Glutamate treatment and p25 transfection increase Cdk5 mediated tau phosphorylation in SH-SY5Y cells

Neurofibrillary tangles (NFT) of hyperphosphorylated tau protein are a major pathological hallmark of Alzheimer's disease (AD). One of the tau phosphorylating kinases with pathological relevance in AD has been suggested to be the cyclin-dependent kinase 5 (Cdk5). The proposed mechanism leading to pathological Cdk5 activity is through induced cleavage of p35 to a proteolytic product, p25. To further study activation of Cdk5 and its role in tau phosphorylation in vitro, we used differentiated SH-SY5Y cells treated with neurotoxic stimuli or transfected with p25. We show that glutamate increased tau phosphorylation, concomitant with an increased Cdk5 activity achieved by upregulation of Cdk5 and p35 protein levels. Treatment with the calcium ionophore A23187 generated the calpain cleaved p25 fragment but only in toxic conditions that caused dephosphorylation and loss of tau. When p25 was transfected to the cells, increased tau phosphorylation was achieved. However, application of the Cdk5 inhibitor Roscovitine did not result in inhibition of tau phosphorylation possibly due to activation of extracellular regulated kinase 1/2 (Erk1/2), which also is capable of phosphorylating tau. Cdk5 and Erk1/2 kinases share some common substrates but impact of their cross talk on tau phosphorylation has not previously been demonstrated. We also show that p25 is degraded via the proteasome in Roscovitine treated cells.     

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.     

SH-SY5Y细胞α-突触核蛋白的过表达可部分抵抗鱼藤酮诱导的氧化应激

帕金森病（Parkinson’s disease,PD）的发病机制涉及到遗传和环境因素。环境因素通过线粒休导致氧化应激和α-突触核蛋白（α—synuclein）聚集,但其确切的作用机制尚不明确。本文利用过表达α-突触核蛋白-增强型绿色荧光蛋白（enhanced green fluorescent protein．EGFP）的人多巴胺能神经母细胞瘤细胞株SH—SY5Y为模型,研究α-突触核蛋白对鱼藤酮诱导氧化应激的影响,从而进一步了解α-突触核蛋白和细胞存活之间的关系。（1）用荧光显微镜观察融合绿色荧光蛋白的α-突触核蛋白的表达情况;（2）用实时定量PCR检测α-突触核蛋白基因的表达;（3）用免疫细胞化学测定α-突触核蛋白的分布;（4）用不同浓度的鱼藤酮作用细胞后,以MTT法测细胞的活力、DCF法检测细胞的氧化应激状态、黄嘌呤氧化酶法检测超氧化物歧化酶的活力,并用流式细胞仪分析细胞的凋亡。实时定量PCR结果显示,α-突触核蛋白基因表达量在α-突触核蛋白过表达的细胞要高于SH—SY5Y细胞,在荧光显微镜下可见绿色荧光蛋白和α-突触核蛋白的表达。鱼藤酮可使细胞活力下降、线粒体complex Ⅰ的活性降低,诱导细胞内氧化应激,而过表达α-突触核蛋白的细胞可以部分抵抗鱼藤酮的毒性作用,表现为细胞抗氧化能力迅速增高（P〈0．05）和鱼藤酮诱导的细胞凋亡数目明显降低。本研究证明α-突触核蛋白对鱼藤酮产生的氧化应激有部分抵抗作用,而使过表达α-突触核蛋白的SH—SY5Y细胞对鱼藤酮的毒性作用表现出一定的耐受性。这种耐受性也可能是细胞对外界损害的一种代偿反应,从而促进细胞的存活。     

Mercury induces cell cytotoxicity and oxidative stress and increases beta-amyloid secretion and tau phosphorylation in SHSY5Y neuroblastoma cells

