Iron modulates the activity of monoamine oxidase B in SH-SY5Y cells

Both monoamine oxidase B (MAO-B) and iron accumulation are associated with neurologic diseases including Parkinson’s disease. However, the association of iron with MAO-B activity was poorly understood. Here we took advantage of highly sensitive and specific fluorescence probes to examine the change in MAO-B activity in human dopaminergic neuroblastoma (SH-SY5Y) cells upon iron exposure. Both ferric and ferrous ions could significantly enhance the activity of MAO-B, instead of MAO-A, in SH-SY5Y cells. In addition, iron-induced increase in MAO-B probe fluorescence could be prevented by pargyline and other newly developed MAO-B inhibitors, suggesting that it was MAO-B activity-dependent. These findings may suggest MAO-B is an important sensor in iron-stressed neuronal cells.     

Tianma modulates proteins with various neuro-regenerative modalities in differentiated human neuronal SH-SY5Y cells

Tianma (Rhizoma gastrodiae) is the dried rhizome of the plant Gastrodia elata Blume (Orchidaceae family). As a medicinal herb in traditional Chinese medicine (TCM) its functions are to control convulsions, pain, headache, dizziness, vertigo, seizure, epilepsy and others. In addition, tianma is frequently used for the treatment of neurodegenerative disorders though the mechanism of action is widely unknown. Accordingly, this study was designed to examine the effects of tianma on the proteome metabolism in differentiated human neuronal SH-SY5Y cells to explore its specific effects on neuronal signaling pathways. Using an iTRAQ (isobaric tags for relative and absolute quantitation)-based proteomics research approach, we identified 2390 modulated proteins, out of which 406 were found to be altered by tianma in differentiated human neuronal SH-SY5Y cells. Based on the observed data, we hypothesize that tianma promotes neuro-regenerative signaling cascades by controlling chaperone/proteasomal degradation pathways (e.g. CALR, FKBP3/4, HSP70/90) and mobilizing neuro-protective genes (such as AIP5) as well as modulating other proteins (RTN1/4, NCAM, PACSIN2, and PDLIM1/5) with various regenerative modalities and capacities related to neuro-synaptic plasticity.     

SH—SY5Y细胞的钙缓冲研究

目的：研究SH－SY5Y神经杂交瘤细胞的钙缓冲能力。方法：通过膜片钳手段,测量未分化的SH－SY5Y细胞钙离子通道电流;并应用显微荧光测量游离钙离子浓度和高钾去极化的方法,研究胞内Ca^2 浓度上升后浓度恢复的动力学过程。结果：未分化的SH－SY5Y细胞存在钙离子通道电流,在刺激时间间隔较短时（＜150s）,胞内钙浓度的恢复过程会由于缓冲机制的饱和而变慢;而时间间隔＞150s时,缓冲物质则可以基本恢复使得胞内钙的恢复过程基本保持不变。结论：钙缓冲蛋白在细胞内钙浓度的调节中起重要作用。     

Interaction between acylphosphatase and SERCA in SH-SY5Y cells

Ca²⁺ transport by sarco/endoplasmic reticulum, tightly coupled with the enzymatic activity of Ca²⁺-dependent ATPase, controls the cell cycle through the regulation of genes operating in the critical G₁ to S checkpoint. Experimental studies demonstrated that acylphosphatase actively hydrolyses the phosphorylated intermediate of sarco/endoplasmic reticulum calcium ATPase (SERCA) and therefore enhances the activity of Ca²⁺ pump. In this study we found that SH-SY5Y neuroblastoma cell division was blocked by entry into a quiescent G₀-like state by thapsigargin, a high specific SERCA inhibitor, highlighting the regulatory role of SERCA in cell cycle progression. Addition of physiological amounts of acylphosphatase to SY5Y membranes resulted in a significant increase in the rate of ATP hydrolysis of SERCA. In synchronized cells a concomitant variation of the level of acylphosphatase isoenzymes opposite to that of intracellular free calcium during the G₁ and S phases occurs. Particularly, during G₁ phase progression the isoenzymes content declined steadily and hit the lowest level after 6 h from G₀ to G₁ transition with a concomitant significant increase of calcium levels. No changes in free calcium and acylphosphatase levels upon thapsigargin inhibition were observed. Moreover, a specific binding between acylphosphatase and SERCA was demonstrated. No significant change in SERCA-2 expression was found. These findings suggest that the hydrolytic activity of acylphosphatase increase the turnover of the phosphoenzyme intermediate with the consequences of an enhanced efficiency of calcium transport across endoplasmic reticulum and a subsequent decrease in cytoplasmic calcium levels. A hypothesis about the modulation of SERCA activity by acylphosphatase during cell cycle in SY5Y cells in discussed.     

