JNK pathway is required for retinoic acid-induced neurite outgrowth of human neuroblastoma,SH-SY5Y

Neurite outgrowth is a central event of neuronal differentiation that proceeds in multiple processes requiring various cellular factors. Here we demonstrated that c-Jun N-terminal kinase 1 (JNK1) plays an essential role in RA-induced neurite outgrowth of SH-SY5Y cells. Treatment of SH-SY5Y cells with RA induced a strong activation of JNK1 within 10 min, and the immediate increase of JNK1 activity returned to the basal level in an hour. The second surge of JNK1 activity was observed around 1 day after RA treatment, which coincided with the period of extensive neurite outgrowth. Interestingly, phospho-JNK was concentrated in the nucleus of cells during the early induction, whereas it was distributed into neurite processes during the delayed second activation period. In SH-SY5Y carrying a dominant negative form of SEK1, an upstream kinase of JNK1, both early and late inductions of JNK1 activity were repressed along with RA-induced neurite outgrowth. These results suggest that JNK1 plays an essential role in RA-induced neuronal differentiation of SH-SY5Y cells.     

Relationship Between Aldose Reductase And Oxidative Stress In Diabetic Peripheral Nerve

It has been suggested that perturbed insulin-like growth factors (IGFs) may be involved in CNS complications of diabetes. We examined IGF messages, hippocampal apoptosis and the effect of C-peptide replacement on encephalopathy in type-1 diabetic BB/Wor-rats. By Northern blot analysis, we found decreased (50%) mRNA expression of IGF-I, IGF-II and IGF-I receptor (IGF-IR) in 2-month diabetic rats. In 6-month diabetic rats, these changes were associated with positive TUNEL staining, nucleosomal DNA fragmentation and an elevated (∼2.5-fold) ratio of Bax/Bcl-xL in hippocampus as assessed by Western blotting and LM-PCR assays. C-peptide replacement (75 ng/kg body weight/day) partially corrected the expression of IGF-I, IGF-II and IGF-IR and eliminated hippocampal apoptosis. To investigate the mechanisms by which C-peptide exerts its antiapoptotic effect, human neuroblastoma SH-SY5Y cells were incubated in high concentrations of glucose (300 mM), which caused significant apoptosis of SH-SY5Y cells as shown by nuclear shrinkage, positive TUNEL staining and DNA laddering. This increased apoptotic activity was delayed in SH-SY5Y cells to which 10 nM C-peptide had been added. In the same cell system, the insulin-induced effect on PI-3 kinase activity was enhanced by C-peptide, in keeping with the notion that PI-kinase plays a role in antiapoptotic signaling. We therefore conclude that insulin and C-peptide deficiencies in type-1 diabetic BB/Wor-rats are associated with hippocampal apoptosis, which can be prevented by C-peptide replacement.     

Nogo-A在维甲酸诱导SH-SY5Y细胞分化后的表达分布

目的研究Nogo-A在维甲酸(RA)诱导SH-SY5Y细胞分化后的表达及分布。方法采用免疫荧光单标记法和免疫荧光双标记法染色,显微镜观察。结果SH-SY5Y细胞经RA诱导后,细胞生长出较长的突起,具有典型的神经元形态。Nogo-A在未分化的SH-SY5Y细胞中主要分布于胞浆中。经RA诱导后,Nogo-A分布于胞浆及突起中,尤以突起末梢生长锥处免疫阳性强度高。结论Nogo-A在RA诱导SH-SY5Y细胞分化后的轴丘和生长锥处的表达提示其在神经元突起生长过程中可能起到重要作用。     

Neuronal protection from glucose deprivation via modulation of glucose transport and inhibition of apoptosis: a role for the insulin-like growth factor system

