest that induction of Myc by neurotoxic agents or other disease factors might be a key mediator in cell cycle activation and consequent cell death that is a feature of neurodegenerative diseases.     

Mutated recombinant human glucagon-like peptide-1 protects SH-SY5Y cells from apoptosis induced by amyloid-β peptide (1–42)

Accumulation and deposition of amyloid β peptide (Aβ) in the brain causes neuronal apoptosis and eventually leads to Alzheimer's disease (AD). A therapeutic target for AD is to block the cascade reaction induced by Aβ. It has been demonstrated that glucagon-like peptide-1 (GLP-1), which is an endogenous insulinotropic peptide secreted from the gut, binds to its receptor in the brain and possesses neuroprotective effects. Using site-directed mutagenesis and gene recombination techniques, we generated a mutated recombinant human glucagon-like peptide-1 (mGLP-1) which has longer half-life as compared with native GLP-1. This present work aims to examine whether mGLP-1 attenuates Aβ_1–42-mediated cytotoxicity in SH-SY5Y cells and to explore the possible mechanisms. Our data indicate that ≥0.02 ng/ml of mGLP-1 facilitated cell proliferation and 0.1 ng/ml and 0.5 ng/ml of mGLP-1 rescued SH-SY5Y cells from Aβ_1–42-induced apoptosis. Moreover, Aβ_1–42 treatment dramatically stimulated the release of Ca²⁺ from internal calcium stores in SH-SY5Y cells, while mGLP-1 helped to maintain the intracellular Ca²⁺ homeostasis. Aβ_1–42 also significantly increased the expression level of TP53 and Bax genes which are involved in apoptotic pathways, and mGLP-1 decreased Aβ_1–42-induced up-regulation of TP53 and Bax. Since mGLP-1 treatment elevated cytosolic cAMP concentration in SH-SY5Y cells, we postulate that mGLP-1 may exert its influence via binding to GLP-1 receptors in SH-SY5Y cells and stimulating the production of cAMP. These results suggest that mGLP-1 exhibited neuronal protection properties, and could potentially be a novel therapeutic agent for intervention in Alzheimer's disease.     

H2O2-stimulated Ca2+ influx via TRPM2 is not the sole determinant of subsequent cell death

Activation of transient receptor potential melastatin 2 (TRPM2), a non-selective, Ca²⁺-permeable cation channel, is implicated in cell death. Channel opening is stimulated by oxidative stress, a feature of numerous disease states. The wide expression profile of TRPM2 renders it a potentially significant therapeutic target in a variety of pathological settings including cardiovascular and neurodegenerative diseases. HEK293 cells transfected with human TRPM2 (HEK293/hTRPM2) were more vulnerable to H₂O₂-mediated cell death than untransfected controls in which H₂O₂-stimulated Ca²⁺ influx was absent. Flufenamic acid partially reduced Ca²⁺ influx in response to H₂O₂ but had no effect on viability. N-(p-Amylcinnamoyl) anthranilic acid substantially attenuated Ca²⁺ influx but did not alter viability. Poly(adenosine diphosphate ribose) polymerase inhibitors (N-(6-oxo-5,6-dihydro-phenanthridin-2-yl)-N,N-dimethylacetamide, 3,4-dihydro-5-[4-(1-piperidinyl)butoxy]-1(2H)-isoquinolinone and nicotinamide) reduced Ca²⁺ influx and provided a degree of protection but also had some protective effects in untransfected controls. These data suggest H₂O₂ triggers cell death in HEK293/hTRPM2 cells by a mechanism that is in part Ca²⁺ independent, as blockade of channel opening (evidenced by suppression of Ca²⁺ influx) did not correlate well with protection from cell death. Determining the underlying mechanisms of TRPM2 activation is pertinent in elucidating the relevance of this channel as a therapeutic target in neurodegenerative diseases and other pathologies associated with Ca²⁺ dysregulation and oxidative stress.     

Sodium pyruvate protects against H2O2 mediated apoptosis in human neuroblastoma cell line-SK-N-MC

Free radicals are involved in neuronal damage. The present study was aimed to investigate the protective effect of sodium pyruvate—a free radical scavenger against hydrogen peroxide (H₂O₂) induced apoptosis in human neuroblastoma cell line-SK-N-MC. On exposure to H₂O₂ (0.025 mM) cells exhibited apoptosis within 24 h, demonstrating a high caspase 3 activity by 3 h followed by cleavage of PARP that was maximum at 24 h. A break down in the mitochondrial membrane potential was observed 3 h onwards. Sodium pyruvate protected cells significantly (P<0.05) against apoptosis in a dose dependent manner as assessed for cell viability by dye exclusion method and apoptosis by TUNEL. Sodium pyruvate significantly inhibited caspase 3 activity, cleavage of PARP and breakdown of mitochondrial membrane potential. These data suggest that sodium pyruvate protects neuronal damage caused by H₂O₂.     