Concentrations of heavy metals, including mercury, have been shown to be altered in the brain and body fluids of Alzheimer's disease (AD) patients. To explore potential pathophysiological mechanisms we used an in vitro model system (SHSY5Y neuroblastoma cells) and investigated the effects of inorganic mercury (HgCl2) on oxidative stress, cell cytotoxicity, beta-amyloid production, and tau phosphorylation. We demonstrated that exposure of cells to 50 microg/L (180 nM) HgCl2 for 30 min induces a 30% reduction in cellular glutathione (GSH) levels (n = 13, p<0.001). Preincubation of cells for 30 min with 1 microM melatonin or premixing melatonin and HgCl2 appeared to protect cells from the mercury-induced GSH loss. Similarly, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) cytotoxicity assays revealed that 50 microg/L HgCl2 for 24 h produced a 50% inhibition of MTT reduction (n = 9, p<0.001). Again, melatonin preincubation protected cells from the deleterious effects of mercury, resulting in MTT reduction equaling control levels. The release of beta-amyloid peptide (Abeta) 1-40 and 1-42 into cell culture supernatants after exposure to HgCl2 was shown to be different: Abeta 1-40 showed maximal (15.3 ng/ml) release after 4 h, whereas Abeta 1-42 showed maximal (9.3 ng/ml) release after 6 h of exposure to mercury compared with untreated controls (n = 9, p<0.001). Preincubation of cells with melatonin resulted in an attenuation of Abeta 1-40 and Abeta 1-42 release. Tau phosphorylation was significantly increased in the presence of mercury (n = 9, p<0.001), whereas melatonin preincubation reduced the phosphorylation to control values. These results indicate that mercury may play a role in pathophysiological mechanisms of AD.     

Quercetin mitigates the deoxynivalenol mycotoxin induced apoptosis in SH-SY5Y cells by modulating the oxidative stress mediators

Deoxynivalenol (DON) is Fusarium mycotoxin that is frequently found in many cereal-based foods, and its ingestion has a deleterious impact on human health. In this investigation, we studied the mechanism of DON-induced neurotoxicity and followed by cytoprotective efficacy of quercetin (QUE) in contradiction of DON-induced neurotoxicity through assessing the oxidative stress and apoptotic demise in the human neuronal model, i.e. SH-SY5Y cells. DON diminished the proliferation of cells in the manner of dose and time-dependent as revealed by cell viability investigations, i.e. MTT and lactate dehydrogenase assays. Additional studies, such as intracellular reactive oxygen species (ROS), lipid peroxidation (LPO), mitochondrial membrane potential (MMP), DNA damage, cell cycle, and neuronal biomarkers (amino acid decarboxylase, tyrosine hydroxylase, and brain-derived neurotrophic factor) demonstrated that DON induces apoptotic demise in neuronal cells through oxidative stress intermediaries. On another hand, pre-treatment of neuronal cells with 1 mM of quercetin (QUE) showed decent viability upon exposure to 100 µM of DON. In detailed studies demonstrated that QUE (1 mM) pre-treated cells show strong attenuation efficiency against DON-induced ROS generation, LPO, MMP loss, DNA impairment, cell cycle arrest, and down-regulation of neuronal biomarkers. The consequences of the investigation concluded that QUE mitigates the DON-induced stress viz., decreased ROS production and LPO generation, upholding MMP and DNA integrity and regulation of neuronal biomarker gene expression in SH-SY5Y cells.     

N-acetyl-L-cysteine protects SHSY5Y neuroblastoma cells from oxidative stress and cell cytotoxicity: effects on beta-amyloid secretion and tau phosphorylation

Redox changes within neurones are increasingly being implicated as an important causative agent in brain ageing and neurodegenerative diseases such as amyotrophic lateral sclerosis (ALS), Parkinson's disease (PD) and Alzheimer's disease (AD). Cells have developed a number of defensive mechanisms to maintain intracellular redox homeostasis, including the glutathione (GSH) system and antioxidant enzymes. Here we examine the effects of N-acetyl-L-cysteine (NAC) on beta-amyloid (A beta) secretion and tau phosphorylation in SHSY5Y neuroblastoma cells after exposure to oxidative stress inducing/cytotoxic compounds (H(2)O(2), UV light and toxic A beta peptides). A beta and tau protein are hallmark molecules in the pathology of AD while the stress factors are implicated in the aetiology of AD. The results show that H(2)O(2), UV light, A beta 1-42 and toxic A beta 25-35, but not the inactive A beta 35-25, produce a significant induction of oxidative stress and cell cytotoxicity. The effects are reversed when cells are pre-treated with 30 mM NAC. Cells exposed to H(2)O(2), UV light and A beta 25-35, but not A beta 35-25, secrete significantly higher amounts of A beta 1-40 and A beta 1-42 into the culture medium. NAC pre-treatment increased the release of A beta 1-40 compared with controls and potentiated the release of both A beta 1-40 and A beta 1-42 in A beta 25-35-treated cells. Tau phosphorylation was markedly reduced by H(2)O(2) and UV light but increased by A beta 25-35. NAC strongly lowered phospho-tau levels in the presence or absence of stress treatment.     