Opioid agonists binding and responses in SH-SY5Y cells.

SH-SY5Y (human neuroblastoma) cultured cells, known to have mu-opioid receptors, have been used to assess and compare the ability of eight representative mu-selective compounds from diverse opioid families to recognize and activate these receptors. A wide range of receptor affinities spanning a factor of 10,000 was found between the highest affinity fentanyl analogs (Ki = 0.1nM) and the lowest affinity analog, meperidine (Ki = 1 microM). A similar range was found for inhibition of PGE1-stimulated cAMP accumulation with a rank order of activities that closely paralleled binding affinities. Maximum inhibition of cAMP accumulation by each compound was about 80%. Maximum stimulation of GTPase activity (approximately 50%) was also similar for all compounds except the lowest affinity meperidine. Both effects were naloxone reversible. These results provide further evidence that mu-receptors are coupled to inhibition of adenylate cyclase and that the SH-SY5Y cell line is a good system for assessment of mu-agonists functional responses.     

Baicalein attenuates 6-hydroxydopamine-induced neurotoxicity in SH-SY5Y cells

It has been suggested that baicalein, a flavonoid obtained from the Scutellaria root, exerts a protective role on neurons against several neuronal insults. However, the protective mechanisms underlying this protective effect remain largely unknown. Our results indicate that baicalein protects SH-SY5Y cells, a dopaminergic neuronal cell line, from 6-hydroxydopamine (6-OHDA)-induced damage by the attenuation of reactive oxygen species (ROS). In order to determine the effects of baicalein on mitochondrial events, mitochondrial membrane potentials (deltapsim) and caspase cascades downstream of mitochondria were assessed. Baicalein inhibited the collapse of deltapsim, suggesting that baicalein reduces the mitochondrial dysfunction associated with 6-OHDA treatment. Baicalein also inhibited caspase-9 and caspase-3 activation, which can be triggered by mitochondrial malfunctions. Furthermore, baicalein induced a significant reduction in the level of phospho-JNK, which is known as an apoptotic mediator in 6-OHDA-induced neuronal cell death. Our results indicate that baicalein protects neurons from the deleterious effects of 6-OHDA via the attenuation of oxidative stress, mitochondrial dysfunction, caspase activity, and JNK activation.     

alpha-Synuclein protects SH-SY5Y cells from dopamine toxicity

Dopaminergic human neuroblastoma SH-SY5Y cells were stably transformed to increase expression of alpha-synuclein, a Parkinson's disease-related protein. Transformed cells were more resistant to oxidative insults, showing a cytoprotective role of alpha-synuclein. The expression of redox chaperonins (DJ-1, HSP70, and 14-3-3) was evaluated by Western blotting. Expression of alpha-synuclein reduced HSP70 levels even in the presence of dopamine, with a twofold increase of DJ-1 in the absence of oxidants. DJ-1 is significantly reduced by dopamine, and even more by dopamine and Cu(II). Increased alpha-synuclein expression did not affect 14-3-3, although dopamine increased its level by 60% in wild-type cells. alpha-Synuclein not only upregulated DJ-1, but also shifted all DJ-1 forms to a single spot at pI=5.7 not observed in wild-type cells. Dopamine gradually restored the distribution of DJ-1 forms to a situation similar to wild-type cells, with the form at pI=6.1 progressively enriched under oxidative conditions.     