Glucose is the brain's major energy source; therefore, loss of neuronal cells is a potential consequence of hypoglycaemia. Since apoptosis is a major mechanism of neuronal loss following a range of insults, we explored potent anti-apoptotic systems (IGF-I and bcl-2) as means of enhancing neuronal survival in the face of glucose deprivation. Human neuroblastoma cells (SH-SY5Y, SHEP and SHEP-bcl-2) were exposed to low glucose as a model of glucopenia-induced neuronal damage. Administration of IGF-I and/or over-expression of the survival gene bcl-2 were exploited to attempt to limit neuronal loss. Neuronal survival mechanisms and interactions between these systems were investigated. Low glucose (0.25-2.5 mM) adversely affected cell growth and survival; however, IGF-I ameliorated these outcomes. Over-expression of bcl-2 blunted low glucose-induced apoptosis and up-regulated IGF-I receptor, with the effect of IGF-I addition being negligible on apoptosis, while significantly enhancing mitochondrial activity. In SH-SY5Y cells, IGF-I significantly changed >two-fold mRNA levels of the apoptosis-related genes gadd45, fas, iNOS, NFkB, TRAIL, without further affecting bcl-2 expression. In low glucose, IGF-I acutely enhanced glucose transport and translocation of GLUT1 protein to the cell membrane. GLUT1 mRNA expression was up-regulated by both IGF-I and bcl-2. The potent anti-apoptotic systems IGF-I and bcl-2 are both thus able to enhance cell survival in a glucose-deprived human neuronal model. Although we clearly show evidence of positive cross-talk via bcl-2 modulation of IGF-I receptor, IGF-I also has enhancing effects on mitochondrial function outside the bcl-2 pathway. The common effect of both systems on enhancement of GLUT-1 expression suggests that this is a key mechanism for enhanced survival. These studies also point to the potential use of IGF-I therapy in prevention or amelioration of hypoglycaemic brain injury.     

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.     

花生四烯酰多巴胺对H2O2诱导SH-SY5Y细胞氧化应激损伤的保护作用

目的 探讨花生四烯酰多巴胺(N-arachidonoyl dopamine,NADA)对H₂O₂诱导的人神经母细胞瘤SH-SY5Y细胞氧化损伤的保护作用。方法 H₂O₂处理SH-SY5Y细胞制作细胞氧化损伤模型,通过CCK8法检测细胞活力,明确不同水平的NADA对细胞存活的影响,分析NADA对SH-SY5Y细胞的保护作用; 利用生化方法检测丙二醛(MDA)和乳酸脱氢酶(LDH)的水平; 运用DCFH-DA荧光探针检测细胞内氧自由基(ROS)水平; Hoechst33342/PI双染法检测NADA对细胞氧化损伤的保护作用; 蛋白免疫印迹实验检测抗凋亡蛋白bcl-2的表达水平。结果 H₂O₂处理SH-SY5Y细胞可导致细胞活力降低,增加MDA水平和LDH活力,促进ROS的产生,降低抗凋亡蛋白bcl-2的表达,加剧细胞凋亡。NADA可剂量依赖性提高H₂O₂诱导的SH-SY5Y细胞的存活率、降低MDA水平和LDH活力以及ROS的产生,促进抗凋亡蛋白bcl-2的表达,降低细胞凋亡。结论 NADA对H₂O₂诱导的细胞氧化损伤模型具有保护作用,其机制可能是通过抑制胞内氧化应激,促进抗凋亡蛋白bcl-2的表达,从而减少神经细胞凋亡。     

Down-regulation of amyloid-β through AMPK activation by inhibitors of GSK-3β in SH-SY5Y and SH-SY5Y-AβPP695 cells