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.     

Effect of lycopene on As2O3 induced oxidative stress in SH-SY5Y cells

It is known that oxidative stress may cause neuronal injury and several experimental models showed that As₂O₃ exposure causes oxidative stress. Lycopene, a carotenoid, has been shown to have protective effect in neurological disease models due to antioxidant activity, but its effect on As₂O₃-induced neurotoxicity is not identified yet. The aim of this study is to investigate the effects of lycopene on As₂O₃-induced neuronal damage and the related mechanisms. Cell viability was determined by the MTT assay. Lycopene was administrated with different concentrations (2, 4, 6 and 8 µM) one hour before 2 µM As₂O₃ exposure in SH-SY5Y human neuroblastoma cells. The anti-oxidant effect of lycopene was determined by measuring superoxide dismutase (SOD), catalase (CAT) hydrogen peroxide (H₂O₂), malondialdehyde (MDA), total antioxidant status (TAS) and total oxidant status (TOS). MTT results and LDH cytotoxicity analyses showed that pretreatment with 8 µM lycopene significantly improved the toxicity due to As₂O₃ exposure in SH?SY5Y neuroblastoma cells. Pretreatment with lycopene significantly increased the activities of anti?oxidative enzymes as well as total antioxidant status and decreased total oxidative status in As₂O₃ exposed cells. The results of this study indicate that lycopene may be a potent neuroprotective against oxidative stress and could be used to prevent neuronal injury or death in several neurological diseases.

     

Neurotoxicity mediated by oxidative stress caused by titanium dioxide nanoparticles in human neuroblastoma (SH-SY5Y) cells

BackgroundTitanium is widely used in biomedicine. Due to biotribocorrosion, titanium dioxide (TiO₂) nanoparticles (NPs) can be released from the titanium implant surface, enter the systemic circulation, and migrate to various organs and tissues including the brain. A previous study showed that 5 nm TiO₂ NPs reached the highest concentration in the brain. Even though TiO₂ NPs are believed to possess low toxicity, little is known about their neurotoxic effects. The aim of the study was to evaluate in vitro the effects of 5 nm TiO₂ NPs on a human neuroblastoma (SH-SY5Y) cell line.MethodsCell cultures were divided into non-exposed and exposed to TiO₂ NPs for 24 h. The following were evaluated: reactive oxygen species (ROS) generation, apoptosis, cellular antioxidant response, endoplasmic reticulum stress and autophagy.ResultsExposure to TiO₂ NPs induced ROS generation in a dose dependent manner, with values reaching up to 10 fold those of controls (p < 0.001). Nrf2 nuclear localization and autophagy, also increased in a dose dependent manner. Apoptosis increased by 4- to 10-fold compared to the control group, depending on the dose employed.ConclusionsOur results show that TiO₂ NPs cause ROS increase, induction of ER stress, Nrf2 cytoplasmic translocation to the nucleus and apoptosis. Thus, neuroblastoma cell response to TiO₂ NPs may be associated with an imbalance of the oxidative metabolism where endoplasmic reticulum-mediated signal pathway seems to be the main neurotoxic mechanism.     

Synergistic induction of ER stress by homocysteine and beta-amyloid in SH-SY5Y cells

