Protective effect of pyrroloquinoline quinone against Aβ-induced neurotoxicity in human neuroblastoma SH-SY5Y cells

The neurotoxicity of aggregated β-amyloid (Aβ) has been implicated as a critical cause in the pathogenesis of Alzheimer's disease (AD). It can cause neurotoxicity in AD by evoking a cascade of oxidative damage-dependent apoptosis to neurons. In the present study, we for the first time investigated the protective effect of pyrroloquinoline quinone (PQQ), an anionic, water soluble compound that acts as a redox cofactor of bacterial dehydrogenases, on Aβ-induced SH-SY5Y cytotoxicity. Aβ_25–35 significantly reduced cell viability, increased the number of apoptotic-like cells, and increased ROS production. All of these phenotypes induced by Aβ_25–35 were markedly reversed by PQQ. PQQ pretreatment recovered cells from Aβ_25–35-induced cell death, prevented Aβ_25–35-induced apoptosis, and decreased ROS production. PQQ strikingly decreased Bax/Bcl-2 ratio, and suppressed the cleavage of caspase-3. These results indicated that PQQ could protect SH-SY5Y cells against β-amyloid induced neurotoxicity.     

Galantamine inhibits calpain–calcineurin signaling activated by beta-amyloid in human neuroblastoma SH-SY5Y cells

Galantamine, which is currently used in the treatment of patients with Alzheimer's disease (AD), has been shown to have a neuroprotective effect against beta-amyloid (Aβ) peptide-induced toxicity, which is involved in the pathogenesis of AD. In this study, we investigated the mechanism underlying the protective effect of galantamine on Aβ-induced toxicity in human neuroblastoma cells (SH-SY5Y). Using MTT and LDH leakage assays, we observed that galantamine pretreatment significantly prevented Aβ1-40-induced cell death. Aβ1-40-induced overexpression and increased cleavage of both calpain and calcineurin were observed by Western blotting and double immunofluorescent staining. Increased calcineurin phosphatase activity and decreased level of pSer112 BAD were also observed in Aβ1-40-damaged cells. However, all these alterations were found to be reversed by galantamine pretreatment. We also found that the neuroprotection of galantamine can be blocked by an α7 nAChR antagonist. Overall, our results suggest that galantamine may prevent the neuronal damage induced by Aβ1-40 through a mechanism related to the regulation of calpain–calcineurin activation and BAD phosphorylation, which may involve the participation of α7 nAChR.     

Indirubin-3-monoxime exhibits anti-inflammatory properties by down-regulating NF-κB and JNK signaling pathways in lipopolysaccharide-treated RAW264.7 cells

Objective  

Indirubin-3-monoxime (I3M), an indirubin analogue that shows favorable inhibitory activity targeting cyclin-dependent kinase and glycogen synthase kinase, exhibits various biological properties, including chemopreventive, antiangiogenic, and neuropreventive activities. In the present study, we investigated the ability of I3M to regulate inflammatory reactions in macrophages.     

Autophagy protects the rotenone-induced cell death in α-synuclein overexpressing SH-SY5Y cells

Loss of dopaminergic cells induced by α-synuclein accumulation in substantia nigra causes the development of Parkinson's disease (PD). To date, although autophagy has been implicated in the pathology of PD, the molecular mechanism is still unclear. To study the role of autophagy in PD pathogenesis, we established stable SH-SY5Y cell lines overexpressing wild-type or mutant α-synuclein proteins (A30P or A53T). Overexpression of mutant α-synuclein induced some protein aggregates and cell death in the absence of drug. LC3-II protein, a critical marker for autophagy, was produced in an autophagy-dependent manner. The rotenone-induced cell death was interrupted by autophagy stimulation. Autophagy activation also restored the mitochondrial membrane potential (MMP) impaired by rotenone in mutant α-synuclein expressing cells. Additionally, autophagy activation significantly relieved rotenone-induced ROS accumulation and HIF-1α expression in neuronal cells expressing mutant α-synuclein proteins. These findings indicate that autophagy plays an important scavenger role against harmful influence of toxic protein aggregates produced in rotenone-treated cells.     