Glycogen synthase kinase 3β (GSK-3β) plays a critical role in the pathogenesis of Alzheimer's disease (AD), implicating amyloid-β (Aβ) production, neurofibrillary tangle formation, and neuronal apoptosis. The activation of 5' AMP-activated protein kinase (AMPK) has been linked to aberrant processing of amyloid-β protein precursor (AβPP), and AMPK signaling controls Aβ metabolism. It is possible that GSK-3β regulated the activation of the AMPK pathway. To test this hypothesis, the influence of GSK-3β on the expression of AβPP cleavage enzyme (BACE), Aβ, and AMPK in the SH-SY5Y and AβPP695 cells line through three inhibitors of GSK-3β was analyzed. Expression of Aβ, AMPK, and pAMPK172 was measured by Western blot, and BACE was tested by Western blot and RT-PCR. This study demonstrated that suppression of GSK-3β activity, through specific inhibitors, dramatically down-regulated Aβ generation in human SH-SY5Y and SH-SY5Y-AβPP695 cells by enhancing AMPK activity to down-regulate Aβ. These results suggest GSK-3β inhibitors may be promising agents in the prevention and treatment of AD.     

Dihydroactinidiolide regulates Nrf2/HO-1 expression and inhibits caspase-3/Bax pathway to protect SH-SY5Y human neuroblastoma cells from oxidative stress induced neuronal apoptosis

Alzheimer’s disease (AD) etiology has been studied for a long time and it is found to be multifaceted involving the accumulation of amyloid β and tau protein. Oxidative stress is an early event in AD associated neurodegeneration provoking neuronal death through mitochondrial dysfunction and activation of caspase-3. Therefore we tested the efficacy of dihydroactinidiolide (DHAc), a monoterpene lactone against the oxidative load involved in AD like pathological conditions induced by sodium dithionite, glutamate, amyloid β and colchicine in SH-SY5Y cells. Some of the indicators of neurotoxicity like acetylcholinesterase activity, intracellular reactive oxygen species (ROS), nitrite content, lipid peroxidation, protein carbonylation, nuclear and membrane damage were found to be significantly high in the toxicant treated cells when compared to the control cells while DHAc pretreatment significantly restored the toxicant induced neuronal damage signatures. Caspase-3 activity was found to be increased in the toxicant treated cells while DHAc significantly reduced it. Western blotting and RT-PCR revealed that DHAc significantly increased anti-apoptotic Bcl-2 expression and mRNA levels of Nrf2 and HO-1. Therefore DHAc was found to protect SH-SY5Y cells from neurotoxicant induced oxidative stress and apoptosis by regulating cellular antioxidant defenses and apoptosis related genes.     

Differential effects of staurosporine and retinoic acid on the vulnerability of the SH-SY5Y neuroblastoma cells: involvement of bcl-2 and p53 proteins.

Human catecholaminergic neuroblastoma cells (SH-SY5Y) have been widely used in different neurochemical investigations. Quite often these cells are induced to differentiation by various agents, such as staurosporine and retinoic acid. Interestingly, even though both staurosporine and retinoic acid induce similar morphological differentiation in SH-SY5Y cells, we found that these two groups of differentiated cells exhibited opposite vulnerability to harmful chemicals and physical insults. In the present study, cisplatin, 5-fluorouracil (5-FU), N-(2-chloroethyl)-N-ethyl-2-bromobenzylamine (DSP-4), 6-hydroxydopamine (6-OHDA), and gamma-radiation were used to assess the tolerance of the differentiated cells. Cell viability was determined by 3-(4,5-dimethylthiazol-2yl)-2, 5-diphenyltetrazolium bromide (MTT) assay. Staurosporine-treated SH-SY5Y cells were more sensitive to these toxic insults than the untreated controls. In contrast, retinoic acid-treated cells became more resistant to the same treatments. The expression of the proteins of the protooncogene Bcl-2 and the tumor suppressor gene p53 following staurosporine or retinoic acid treatment was assessed by Western blot and immunocytochemistry. Retinoic acid increased Bcl-2 and decreased p53 levels, whereas staurosporine decreased Bcl-2 and increased p53 levels. The opposite alteration of Bcl-2 (anti-apoptotic) and p53 (apoptotic) contents in SH-SY5Y cells with retinoic acid and staurosporine are attributed to the changes in cell vulnerability. These observations also indicate that caution should be taken when chemically induced differentiated neuroblastoma cells are to be used as an in vitro model for studying neuronal survival.     