Clinical studies have raised the possibility that elevated plasma levels of homocysteine increase the risk of atherosclerosis, stroke and possibly neurodegenerative diseases such as Alzheimer's disease (AD); however, the direct impact of homocysteine on neuron cells and the mechanism by which it could induce neurodegeneration have yet to be clearly demonstrated. Here, we investigated the effect of homocysteine on endoplasmic reticulum (ER) stress, the suggested mechanism of neurotoxicity, in human neuroblastoma SH-SY5Y cells. The effect of homocysteine on amyloid-beta (Abeta)-induced neurotoxicity and the protective activity of folate were also investigated. Homocysteine led to increased expressions of the binding protein (BiP) and the spliced form of X-box-protein (XBP)-1 mRNAs, suggesting activation of the unfolded-protein response and an increase in apoptosis. When cells were cotreated with homocysteine and Abeta, caspase-3 activity was significantly increased, and expressions of BiP and the spliced form of XBP-1 mRNAs were significantly induced. The neurotoxicity of homocysteine was attenuated by the treatment of cells with folate, as determined by caspase-3 activity and apoptotic body staining. These findings indicate that homocysteine induces ER stress and, ultimately, apoptosis and sensitizes neurons to amyloid toxicity via the synergistic induction of ER stress. Furthermore, a neuroprotective effect of folate against homocysteine-induced toxicity was also observed. Therefore, the findings of our study suggest that ER stress-induced homocysteine toxicity may play an important physiological role in enhancing the pathogenesis of Abeta-induced neuronal degeneration.     

Phosphoproteomic profiling of human SH-SY5Y neuroblastoma cells during response to 6-hydroxydopamine-induced oxidative stress

Dopaminergic neurons are known to be vulnerable to age-related neuronal disorders due to reactive oxygen species (ROS) generated during dopamine metabolism. However, it remains unclear what kinds of proteins are involved in the response to oxidative stress. We examined changes in whole proteins and phosphoproteins in the human dopaminergic neuroblastoma cell line SH-SY5Y under oxidative stress induced by the dopaminergic neurotoxin 6-hydroxydopamine (6-OHDA). Proteins of SH-SY5Y cells at various stages of oxidative stress were separated by two-dimensional gel electrophoresis for comparative analysis. Increase in glutathione-S-transferase pi was detected on SYPRO Ruby-stained gels by computer-aided image analysis. Stress-induced alterations in phosphoproteins were detected by Pro-Q Diamond staining. Elongation factor 2, lamin A/C, T-complex protein 1, and heterogeneous nuclear ribonucleoprotein H3 were identified by MALDI-TOF mass spectrometry as stress-responsive elements.     

The retinoic acid and brain-derived neurotrophic factor differentiated SH-SY5Y cell line as a model for Alzheimer's disease-like tau phosphorylation

The paired helical filaments of highly phosphorylated tau protein are the main components of neurofibrillary tangles (NFT) in Alzheimer's disease (AD). Protein kinases including glycogen synthase kinase 3 beta (GSK3beta), cyclin-dependent kinase 5 (Cdk5), and c-Jun N-terminal kinase (JNK) have been implicated in NFT formation making the use of selective kinase inhibitors an attractive treatment possibility in AD. When sequentially treated with retinoic acid (RA) and brain-derived neurotrophic factor (BDNF), the human neuroblastoma SH-SY5Y differentiates to neuron-like cells. We found that coincident with morphologically evident neurite outgrowth, both the content and phosphorylation state of tau increased in RA-BDNF differentiated SH-SY5Y cells. Tau phosphorylation increased at all the examined sites ser-199, ser-202, thr-205, ser-396, and ser-404, all of which are hyperphosphorylated in AD brain. We also investigated whether GSK3beta, Cdk5 or JNK was involved in tau phosphorylation in the differentiated SH-SY5Y cells. We found that GSK3beta contributed most and that Cdk5 made a minor contribution. JNK was not involved in tau phosphorylation in this system. The GSK3beta-inhibitor, lithium, inhibited tau phosphorylation in a concentration-dependent manner and with good reproducibility, which enables ranking of substances in this cell model. RA-BDNF differentiated SH-SY5Y cells could serve as a suitable model for studying the mechanisms of tau phosphorylation and for screening potential GSK3beta inhibitors.     