Gonadotropin-releasing hormone-agonist inhibits synthesis of nitric oxide and steroidogenesis by luteal cells in the pregnant rat

We have demonstrated that continuous administration of a gonadotropin-releasing hormone agonist (GnRH-Ag) in vivo suppressed progesterone production and induced apoptosis in the corpus luteum (CL) of the pregnant rat. To investigate the mechanism(s) by which progesterone secretion is suppressed and apoptosis is induced in the luteal cells, we studied nitric oxide (NO) as a messenger molecule for GnRH action. Rats were treated individually on Day 8 of pregnancy with 5 microg/day of GnRH-Ag for 4, 8, and 24 h. GnRH-Ag decreased the production of progesterone and pregnenolone 8 and 24 h after the administration. Corresponding with the reduction in these steroid hormones, luteal NO concentrations decreased at 8 and 24 h. Western blotting and immunohistochemical studies of endothelial nitric oxide synthase (eNOS), inducible nitric oxide synthase (iNOS), and neuronal nitric oxide synthase (nNOS) in the CL demonstrated that administration of GnRH-Ag was associated with a marked decrease in eNOS and iNOS compared with sham controls at 4 and 8 h, but nNOS did not change throughout the experimental period. We demonstrated, for the first time, the presence of nNOS protein in the CL of the pregnant rat. To determine if this suppressive action of GnRH-Ag is directly on the CL, luteal cells were treated with GnRH-Ag for 4, 8, 12, and 24 h in vitro. Progesterone and NO concentrations in the media decreased at 8 and 12 h after the treatment and recovered at 24 h. Western blots revealed that eNOS and iNOS decreased in luteal cells treated with GnRH-Ag compared with controls at 4 and 8 h. These results demonstrate that suppression of luteal NO synthesis by GnRH-Ag is direct and leads to a decrease in the luteal production and release of progesterone and pregnenolone and thus suggest that GnRH could induce luteolysis in pregnant rats via NO.     

Prostaglandin A1 inhibits rotenone-induced apoptosis in SH-SY5Y cells

The degeneration of nigral dopamine neurons in Parkinson's disease (PD) reportedly involves a defect in brain mitochondrial complex I in association with the activation of nuclear factor-kappaB (NF-kappaB) and caspase-3. To elucidate molecular mechanisms possibly linking these events, as well as to evaluate the neuroprotective potential of the cyclopentenone prostaglandin A1 (PGA1), an inducer of heat shock proteins (HSPs), we exposed human dopaminergic SH-SY5Y cells to the complex I inhibitor rotenone. Dose-dependent apoptosis was preceded by the nuclear translocation of NF-kappaB and then the activation of caspase-3 over the ensuing 24 h. PGA1 increased the expression of HSP70 and HSP27 and protected against rotenone-induced apoptosis, without increasing necrotic death. PGA1 blocked the rotenone-induced nuclear translocation of NF-kappaB and attenuated, but did not abolish, the caspase-3 elevation. Unexpectedly, the caspase-3 inhibitor, Ac-DEVD.CHO (DEVD), at a concentration that completely prevented the caspase-3 elevation produced by rotenone, failed to protect against apoptosis. These results suggest that complex I deficiency in dopamine cells can induce apoptosis by a process involving early NF-kappaB nuclear translocation and caspase-3 activation. PGA1 appears to protect against rotenone-induced cell death by inducing HSPs and blocking nuclear translocation of NF-kappaB in a process that attenuates caspase-3 activation, but is not mediated by its inhibition.     

Copper-dependent toxicity in SH-SY5Y neuroblastoma cells involves mitochondrial damage

Treatment of SH-SY5Y human neuroblastoma cells with copper sulphate (50-300microM) in complete medium for 24h caused an increase in the level of the metal both in whole cells and in isolated mitoplasts. Toxic effects of copper resulted in the impairment of the capability of mitochondrial dehydrogenases to reduce a tetrazolium salt, and, to a lesser extent, in the loss of the integrity of the plasma membrane. The mechanism of toxicity involved the production of reactive oxygen species, amplified by the presence of ascorbate. Decreases in the levels of several mitochondrial proteins (subunits of complex I, complex V, and of the pyruvate dehydrogenase complex) were observed. These findings demonstrate that mitochondria are an early and susceptible target of copper-mediated oxidative stress in neuronal cells and support the hypothesis that mitochondrial damage triggers the neurodegenerative processes associated with copper overload in Wilson's disease.     