Gas1 reduces Ret tyrosine 1062 phosphorylation and alters GDNF-mediated intracellular signaling

The present results show that the expression of Growth Arrest Specific1 (Gas1) in SH-SY5Y neuroblastoma cells significantly inhibits the increased phosphorylation of tyrosine 1062 of the Ret receptor tyrosine kinase induced by glial-cell-line-derived neurotrophic factor (GDNF). We also observed that Gas1 significantly reduces the activation of Akt. GDNF and members of its family of ligands (GFLs), signal through a molecular complex consisting of one of its receptors (GFRs) and the Ret receptor tyrosine kinase. GDNF is a key component to preserve several cell populations in the nervous system, including dopaminergic and motor neurons, and also participates in the survival and differentiation of peripheral neurons such as enteric, sympathetic and parasympathetic. On the other hand, Gas1 is a molecule involved in cell arrest that can induce apoptosis when over-expressed in different cell lines, including cells of neuronal and glial origin. Although, Gas1 is widely expressed during development, its role in vivo has not yet been clearly defined. We recently showed the structural homology between Gas1 and GFRs, thus suggesting that the physiological role of Gas1 is that of modulating the biological responses induced by GDNF and/or other members of this family of signaling molecules. The results of this work are consistent with the hypothesis of Gas1 acting as a negative modulator of GDNF signaling.     

Protein kinase C-α and -ε are enriched in growth cones of differentiating SH-SY5Y human neuroblastoma cells

SH-SY5Y cells differentiate into neuronal-like cells and express marker proteins like growth-associated protein (GAP-43) and neuropeptide tyrosine when treated with a low concentration (16 nM) of the phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA) in the presence of growth factors or serum. Both control and differentiated cells expressed protein kinase C-α (PKC-α), PKC-ε, and PKC-ζ as revealed by Western blot analyses, but the subcellular distribution of the three isoforms was not uniform, indicating specific localized functions of the enzymes. In growth cones prepared from differentiating cells PKC-α and PKC-ε were enriched. In contrast, PKC-ζ was more evenly distributed within the differentiating cell. Cells treated with a high concentration of TPA (1.6 μM) differentiate poorly and continue to proliferate. In those cells, PKC-α and PKC-ε were found to be down-regulated while PKC-ζ remained present. Thus, down-regulation of PKC-α and PKC-ζ appears to be incompatible with neuronal differentiation of SH-SY5Y cells. These cells also differentiate when treated with a combination of basic fibroblast growth factor and insulin-like growth factor I. Growth cones isolated from such cells are also enriched in PKC-α and PKC-ε, but not in PKC-ζ. Based on the subcellular distribution of PKC-α and ε, and that PKC substrates like GAP-43 and pp60^c-src are enriched in SH-SY5Y growth cones, a role during neurite growth is suggested. © 1995 Wiley-Liss, Inc.     

Vasoactive intestinal peptide-induced neuritogenesis in neuroblastoma SH-SY5Y cells involves SNAP-25

Vasoactive intestinal peptide (VIP) is a neuropeptide known to regulate proliferation and differentiation in normal and tumoral cells. We previously reported that VIP induced neuritogenesis in human neuroblastoma SH-SY5Y cells cultured in serum-free medium. This neuritogenesis was associated with a regulated expression of neuronal cytoskeleton markers. To further characterize the neuroblastic cell differentiation induced by VIP in human SH-SY5Y cells, we investigated expression of synaptosomal-associated protein of 25 kDa (SNAP-25), a protein implicated in exocytosis associated with different processes, including neurite outgrowth. Western immunoblotting and real-time RT-PCR analyses revealed that VIP increased expression of the SNAP-25 protein and the level of both SNAP-25a and SNAP-25b mRNA isoforms. Immunofluorescence experiments indicated that SNAP-25 was mainly located in neurites and at the plasma membrane in SH-SY5Y cells treated with VIP. RNA interference experiments demonstrated that SNAP-25 was involved in VIP-induced neuritogenesis. In conclusion, SNAP-25 is up-regulated and implicated in neuritogenesis in human neuroblastoma SH-SY5Y cells treated with the neuropeptide VIP.     