The marijuana component cannabidiol inhibits β-amyloid-induced tau protein hyperphosphorylation through Wnt/β-catenin pathway rescue in PC12 cells

Alzheimer’s disease (AD) is the most common age-related neurodegenerative disorder. A massive accumulation of β-amyloid (Aβ) peptide aggregates has been proposed as pivotal event in AD. Aβ-induced toxicity is accompanied by a variegated combination of events including oxidative stress. The Wnt pathway has multiple actions in the cascade of events triggered by Aβ, and drugs that rescue Wnt activity may be considered as novel therapeutics for AD treatment. Cannabidiol, a non-psychoactive marijuana component, has been recently proposed as an antioxidant neuroprotective agent in neurodegenerative diseases. Moreover, it has been shown to rescue PC12 cells from toxicity induced by Aβ peptide. However, the molecular mechanism of cannabidiol-induced neuroprotective effect is still unknown. Here, we report that cannabidiol inhibits hyperphosphorylation of tau protein in Aβ-stimulated PC12 neuronal cells, which is one of the most representative hallmarks in AD. The effect of cannabidiol is mediated through the Wnt/β-catenin pathway rescue in Aβ-stimulated PC12 cells. These results provide new molecular insight regarding the neuroprotective effect of cannabidiol and suggest its possible role in the pharmacological management of AD, especially in view of its low toxicity in humans.     

The use of nitroxide radical-containing nanoparticles coupled with piperine to protect neuroblastoma SH-SY5Y cells from Aβ-induced oxidative stress

The antioxidant effect and potential mechanism of nitroxide radical-containing nanoparticles (RNPs) coupled with piperine (PI) were investigated in human neuroblastoma SH-SY5Y cells. The effects of RNP/PI on SH-SY5Y cell lines was determined by WST assay for cell viability, nitroblue tetrazolium and deoxyribose assay for reactive oxygen species generation, ELISA assay for reactive oxygen species products and apoptotic cell death, and biochemical techniques for catalase and glutathione peroxidase activity. The RNP/PI significantly reduced the reactive oxygen species level and reactive oxygen species products compared with those of cells treated with RNPs alone. The RNP/PI treatment enhanced catalase and glutathione peroxidase activity. The combination of RNP/PI has been found to have an augmented antioxidant effect on an Alzheimer's model in vitro. The mechanism of the protective effect of this combination therapy was correlated in this study with its ability to reduce the generation of reactive oxygen species and prevent apoptosis via scavenging enzyme action pathways.     

Mobile phone electromagnetic radiation affects Amyloid Precursor Protein and α-synuclein metabolism in SH-SY5Y cells

In this study, the effects of low-level, GSM emitted ElectroMagnetic Field (EMF) on Amyloid Precursor Protein (APP) and alpha-synuclein (α-syn) in human neuroblastoma cells was investigated. Our data indicated alterations on APP processing and cellular topology, following EMF exposure (ℇ = 10.51 V/m, SAR = 0.23 W/kg, exposure time: 3 times, for 10 min, for 2 days). Furthermore, changes in monomeric α-syn accumulation and multimerization, as well as induction of oxidative stress and cell death, were documented. The results presented here require further investigation to determine potential links of EMF with the molecular pathogenic mechanisms in Alzheimer’s and Parkinson’s Diseases.     

Baicalin prevents the production of hydrogen peroxide and oxidative stress induced by Aβ aggregation in SH-SY5Y cells

This study examined whether rats can simultaneously learn to associate lithium chloride (LiCl)-induced nausea with both contextual and intravascular taste cues. During the conditioning phase (4 days, 72 h apart), 32 male Long Evans rats were injected intraperitoneally with either isotonic saline (NaCl), lithium chloride (LiCl, 127 mg/kg), saline plus 2% saccharin (NaCl + Saccharin), or lithium chloride plus 2% saccharin (LiCl + Saccharin) immediately prior to a 30 min exposure to a novel context. 72 h following the final conditioning day, each animal was re-exposed to the context on a drug-free test day. The next day, animals received a 24 h 2-bottle preference test with a choice between water and a palatable saccharin solution. Results showed that animals treated with LiCl during conditioning, with or without saccharin, displayed significantly higher levels of conditioned gaping responses, indicative of nausea, upon re-exposure to the context, relative to NaCl and NaCl + Saccharin controls. Animals administered LiCl + Saccharin during conditioning also displayed significant conditioned taste avoidance to the saccharin solution during the two bottle choice test. These results indicate that systemic administration (intraperitoneal) of a LiCl + Saccharin solution is effective in simultaneously conditioning toxin elicited nausea to both internal (taste) and external (context) cues.     

