α-Synuclein Overexpression Induces Lysosomal Dysfunction and Autophagy Impairment in Human Neuroblastoma SH-SY5Y

Neurochemical Research - Although the etiology of Parkinson's disease (PD) is multifactorial, it has been linked to abnormal accumulation of α-synuclein (α-syn) in dopaminergic...     

Effects of 2‐Chloro‐2′‐Deoxyadenosine on the Cell Cycle in the Human Leukemia EHEB Cell Line

To explain why 2‐chloro‐2′‐deoxyadenosine (CdA) is unable to block DNA synthesis and cell cycle progression, and paradoxically enhances progression from G1 into S phase in the CdA‐resistant leukemia EHEB cell line, we studied its metabolism and effects on proteins regulating the transition from G1 to S phase. A low deoxycytidine kinase activity and CdATP accumulation, and a lack of p21 induction despite p53 phosphorylation and accumulation may account for the inability of CdA to block the cell cycle. An alternative pathway involving pRb phosphorylation seems implicated in the CdA‐induced increase in G1 to S phase progression.     

Retinol (Vitamin A) Increases α-Synuclein, β-Amyloid Peptide,Tau Phosphorylation and RAGE Content in Human SH-SY5Y Neuronal Cell Line

Retinoids (vitamin A and derivatives) are recognized as essential factors for central nervous system (CNS) development. Retinol (vitamin A) also was postulated to be a major antioxidant component of diet as it modulates reactive species (RS) production and oxidative stress in biological systems. Oxidative stress plays a major role either in pathogenesis or development of neurodegenerative diseases, or even in both. Here we investigate the role of retinol supplementation to human neuron-derived SH-SY5Y cells over RS production and biochemical markers associated to neurodegenerative diseases expressed at neuronal level in Parkinson’s disease and Alzheimer’s disease: α-synuclein, β-amyloid peptide, tau phosphorylation and RAGE. Retinol treatment (24 h) impaired cell viability and increased intracellular RS production at the highest concentrations (7 up to 20 µM). Antioxidant co-treatment (Trolox 100 µM) rescued cell viability and inhibited RS production. Furthermore, retinol (10 µM) increased the levels of α-synuclein, tau phosphorylation at Ser396, β-amyloid peptide and RAGE. Co-treatment with antioxidant Trolox inhibited the increased in RAGE, but not the effect of retinol on α-synuclein, tau phosphorylation and β-amyloid peptide accumulation. These data indicate that increased availability of retinol to neurons at levels above the cellular physiological concentrations may induce deleterious effects through diverse mechanisms, which include oxidative stress but also include RS-independent modulation of proteins associated to progression of neuronal cell death during the course of neurodegenerative diseases.     

Impact of Inhomogeneous Static Magnetic Field (31.7–232.0 mT) Exposure on Human Neuroblastoma SH-SY5Y Cells during Cisplatin Administration

Beneficial or adverse effects of Static Magnetic Fields (SMFs) are a large concern for the scientific community. In particular, the effect of SMF exposure during anticancer therapies still needs to be fully elucidated. Here, we evaluate the effects of SMF at induction levels that cisPt-treated cancer patients experience during the imaging process conducted in Low field (200–500 mT), Open field (300–700 mT) and/or inhomogeneous High field (1.5–3 T) Magnetic Resonance Imaging (MRI) machines. Human adrenergic neuroblastoma SH-SY5Y cells treated with 0.1 µM cisPt (i.e. the lowest concentration capable of inducing apoptosis) were exposed to SMF and their response was studied in vitro. Exposure of 0.1 µM cisPt-treated cells to SMF for 2 h decreased cell viability (30%) and caused overexpression of the apoptosis-related cleaved caspase-3 protein (46%). Furthermore, increase in ROS (Reactive Oxygen Species) production (23%) and reduction in the number of mitochondria vs controls were seen. The sole exposure of SMF for up to 24 h had no effect on cell viability but increased ROS production and modified cellular shape. On the other hand, the toxicity of cisPt was significantly prevented during 24 h exposure to SMF as shown by the levels of cell viability, cleaved caspase-3 and ROS production. In conclusion, due to the cytoprotective effect of 31.7–232.0 mT SMF on low-cisPt-concentration-treated SH-SY5Y cells, our data suggest that exposure to various sources of SMF in cancer patients under a cisPt regimen should be strictly controlled.     