RA Differentiation Enhances Dopaminergic Features,Changes Redox Parameters,and Increases Dopamine Transporter Dependency in 6-Hydroxydopamine-Induced Neurotoxicity in SH-SY5Y Cells

Research on Parkinson’s disease (PD) and drug development is hampered by the lack of suitable human in vitro models that simply and accurately recreate the disease conditions. To counteract this, many attempts to differentiate cell lines, such as the human SH-SY5Y neuroblastoma, into dopaminergic neurons have been undertaken since they are easier to cultivate when compared with other cellular models. Here, we characterized neuronal features discriminating undifferentiated and retinoic acid (RA)-differentiated SH-SYSY cells and described significant differences between these cell models in 6-hydroxydopamine (6-OHDA) cytotoxicity. In contrast to undifferentiated cells, RA-differentiated SH-SY5Y cells demonstrated low proliferative rate and a pronounced neuronal morphology with high expression of genes related to synapse vesicle cycle, dopamine synthesis/degradation, and of dopamine transporter (DAT). Significant differences between undifferentiated and RA-differentiated SH-SY5Y cells in the overall capacity of antioxidant defenses were found; although RA-differentiated SH-SY5Y cells presented a higher basal antioxidant capacity with high resistance against H₂O₂ insult, they were twofold more sensitive to 6-OHDA. DAT inhibition by 3α-bis-4-fluorophenyl-methoxytropane and dithiothreitol (a cell-permeable thiol-reducing agent) protected RA-differentiated, but not undifferentiated, SH-SY5Y cells from oxidative damage and cell death caused by 6-OHDA. Here, we demonstrate that undifferentiated and RA-differentiated SH-SY5Y cells are two unique phenotypes and also have dissimilar mechanisms in 6-OHDA cytotoxicity. Hence, our data support the use of RA-differentiated SH-SY5Y cells as an in vitro model of PD. This study may impact our understanding of the pathological mechanisms of PD and the development of new therapies and drugs for the management of the disease.     

Differential expression and function of apoptosis-associated tyrosine kinase (AATYK) in the developing mouse brain

Apoptosis-associated tyrosine kinase (AATYK) is a non-receptor type tyrosine kinase that is predominantly expressed in adult mouse brain. Although it is also expressed in developing brains, its expression pattern and physiological functions are unclear. In the present study, we analyzed expression profiles of AATYK in developing mouse brains and its functional role and subcellular localization in cultured cerebellar granule cells. Expression of AATYK mRNA and protein increased during postnatal brain development. Immunohistochemical analysis indicated that the protein was differentially expressed in postmitotic neurons within various brain areas including the olfactory bulb, cerebral cortex, hippocampus, thalamus, colliculus, cerebellum, and brain stem. Developmental increases in its expression were also observed in cultured cerebellar granule cells. AATYK protein was largely fractionated into the microsomal fraction and was immunocytochemically distributed in an ER-like meshwork of the granule cell soma, suggesting a possible association with the ER membrane. AATYK protein was also present in neurites. In immature granule cells, overexpression of wild-type AATYK promoted neurite outgrowth, whereas that of tyrosine kinase-defective mutant significantly inhibited it. These results suggest that, in addition to its role in cell death in mature neurons, AATYK has a unique role in promoting neurite extension through its tyrosine kinase activity in developing neurons.     