Activation of nucleotide receptors inhibits M-type K current [I K(M)] in neuroblastoma × glioma hybrid cells

A phospholipase-C-linked nucleotide receptor, sensitive to both uridine and adenosine triphosphate (UTP and ATP) has been cloned from NG108-15 neuroblastoma × glioma hybrid cells. We have tested whether activation of this receptor could inhibit the voltage-dependent K⁺ current [I _K(M) or M-current] in NG108-15 cells recorded using whole-cell patch-clamp methods. Both UTP and ATP inhibited I _K(M) by 44% and 42%, respectively, at 100 M. Mean IC₅₀ values were: UTP, 0.77±0.27 M; ATP, 1.81±0.82 M. The order of nucleotide and nucleoside activity at 100 M was: UTP = ATP > ATP[S] = ITP > 2 MeSATP > ADP = GTP AMP-CPP, adenosine, where ATP[S] is adenosine 5-O-(3-thiotriphosphate), ITP is inosine 5-triphosphate, 2-MeSATP is 2-methylthio ATP and AMP-CPP is , methylene ATP. This rank order accords with their activities at the cloned P_2U receptor. Effects were not inhibited by suramin (up to 500 M) or by pre-incubation for 12 h in 500 ng·ml^–1 Pertussis toxin. Inhibition of I_K(M) was frequently preceded by a transient outward current, probably a Ca²⁺-activated K⁺ current, responding to Ca²⁺ mobilization. No effect on the delayed rectifier K⁺ current was observed. These observations match those expected from stimulating other phospholipase-C-linked receptors in NG108-15 cells.Shemyakin Institute of Bio-organic Chemistry, on leave from the Russian Academy of Sciences, 142292 Pushchino, Moscow Region, Russia     

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.     

Leptin inhibits glycogen synthase kinase-3β to prevent tau phosphorylation in neuronal cells

We have previously demonstrated that Leptin reduces extracellular amyloid β (Aβ) protein both in vitro and in vivo, and intracellular tau phosphorylation in vitro. Further, we have shown that these effects are dependent on activation of AMP-activated protein kinase (AMPK) in vitro. Herein, we investigated downstream effectors of AMPK signaling directly linked to tau phosphorylation. One such target, of relevance to Alzheimer's disease (AD), may be GSK-3β, which has been shown to be inactivated by Leptin. We therefore dissected the role of GSK-3β in mediating Leptin's ability to reduce tau phosphorylation in neuronal cells. Our data suggest that Leptin regulates tau phosphorylation through a pathway involving both AMPK and GSK-3β. This was based on the following: Leptin and the cell-permeable AMPK activator, 5-aminoimidazole-4-carboxyamide ribonucleoside (AICAR), reduced tau phosphorylation at AD-relevant sites similarly to the GSK-3β inhibitor, lithium chloride (LiCl). Further, this reduction of tau phosphorylation was mimicked by the downregulation of GSK-3β, achieved using siRNA technology and antagonized by the ectopic overexpression of GSK-3β. These studies provide further insight into Leptin's mechanism of action in suppressing AD-related pathways.     

Calcineurin inhibition eliminates the normal inverted U curve,enhances acquisition and prolongs memory in a mammalian 3′-5′-cyclic AMP–dependent learning paradigm