The sulfite molecule enhances homocysteine toxicity in SH-SY5Y cells

Molecular Biology Reports - Homocysteine (hcy) is an amino acid that contains sulfur species. In healthy individuals, plasma hcy levels are low. The aim of this study was to investigate the...     

Modulation of innate immune-related pathways in nicotine-treated SH-SY5Y cells

Although nicotine has a broad impact on both the central and peripheral nervous systems, the molecular mechanisms remain largely unknown, especially at the signaling pathway level. To investigate that aspect, we employed both conventional molecular techniques, such as quantitative real-time PCR and Western blotting analysis, and high-throughput microarray approach to identify the genes and signaling pathways that are modulated by nicotine. We found 14 pathways significantly altered in SH-SY5Y neuroblastoma cells. Of these, the Toll-like receptor pathway (TLR; p?=?2.57?×?10(-4)) is one of the most important innate immune pathways. The death receptor pathway (DR; p?=?8.71?×?10(-4)), whose transducers coordinate TLR signals and help conduct the host immune response to infection, was also significantly changed by nicotine. Furthermore, we found that several downstream pathways of TLR and DR signaling, such as PI3K/AKT signaling (p?=?9.55?×?10(-6)), p38 signaling (p?=?2.40?×?10(-6)), and ERK signaling (p?=?1.70?×?10(-4)), were also significantly modulated by nicotine. Interestingly, most of the differentially expressed genes in these pathways leading to nuclear factor κB (NF-κB) activation and those important inhibitors of pathways leading to apoptosis, including FLIP and Bcl-2, were up-regulated by nicotine. Taken together, our findings demonstrate that nicotine can regulate multiple innate immune-related pathways, and our data thus provide new clues to the molecular mechanisms underlying nicotine's regulatory effects on neurons.     

Glutathione intensifies gliotoxin-induced cytotoxicity in human neuroblastoma SH-SY5Y cells

Gliotoxin is a fungal second metabolite produced by diverse species that can be found in compost, stored crops, moist animal feed and sawdust. The role of glutathione in gliotoxin-induced toxicity was studied in order to elucidate the toxic mechanisms leading to neurite degeneration and cell death in differentiated human neuroblastoma (SH-SY5Y) cells. After 72 h of exposure to gliotoxin, moderate cytotoxicity was induced at 0.1 μmol/L, which was more severe at higher concentrations. A reduction in the number of neurites per cell was also observed. By decreasing the level of intracellular glutathione with l-buthionine-sulfoxamine (BSO) a specific inhibitor of glutathione synthesis, the cytotoxic effect of gliotoxin was significantly attenuated. The gliotoxin-induced cytotoxicity was also slightly reduced by the antioxidant vitamin C. However, the neurite degenerative effect was not altered by BSO, or by vitamin C. A concentration-dependent increase in the ratio between oxidized and reduced forms of glutathione, as well as the total intracellular glutathione levels, was noted after exposure to gliotoxin. The increase of glutathione was also reflected in western blot analyses showing a tendency for the regulatory subunit of γ-glutamylcysteine synthetase to be upregulated. In addition, the activity of glutathione reductase was slightly increased in gliotoxin-exposed cells. These results indicate that glutathione promotes gliotoxin-induced cytotoxicity, probably by reducing the ETP (epipolythiodioxopiperazine) disulfide bridge to the dithiol form.     