Presenilin 1 mediates retinoic acid-induced differentiation of SH-SY5Y cells through facilitation of Wnt signaling

Presenilin 1 interacts with beta-catenin, an essential component of the Wnt signaling pathway. To elucidate the role of presenilin 1-beta-catenin interaction in neuronal differentiation, we established SH-SY5Y cells stably expressing wild-type presenilin 1, P117L mutant presenilin 1, which is linked to the early-onset familial form of Alzheimer's disease, and D385A mutant presenilin 1, which has no aspartyl proteinase activity. We demonstrate that SH-SY5Y cells stably expressing D385A mutant presenilin 1 failed to differentiate in response to retinoic acid treatment. Retinoic acid caused an increase in nuclear beta-catenin levels in SH-SY5Y cells, which was followed by an increase in cyclin D1 protein levels. Abnormal cellular accumulation of beta-catenin was observed in D385A mutant transfected cells, whereas nuclear beta-catenin and cellular cyclin D1 levels failed to increase. Conversely, SH-SY5Y cells expressing the P117L mutant differentiated normally and showed increased nuclear beta-catenin and cellular cyclin D1 levels. These findings suggest that neuronal differentiation of SH-SY5Y cells involves the Wnt signaling pathway and that presenilin 1 plays a crucial role in Wnt signal transduction by regulating the nuclear translocation of beta-catenin.     

Effects of optineurin mutants on SH-SY5Y cell survival

Mutations in the optineurin gene (OPTN) have been found to be associated with glaucoma and amyotrophic lateral sclerosis (ALS). However, the mechanism by which this single gene mutation leads to neurodegeneration in those two diseases remains unrevealed. To study the roles of wild-type (WT) OPTN and its pathogenic mutants in neuronal survival, here we overexpressed SH-SY5Y cells with WT OPTN or its four mutants (E50K, M98K, Q398X and E478G), and detected their effects on neuronal viability under normal or oxidative stress conditions. We found that overexpression of WT OPTN or its glaucoma-linked mutants (E50K and M98K) causes little harm in SH-SY5Y cells, while ALS-associated OPTN mutants (Q398X and E478G) leads to remarkably increased oxidative status and decreased antioxidase activity, which might result in severe mitochondrial dysfunction and neuronal injury. Further investigation suggests that overexpression of WT OPTN promotes endogenous antioxidase activation in the SH-SY5Y cells under oxidative stress and increases neuronal survival. Nevertheless, this neuroprotective effect of WT OPTN is abolished by its four mutants. Our results indicate that oxidative stress may play a central role in the pathogenesis of glaucoma and ALS caused by OPTN mutation.     

Akt activity in presenilin 1 wild-type and mutation transfected human SH-SY5Y neuroblastoma cells after serum deprivation and high glucose stress.

The majority of early-onset familial Alzheimer disease cases are caused by mutations in the genes encoding presenilin 1 (PS1) and presenilin 2 (PS2). Presenilin mutations have been hypothesised to cause Alzheimer disease either by altering amyloid precursor protein metabolism or by increasing the vulnerability of neurons to undergo death by apoptosis. We showed previously that PS1 exon 9 deletion (PS1 DeltaE9) and L250S mutations predispose SH-SY5Y neuroblastoma cells to high glucose stress-induced apoptosis and that the anti-apoptotic effect of insulin-like growth factor I (IGF-I) is compromised by these mutations. The present study investigates whether the susceptibility of PS1 mutation transfected SH-SY5Y cells to undergo apoptosis is likely due to a downregulation of Akt/protein kinase B (Akt), a key intermediate in the phosphatidylinositol 3 (PI3)-kinase arm of the IGF-I signaling pathway. We used two methods to determine the regulation of Akt in response to the pro-apoptotic stimuli of serum deprivation and high glucose stress, as well as treatment with IGF-I. We also looked at the phosphorylatiom state of GSK-3beta at Ser9. Using a kinase assay with immunoprecipitated Akt, we detected an increased Akt activity in PS1 L250S cells at 1 hr after the combination of 20 mM glucose plus 10 nM IGF-I, when compared to the other cell types. This effect, however, was transient in that no mutation related differences were seen at either 6- or 24-hr post-treatment. Immunoblotting for Phospho-Akt as a ratio of total Akt, as well as for GSK-3beta phosphorylated at Ser9 revealed no apparent between cell type and treatment differences. This data strongly indicates that PS1 wt and mutant cells show no major differences in the pattern of Akt regulation after exposure to the pro-apoptotic stimuli of either serum deprivation or high glucose stress, or treatment with IGF-I. It is suggested that another component of IGF-I signaling is likely disrupted in these cells to increase their vulnerability to undergo death by apoptosis.     