The role protein phosphatase 2B (calcineurin, CaN) plays in learning and memory has received a significant amount of attention due to its promotion of the dephosphorylation of 3′-5′-cyclic AMP response element binding protein (CREB). Researchers have ascertained that overexpression of CaN is associated with memory retention deficits [Foster TC, Sharrow KM, Masse JR, Norris CM, Kumar A (2001) Calcineurin links Ca²⁺ dysregulation with brain aging. J Neurosci 21:4066–4073; Mansuy IM, Mayford M, Jacob B, Kandel ER, Bach ME (1998) Restricted and regulated overexpression reveals calcineurin as a key component in the transition from short-term to long-term memory. Cell 92:39–49], while CaN inhibition enhances learning and memory [Gerdjikov TV, Beninger RJ (2005) Differential effects of calcineurin inhibition and protein kinase A activation on nucleus accumbens amphetamine-produced conditioned place preference in rats. Eur J Neurosci 22:697–705; Ikegami S, Inokuchi K (2000) Antisense DNA against calcineurin facilitates memory in contextual fear conditioning by lowering the threshold for hippocampal long-term potentiation induction. Neuroscience 98:637–646]. The present study hypothesized that infusion of a CaN inhibitor (FK506) bilaterally into the olfactory bulbs of postnatal day 6 Sprague Dawley rat pups would prolong the duration of a conditioned odor preference and retard cyclic AMP response element binding protein dephosphorylation. A 2 mg/kg s.c. injection of isoproterenol (ISO, β-adrenoceptor agonist) was paired with a 10 min exposure to peppermint and subsequently an infusion of FK506. Immunohistochemistry for phosphorylated 3′-5′-cyclic AMP response element binding protein (pCREB) revealed that unilateral infusion of FK506 resulted in an amplification of phosphorylated CREB in the olfactory bulb 40 min after training compared with saline-infused bulbs. Pups infused bilaterally with FK506 maintained a learned preference for peppermint 48, 72 and 96 h after training. CaN inhibition also modified the conventional inverted U curve obtained when ISO is used to replace stroking, as the unconditioned stimulus. When pups were infused with FK506, learning occurred with sub- and supra-optimal doses of ISO indicating that CaN overcomes non-optimal effects ISO may have on learning. We demonstrate that CaN inhibition can extend the duration of conditioned olfactory memory and may provide a target for memory prolongation that is superior to even phosphodiesterase inhibition observed in previous studies.     

Protective effect of (−)clausenamide against Aβ-induced neurotoxicity in differentiated PC12 cells

The neurotoxicity of aggregated β-amyloid (Aβ) has been implicated as a critical cause in the pathogenesis of Alzheimer's disease (AD). In the present study, we investigated the effect of (−)clausenamide ((−)Clau), an aqueous extract of leaves of Clausena lassium (lour) skeel, on the neurotoxicity of Aβ_25–35. The viability of differentiated PC12 cells was determined by MTT assay. Apoptosis was detected by flow cytometry. DCFH-DA was used for assessment of intracellular ROS generation, JC-1 and Rhodamine 123 for measurement of mitochondrial transmembrane potential (MMP). The intracellular calcium was determined with Fluo-3. The phosphorylation of p38 MAPK and the expression of Bcl-2, Bax, P53, Caspase 3 were examined by Western blot. The results showed that (−)Clau significantly elevated cell viability. Furthermore, (−)Clau arrested the apoptotic cascade by reversing overload of calcium, preventing ROS generation, moderated the dissipation of MMP and the misbalance of Bcl-2 and Bax, inhibiting the activation of p38 MAPK and the expression of P53 and cleaved Caspase 3. Our results suggested that (−)Clau may be a therapeutic agent for AD.     

Modulation of Nav1.5 by β1- and β3-subunit co-expression in mammalian cells

Cardiac sodium channels (Na_v1.5) comprise a pore-forming -subunit and auxiliary -subunits that modulate channel function. In the heart, 1 is expressed throughout the atria and ventricles, whilst 3 is present only in the ventricles and Purkinje fibers. In view of this expression pattern, we determined the effects of 3 and 1 co-expression alone, and in combination, on Na_v1.5 stably expressed in Chinese hamster ovary cells. The current/voltage relationship was shifted –5 mV with either 1 or 3 co-expression alone and –10 mV with co-expression of both 1 and 3. In addition, 3 and 1/3 co-expression accelerated macroscopic current decay. There were significant hyperpolarizing shifts in equilibrium gating relationships with co-expression of 1 and 3 alone and in combination. Co-expression of 1/3 together resulted in a greater hyperpolarizing shift in channel availability, and an increase in the slopes of equilibrium gating relationships. Co-expression of 3 and 1/3, but not 1, slowed recovery from inactivation at –90 mV. Development of inactivation at –70 and –50 mV was accelerated by -subunit co-expression alone and in combination. -Subunit co-expression also reduced the late Na current measured at 200 ms. In conclusion, -subunits modulate Na_v1.5 gating with important differences between co-expression of 1 and 3 alone and 1/3 together.     