Hydrogen peroxide induces autophagic cell death in C6 glioma cells via BNIP3-mediated suppression of the mTOR pathway

Oxidative stress by exposure to H₂O₂ induces various types of cell death depending on cell type and conditions. We report herein on a study of the mechanisms underlying H₂O₂-induced cell death in C6 glioma cells. The findings show that H₂O₂ triggers a caspase-independent autophagic cell death in these cells. The findings also show that H₂O₂ induces the dephosphorylation of the mammalian target of rapamycin (mTOR) at Ser 2481 and the p70 ribosomal protein S6 kinase (p70S6K) at Thr389 in a Bcl-2/E1B 19 kDa interacting protein 3 (BNIP3)-dependent manner. BNIP3 has the capacity to inhibit mTOR activity and mTOR inhibition plays a role in autophagic induction. This suggests that BNIP3 may mediate H₂O₂-induced autophagic cell death through the suppression of mTOR. The findings show that the down-regulation of BNIP3 by BNIP3 siRNA prevents C6 cells from undergoing H₂O₂-induced autophagic cell death. Collectively, these results suggest that H₂O₂ induces autophagic cell death in C6 cells via the BNIP3-mediated suppression of the mTOR pathway.     

Limited protection of TAT-Bcl-X(L) against pneumolysin-induced neuronal cell death

Severe brain damage in patients with pneumococcal meningitis is in part caused by the cytosolic pneumococcal protein pneumolysin. The devastating effect of this neurotoxin might be alleviated by interfering with the cell death pathways that it sets in motion. An important player in these pathways is Bcl-X(L), an antiapoptotic protein of the Bcl-2 family, which is neuroprotective in various in vitro and in vivo models of cell death. We investigated whether its membrane-permeable form, the TAT-Bcl-X(L) fusion protein, is capable of protecting human SH-SY5Y neuroblastoma cells against pneumolysin-induced cell death. Under mild pneumolysin-induced neuronal injury, TAT-Bcl-X(L) increased cell viability significantly by approximately 40% (82.7 +/- 16.1% versus 70.0+/-8.2%; p = 0.04). When the cells were exposed to a more rigorous pneumolysin treatment, TAT-Bcl-X(L) had no protective effects. This suggests the involvement of additional neuronal death pathways in pneumolysin-induced cell death, which are not controlled by Bcl-X(L). Therefore, Bcl-X(L), a promising therapeutic candidate for ischemia and neurodegenerative diseases, is only of partial efficacy in preventing the direct neurotoxicity of pneumolysin.     

Prion peptide-mediated cellular prion protein overexpression and neuronal cell death can be blocked by aspirin treatment

Prion diseases are infectious neurodegenerative disorders characterized by the conversion of the cellular prion protein (PrPc) to the misfolded isoform (PrPsc). Prion peptide PrP 106-126 [PrP (106-126)] shares many physiological properties with PrPsc; it is neurotoxic in vitro and in vivo. PrP (106-126) induces neurotoxicity by the overexpression of PrPc and activation of the mitogen-activated protein (ERK1/2). Aspirin, an anti-inflammatory drug, is a known ERK inhibitor and prevents neurodegenerative disorders including prion diseases. The influence of aspirin treatment on prion protein-mediated neurotoxicity and expression of PrPc were the focus of this study. Cell viability and apoptosis were assessed by crystal violet staining and the TUNEL and DNA fragmentation assays. Apoptosis-associated protein expression of PrPc, p-53, p-ERK1/2, p-p38, Bcl-2 and cleaved-caspase-3 was examined by Western blotting and immunocytochemistry. Aspirin treatment inhibited PrP (106-126)-induced neuronal cell death in SH-SY5Y neuroblastoma cells. In addition, the PrP (106-126)-mediated increase of p-p38, p53, cleaved-caspase-3 and decrease of Bcl-2 expressions were blocked by aspirin and the ERK inhibitor, PR98059. Furthermore, we showed that the PrP (106-126)-mediated increase of PrPc and p-ERK1/2 were inhibited by PD98059 and aspirin. Taken together, these results demonstrate that ERK1/2 is a key modulator of the protective effect of aspirin on PrP-106-126-mediated cellular prion protein overexpression and neurotoxicity and also suggest that aspirin may prevent neuron cell damages caused by the prion peptide.     