Autophagy Activation Alleviates Amyloid-β-Induced Oxidative Stress,Apoptosis and Neurotoxicity in Human Neuroblastoma SH-SY5Y Cells

Autophagy is an evolutionary conserved catabolic process that ensures continuous removal of damaged cell organelles and long-lived protein aggregates to maintain cellular homeostasis. Although autophagy has been implicated in amyloid-β (Aβ) production and deposition, its role in pathogenesis of Alzheimer’s disease remains elusive. Thus, the present study was undertaken to assess the cytoprotective and neuroprotective potential of autophagy on Aβ-induced oxidative stress, apoptosis and neurotoxicity in human neuroblastoma SH-SY5Y cells. The treatment of Aβ1-42 impaired the cell growth and redox balance, and induced apoptosis and neurotoxicity in SH-SY5Y cells. Next, the treatment of rapamycin (RAP) significantly elevated the expression of autophagy markers such as microtubule-associated protein-1 light chain-3 (LC3), sequestosome-1/p62, Beclin-1, and unc-51-like kinase-1 (ULK1) in SH-SY5Y cells. RAP-induced activation of autophagy notably alleviated the Aβ1-42-induced impairment of redox balance by decreasing the levels of pro-oxidants such as reactive oxygen species, lipid peroxidation and Ca²⁺ influx, and concurrently increasing the levels of antioxidant enzymes such as superoxide dismutase and catalase. The RAP-induced autophagy also ameliorated Aβ1-42-induced loss of mitochondrial membrane potential and apoptosis. Additionally, the activated autophagy provided significant neuroprotection against Aβ1-42-induced neurotoxicity by elevating the expression of neuronal markers such as synapsin-I, PSD95, NCAM, and CREB. However, 3-methyladenine treatment significantly exacerbated the neurotoxic effects of Aβ1-42. Taken together, our study demonstrated that the activation of autophagy provided possible neuroprotection against Aβ-induced cytotoxicity, oxidative stress, apoptosis, and neurotoxicity in SH-SY5Y neuronal cells.     

Magnolol Protects Against Oxidative Stress-Mediated Neural Cell Damage by Modulating Mitochondrial Dysfunction and PI3K/Akt Signaling

Magnolol, an orally available compound from Magnolia officinalis used widely in traditional herbal medicine against a variety of neuronal diseases, possesses potent antioxidant properties and protects the brain against oxidative damage. The aim of the work is to examine the protective mechanisms of magnolol on human neuroblastoma SH-SY5Y cells against apoptosis induced by the neurotoxin acrolein, which can cause neurodegenerative disorders by inducing oxidative stress. By investigating the effect of magnolol on neural cell damage induced by the neurotoxin acrolein, we found that magnolol pretreatment significantly attenuated acrolein-induced oxidative stress through inhibiting reactive oxygen species accumulation caused by intracellular glutathione depletion and nicotinamide adenine dinucleotide phosphate oxidase activation. We next examined the signaling cascade(s) involved in magnolol-mediated antiapoptotic effects. The results showed that acrolein induced SH-SY5Y cell apoptosis by activating mitochondria/caspase and MEK/ERK signaling pathways. Our findings provide the first evidence that magnolol protects SH-SY5Y cells against acrolein-induced oxidative stress and prolongs SH-SY5Y cell survival through regulating JNK/mitochondria/caspase, PI3K/MEK/ERK, and PI3K/Akt/FoxO1 signaling pathways.