Differentiation of SH-SY5Y cells to a neuronal phenotype changes cellular bioenergetics and the response to oxidative stress

Cell differentiation is associated with changes in metabolism and function. Understanding these changes during differentiation is important in the context of stem cell research, cancer, and neurodegenerative diseases. An early event in neurodegenerative diseases is the alteration of mitochondrial function and increased oxidative stress. Studies using both undifferentiated and differentiated SH-SY5Y neuroblastoma cells have shown distinct responses to cellular stressors; however, the mechanisms remain unclear. We hypothesized that because the regulation of glycolysis and oxidative phosphorylation is modulated during cellular differentiation, this would change bioenergetic function and the response to oxidative stress. To test this, we used retinoic acid (RA) to induce differentiation of SH-SY5Y cells and assessed changes in cellular bioenergetics using extracellular flux analysis. After exposure to RA, the SH-SY5Y cells had an increased mitochondrial membrane potential, without changing mitochondrial number. Differentiated cells exhibited greater stimulation of mitochondrial respiration with uncoupling and an increased bioenergetic reserve capacity. The increased reserve capacity in the differentiated cells was suppressed by the inhibitor of glycolysis 2-deoxy-d-glucose. Furthermore, we found that differentiated cells were substantially more resistant to cytotoxicity and mitochondrial dysfunction induced by the reactive lipid species 4-hydroxynonenal or the reactive oxygen species generator 2,3-dimethoxy-1,4-naphthoquinone. We then analyzed the levels of selected mitochondrial proteins and found an increase in complex IV subunits, which we propose contributes to the increase in reserve capacity in the differentiated cells. Furthermore, we found an increase in MnSOD that could, at least in part, account for the increased resistance to oxidative stress. Our findings suggest that profound changes in mitochondrial metabolism and antioxidant defenses occur upon differentiation of neuroblastoma cells to a neuron-like phenotype.     

Src family kinase involvement in muscarinic receptor-induced tyrosine phosphorylation in differentiated SH-SY5Y cells

Muscarinic receptor-mediated changes in protein tyrosine phosphorylation were examined in differentiated human neuroblastoma SH-SY5Y cells. Treatment of differentiated cells with 1 mM carbachol caused rapid increases in the tyrosine phosphorylation of focal adhesion kinase (FAK), Cas, and paxillin. The src family kinase-selective inhibitor PP1 reduced carbachol-stimulated tyrosine phosphorylation of FAK, Cas, and paxillin by 50 to 75%. In contrast, carbachol-stimulated activation of ERK1/2 was unaffected by PP1. Src family kinase activation by carbachol was further demonstrated by increased carbachol-induced tyrosine phosphorylation of the src-substrate, p120, and tyrosine phosphorylation of the src family kinase activation-associated autophosphorylation site. Site-specific FAK phosphotyrosine antibodies were used to determine that the carbachol-stimulated increase in the autophosphorylation of FAK was unaffected by pretreatment with PP1, whereas the carbachol-stimulated increase in the src family kinase-mediated phosphotyrosine of FAK was completely blocked by pretreatment with PP1. In SH-SY5Y cell lines stably overexpressing Fyn, the phosphotyrosine immunoreactivity of FAK was 625% that of control cells. Thus, muscarinic receptors activate protein tyrosine phosphorylation in differentiated cells, and the tyrosine phosphorylation of FAK, Cas, and paxillin, but not ERK1/2, is mediated by a src family tyrosine kinase activated in response to stimulation of muscarinic receptors.     

Increased levels of tau protein in SH-SY5Y cells after treatment with cholinesterase inhibitors and nicotinic agonists

Several cholinesterase inhibitors used in the treatment of Alzheimer's disease (AD) have been shown to interact with an allosteric site on the nicotinic acetylcholine receptor (nAChR). A possible linkage between the phosphorylation state of tau, the major component of paired helical filaments found in AD brain, and stimulation of nAChRs by cholinesterase inhibitors and nicotinic agonists was investigated. Western blot analysis showed that treatment of SH-SY5Y cells for 72 h with the cholinesterase inhibitors tacrine (10(-5) M), donepezil (10(-5) M), and galanthamine (10(-5) M), nicotine (10(-5) M), and epibatidine (10(-7) M) increased tau levels as detected with Tau-1, AT 8, and AT 270 monoclonal antibodies and binding of [3H]epibatidine. The increase in tau immunoreactivity induced by nicotine, epibatidine, and tacrine, but not the up-regulation of nAChRs, was prevented by the antagonists d-tubocurarine and mecamylamine. Both antagonists were synergistic with the nicotinic agonists in causing up-regulation, but only d-tubocurarine showed a synergistic effect with tacrine. The increased tau immunoreactivity induced by tacrine was not prevented by atropine, indicating that in terms of cholinergic receptors, tacrine modulates tau levels mainly through interactions with nAChRs and not with muscarinic receptors. Additional work is needed to determine the exact mechanism by which cholinesterase inhibitors and nicotinic agonists modulate phosphorylation and levels of tau protein.     