Sigma 1 receptor stimulation protects against oxidative damage through suppression of the ER stress responses in the human lens

Stimulation of sigma-1 receptors is reported to protect against oxidative stress. The present study uses cells and tissue from the human lens to elucidate the relationship between the sigma 1 receptor, ER stress and oxidative stress-induced damage. Exposure of the human lens cell line FHL124 to increasing concentrations of H₂O₂ led to reduced cell viability and increased apoptosis. In response to 30 μM H₂O₂, levels of the ER stress proteins BiP, ATF6 and pEIF2α were significantly increased within 4 h of exposure. Expression of the sigma 1 receptor was markedly increased in response to H₂O₂. Application of 10 and 30 μM (+)-pentazocine, a sigma 1 receptor agonist, significantly inhibited the H₂O₂ induced cell death. (+)-Pentazocine also suppressed the oxidative stress induced reduction of pro-caspase 12 and suppressed the induction of the ER stress proteins BiP and EIF2α. When applied to cultured human lenses, (+)-pentazocine protected against apoptotic cell death, LDH release and against H₂O₂ induced opacification. These data demonstrate that stimulation of the sigma 1 receptor provides significant protection from oxidative damage and is, therefore, a putative therapeutic approach to delay the onset of diseases that may be triggered by oxidative damage, including cataract formation.     

Insulin-like growth factor-1 protects against prion peptide-induced cell death in neuronal cells via inhibition of Bax translocation

Insulin-like growth factor-1 (IGF-1) is one of the most important components of bovine colostrum. It exhibits antiapoptotic and antioxidative activities. Prion diseases are neurodegenerative disorders caused by cell death through mitochondrial dysfunction and increasing generation of reactive oxygen species (ROS). This study examined the protective effect of IGF-1 on residues 106-126 of the cellular prion protein [PrP (106-126)]-mediated mitochondrial neurotoxicity and oxidative stress. In SH-SY5Y human neuronal cells, treatment with PrP (106-126) decreased the cell viability and IGF-1 pretreatment markedly blocked the PrP?(106-126)-induced neuronal cell death. IGF-1 inhibited PrP?(106-126)-induced intracellular ROS generation and mitochondrial oxidative stress. In addition, IGF-1 blocked the translocation of the Bax protein to the mitochondria induced by PrP (106-126). These results demonstrate that IGF-1 protects neuronal cells against PrP (106-126)-mediated neurotoxicity through an antioxidative effect and blockage of mitochondrial Bax translocation. The results also suggest that regulation of IGF-1 secretion may have a therapeutic potential in the management of mitochondrial dysfunction and oxidative stress-induced neurodegeneration.     

Pigment epithelium-derived factor (PEDF) protects cortical neurons in vitro from oxidant injury by activation of extracellular signal-regulated kinase (ERK) 1/2 and induction of Bcl-2

Mitigating oxidative stress-induced damage is critical to preserve neuronal function in diseased or injured brains. This study explores the mechanisms contributing to the neuroprotective effects of pigment epithelium-derived factor (PEDF) in cortical neurons. Cultured primary neurons are exposed to PEDF and H₂O₂ as well as inhibitors of phosphoinositide-3 kinase (PI3K) or extracellular signal-regulated kinase 1/2 (ERK1/2). Neuronal survival, cell death and levels of caspase 3, PEDF, phosphorylated ERK1/2, and Bcl-2 are measured. The data show cortical cultures release PEDF and that H₂O₂ treatment causes cell death, increases activated caspase 3 levels and decreases release of PEDF. Exogenous PEDF induces a dose-dependent increase in Bcl-2 expression and neuronal survival. Blocking Bcl-2 expression by siRNA reduced PEDF-induced increases in neuronal survival. Treating cortical cultures with PEDF 24 h before H₂O₂ exposure mitigates oxidant-induced decreases in neuronal survival, Bcl-2 expression, and phosphorylation of ERK1/2 and also reduces elevated caspase 3 level and activity. PEDF pretreatment effect on survival is blocked by inhibiting ERK or PI3K. However, only inhibition of ERK reduced the ability of PEDF to protect neurons from H₂O₂-induced Bcl-2 decrease and neuronal death. These data demonstrate PEDF-mediated neuroprotection against oxidant injury is largely mediated via ERK1/2 and Bcl-2 and suggest the utility of PEDF in preserving the viability of oxidatively challenged neurons.