RUNX3小干扰RNA对SH-SY5Y细胞生长和药物敏感性的作用

目的：探讨RUNX3基因对人神经母细胞瘤细胞生长和药物敏感性的调节作用。方法:构建RUNX3基因的小干扰RNA载体并将其转导入SH-SY5Y细胞，G418筛选后获得稳定转染的阳性克隆后，应用RT-PCR和Western blotitng进行鉴定；MTT法和流式细胞仪检测细胞转染前后生长速度和细胞周期的变化；MTT法和流式细胞仪检测细胞转染前后对阿霉素敏感性的变化；Western blotting检测细胞转染前后细胞周期相关蛋白cyclin D1、CDK4、CDK6、p21、 p27和凋亡相关蛋白Bcl-2、Bax以及耐药相关蛋白P-gp、MRP的表达变化。结果:成功构建了RUNX3的小干扰RNA载体并将其转染SH-SY5Y细胞；筛选到稳定的RUNX3低表达的神经母细胞瘤细胞模型；MTT和流式细胞仪检测结果显示，转染RUNX3小干扰RNA后的细胞的生长速度显著快于对照组（P<0.05），且G1期的细胞比率显著低于对照组（P<0.05）；MTT法和流式细胞仪结果显示, 转染RUNX3小干扰RNA后的细胞对化疗药物的敏感性降低，细胞内的阿霉素蓄积量显著减少（P<0.05）；Western blotting显示，转染RUNX3小干扰RNA后的细胞中Bcl-2、P-gp和cyclin D1的表达明显增高，p21的表达明显降低。结论:下调RUNX3基因能促进神经母细胞瘤细胞生长，降低细胞对化疗药物的敏感性，提示RUNX3在神经母细胞瘤的发生和发展中可能扮演重要角色。     

γ-terpineol inhibits cell growth and induces apoptosis in human liver cancer BEL-7402 cells in vitro

To investigate the effect of γ-terpineol on cell proliferation and apoptosis of human hepatoma BEL-7402 cells to elucidate its molecular mechanism. Here, BEL-7402 cells were treated with various concentrations (40, 80, 160, 320 and 640 μg/ml) of γ-terpineol for 48 h, cell proliferation was determined by 3-(4,5-dimethyl-thiazolyl-2)-2,5-diphenyl tetrazolium bromides (MTT) assay. Cell colony inhibition was determined by soft agar assay. Apoptosis and possible molecular mechanisms were evaluated by morphological observation, flow cytometry analysis, and DNA fragmentation assay. The γ-terpineol significantly suppressed BEL-7402 cell proliferation in a dose-dependent manner. Characteristic morphological and biochemical changes associated with apoptosis such as cells shrinkage, deformation and vacuolization of mitochondria, nuclear chromatin condensation and fragmentation, formation of apoptotic bodies were observed after BEL-7402 cells treated with γ-terpineol for 24 h and 48 h. Cell cycle were displayed by flow cytometry analysis, the γ-terpineol treatment resulted in accumulation of cells at G₁ or S phase and a blockade of cell proliferation compared to control group. Treating BEL-7402 cells with 320 μg/ml of γ-terpineol for 36 h and 48 h, a typical apoptotic “DNA ladder” was observed using DNA fragmentation assay. The present study demonstrated that possible anti-cancer mechanism of γ-terpineol on human hematomas cells is through inducing cell apoptosis to suppress tumor cell growth.     

P38MAPK的激活上调SH-SY5Y细胞APP表达及组蛋白H3乙酰化水平

目的研究P38MAPK信号通路的激活对淀粉前体蛋白(APP)表达的影响及其相关表观遗传学机制。方法体外培养神经母细胞瘤细胞(SH-SY5Y),Western blot法检测SH-SY5Y的APP蛋白表达及组蛋白乙酰化酶(HAT)和组蛋白去乙酰化酶(HDAC)的表达;吸光度值法检测组蛋白3(H3)和组蛋白4(H4)整体乙酰化水平。结果 P38MAPK信号通路经特异性激动剂激活SH-SY5Y 72 h后,APP表达明显增高至对照组的1.5倍(0.41±0.09vs0.28±0.08,P<0.01);同时组蛋白H3整体乙酰化水平增高(0.20±0.04vs0.06±0.03,P<0.01),但H4乙酰化水平无明显改变;组蛋白乙酰化酶CBP表达增高至对照组的2.5倍(0.30±0.03vs0.11±0.05,P<0.01),而组蛋白去乙酰化酶HDAC3表达下降至对照组的40%(0.19±0.05vs0.49±0.03,P<0.01)。结论 P38MAPK信号通路可能通过上调组蛋白乙酰化水平增加SH-SY5Y的APP蛋白表达。