Methadone induces necrotic-like cell death in SH-SY5Y cells by an impairment of mitochondrial ATP synthesis

Methadone is a widely used therapeutic opioid in narcotic addiction and neuropathic pain syndromes. Oncologists regularly use methadone as a long-lasting analgesic. Recently it has also been proposed as a promising agent in leukemia therapy, especially when conventional therapies are not effective. Nevertheless, numerous reports indicate a negative impact on human cognition with chronic exposure to opiates. Thus, clarification of methadone toxicity is required. In SH-SY5Y cells we found that high concentrations of methadone were required to induce cell death. Methadone-induced cell death seems to be related to necrotic processes rather than typical apoptosis. Cell cultures challenged with methadone presented alterations in mitochondrial outer membrane permeability. A mechanism that involves Bax translocation to the mitochondria was observed, accompanied with cytochrome c release. Furthermore, no participation of known protein regulators of apoptosis such as Bcl-X_L and p53 was observed. Interestingly, methadone-induced cell death took place by a caspases-independent pathway; perhaps due to its ability to induce a drastic depletion in cellular ATP levels. Therefore, we studied the effect of methadone on isolated rat liver mitochondria. We observed that methadone caused mitochondrial uncoupling, coinciding with the ionophoric properties of methadone, but did not cause swelling of the organelles. Overall, the effects observed for cells in the presence of supratherapeutic doses of methadone may result from a “bioenergetic crisis.” A decreased level of cellular energy may predispose cells to necrotic-like cell death.     

Valproic acid-mediated Hsp70 induction and anti-apoptotic neuroprotection in SH-SY5Y cells

Valproic acid (VPA), an anticonvulsant and mood-stabilizing drug, has been reported to exert neuroprotection against a variety of insults. We now show that VPA attenuates rotenone (a potent complex I inhibitor)-induced apoptosis through the induction of heat shock protein 70, which may interact with apoptotic-protease-activating factor 1. Activation of p-Akt, p-Bcl-2, as well as p-Erk1/2 by VPA may be co-contributors to the protection.     

Cytokine action and oxidative stress response in differentiated neuroblastoma SH-SY5Y cells

In the retinoic acid-differentiated neuroblastoma SH-SY5Y cells, IL-1 induced binding activity of NFkappaB and up-regulated the expression and activity of MnSOD. The IL-1-elicited effects were partly reversed by IL-4 and IL-6. It is proposed that IL-4 and IL-6 may participate in the regulation of the imbalanced oxidant status induced by IL-1 in differentiated neuroblastoma cells. In the SH-SY5Y cell line, TNFalpha neither activated NFkappaB nor induced MnSOD expression and activity, but was capable of modulating the IL-1 effects. Pyrrolidine dithiocarbamate (PDTC), an inhibitor of NFkappaB activation, down-regulated the expression and activity of MnSOD, which may suggest that the regulation of MnSOD by IL-1 in retinoic acid-differentiated neuroblastoma cells was mediated by the nuclear factor kappaB.     

Antipsychotic drugs increase N-acetylaspartate and N-acetylaspartylglutamate in SH-SY5Y human neuroblastoma cells

N -Acetylaspartate (NAA) and N -acetylaspartylglutamate (NAAG) are related neuronal metabolites associated with the diagnosis and treatment of schizophrenia. NAA is a valuable marker of neuronal viability in magnetic resonance spectroscopy, a technique which has consistently shown NAA levels to be modestly decreased in the brains of schizophrenia patients. However, there are conflicting reports on the changes in brain NAA levels after treatment with antipsychotic drugs, which exert their therapeutic effects in part by blocking dopamine D₂ receptors. NAAG is reported to be an agonist of the metabotropic glutamate 2/3 receptor, which is linked to neurotransmitter release modulation, including glutamate release. Alterations in NAAG metabolism have been implicated in the development of schizophrenia possibly via dysregulation of glutamate neurotransmission. In the present study we have used high performance liquid chromatography to determine the effects of the antipsychotic drugs haloperidol and clozapine on NAA and NAAG levels in SH-SY5Y human neuroblastoma cells, a model system used to test the responses of dopaminergic neurons in vitro . The results indicate that the antipsychotic drugs haloperidol and clozapine increase both NAA and NAAG levels in SH-SY5Y cells in a dose and time dependant manner, providing evidence that NAA and NAAG metabolism in neurons is responsive to antipsychotic drug treatment.