The role of insulin against hydrogen peroxide-induced oxidative damages in differentiated SH-SY5Y cells

Abstract

Exogenous hydrogen peroxide (H₂O₂) can easily penetrate into biological membranes and enhance the formation of other reactive oxygen species (ROS). In the present study, we have investigated the neuroprotective effects of insulin on H₂O₂-induced toxicity of retinoic acid (RA)-differentiated SH-SY5Y cells. To measure the changes in the cell viability of SH-SY5Y cells at different concentrations of H₂O₂ for 24?h, a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT)-based assay was used and a 100?µM H₂O₂ was selected to establish a model of H₂O₂-induced oxidative stress. Further assays showed that 24?h of 100?µM H₂O₂-induced significant changes in the levels of lactate dehydrogenase (LDH), nitric oxide (NO), ROS, and calcium ion (Ca²⁺) in neuronal cells, but insulin can effectively diminish the H₂O₂-induced oxidative damages to these cells. Moreover, cells treated with insulin increased H₂O₂-induced suppression of glutathione levels and exerted an apparent suppressive effect on oxidative products. The results of insulin treatment with SH-SY5Y cells increased the Bcl-2 levels and decreased the Akt levels. The treatment of insulin had played a protective effect on H₂O₂-induced oxidative stress related to the Akt/Bcl-2 pathways.     

Modulation of heat shock protein response in SH-SY5Y by mobile phone microwaves

AIM: To investigate putative biological damage caused by GSM mobile phone frequencies by assessing electromagnetic fields during mobile phone working. METHODS: Neuron-like cells, obtained by retinoicacid-induced differentiation of human neuroblastoma SH-SY5Y cells, were exposed for 2 h and 4 h to microwaves at 1800 MHz frequency bands. RESULTS: Cell stress response was evaluated by MTT assay as well as changes in the heat shock protein expression (Hsp20, Hsp27 and Hsp70) and caspase-3 activity levels, as biomarkers of apoptotic pathway. Under our experimental conditions, neither cell viability nor Hsp27 expression nor caspase-3 activity was significantly changed. Interestingly, a significant decrease in Hsp20 expression was observed at both times of exposure, whereas Hsp70 levels were significantly increased only after 4 h exposure. CONCLUSION: The modulation of the expression of Hsps in neuronal cells can be an early response to radiofrequency microwaves.     

Iron-induced oxidative stress modify tau phosphorylation patterns in hippocampal cell cultures

Oxidative stress phenomena have been related with the onset of neurodegenerative diseases. Particularly in Alzheimer Disease (AD), oxygen reactive species (ROS) and its derivatives can be found in brain samples of postmortem AD patients. However, the mechanisms by which oxygen reactive species can alter neuronal function are still not elucidated. There is a growing amount of evidence pointing to a role for mitochondrial damage as the source of free radicals involved in oxidative stress. Among the species that participate in the production of oxygen reactive radicals, transition metals are one of the most important. Several reports have implicated the involvement of redox-active metals with the onset of different neurodegenerative diseases such as Alzheimer's Disease (AD), Progressive Supranuclear Palsy (PSP), Amyotrophic Lateral Sclerosis (ALS) and Parkinson's Disease (PD). On the other hand, our previous studies have indicated that A-induced deregulation of the protein kinase Cdk5 associated with tau protein hyperphosphorylation constitute a critical pathway toward neurodegeneration. In the current paper we have shown that iron induces an imbalance in the function of Cdk5/p25 system of hippocampal neurons, resulting in a marked decrease in tau phosphorylation at the typical Alzheimer's epitopes. The loss of phosphorylated tau epitopes correlated with an increase in 4-hydroxy-nonenal (HNE) adducts revealing damage by oxidative stress. This effects on tau phosphorylation patterns seems to be a consequence of a decrease in the Cdk5/p25 complex activity that appears to result from a depletion of the activator p25, a mechanism in which calcium transients could be implicated.