The Neuroprotective Effects of Justicidin A on Amyloid Beta25–35-Induced Neuronal Cell Death Through Inhibition of Tau Hyperphosphorylation and Induction of Autophagy in SH-SY5Y Cells

Justicidin A is a structurally defined arylnaphthalide lignan, which has been shown anti-cancer activity; however, the neuroprotective effect of justicidin A is still untested. In this study, we investigated the action of justicidin A on amyloid beta (Aβ)_25–35-induced neuronal cell death via inhibition of the hyperphosphorylation of tau and induction of autophagy in SH-SY5Y cells. Pretreatment with justicidin A significantly elevated cell viability in cells treated with Aβ_25–35. Western blot data demonstrated that justicidin A inhibited the Aβ_25–35-induced up-regulation the levels of hyperphosphorylation of tau in SH-SY5Y cells. In addition, treatment with justicidin A significantly induced autophagy as measured by the increasing LC3 II/I ratio, an important autophagy marker. These studies showed that justicidin A inhibited activity of glycogen synthase kinase-3beta (GSK-3β), which is an important kinase in up-stream signaling pathways; inhibited hyperphosphorylation of tau in AD; and enhanced activity of AMP-activated protein kinase (AMPK), which is the key molecule for both hyperphosphorylation of tau and induction of autophagy. These data provide the first evidence that justicidin A protects SH-SY5Y cells from Aβ_25–35-induced neuronal cell death through inhibition of hyperphosphorylation of tau and induction of autophagy via regulation the activity of GSK-3β and AMPK, and they also provide some insights into the relationship between tau protein hyperphosphorylation and autophagy. Thus, we conclude that justicidin A may have a potential role for neuroprotection and, therefore, may be used as a therapeutic agent for AD.

     

Pinocembrin Attenuates Mitochondrial Dysfunction in Human Neuroblastoma SH-SY5Y Cells Exposed to Methylglyoxal: Role for the Erk1/2–Nrf2 Signaling Pathway

Pinocembrin (PB; 5,7-dihydroxyflavanone) is found in propolis and exhibits antioxidant activity in several experimental models. The antioxidant capacity of PB is associated with the activation of the nuclear factor erythroid 2-related factor 2/antioxidant response element (Nrf2/ARE) signaling pathway. The Nrf2/ARE axis mediates the expression of antioxidant and detoxifying enzymes, such as glutathione peroxidase (GPx), glutathione reductase (GR), heme oxygenase-1 (HO-1), and the catalytic (GCLC) and regulatory (GCLM) subunits of the rate-limiting enzyme in the synthesis of glutathione (GSH), γ-glutamate-cysteine ligase (γ-GCL). Nonetheless, it is not clear how PB exerts mitochondrial protection in mammalian cells. Human neuroblastoma SH-SY5Y cells were pretreated (4 h) with PB (0–25 µM) and then exposed to methylglyoxal (MG; 500 µM) for further 24 h. Mitochondria were isolated by differential centrifugation. PB (25 µM) provided mitochondrial protection (decreased lipid peroxidation, protein carbonylation, and protein nitration in mitochondrial membranes; decreased mitochondrial free radical production; enhanced the content of GSH in mitochondria; rescued mitochondrial membrane potential—MMP) and blocked MG-triggered cell death by a mechanism dependent on the activation of the extracellular-related kinase (Erk1/2) and consequent upregulation of Nrf2. PB increased the levels of GPx, GR, HO-1, and mitochondrial GSH. The PB-induced effects were suppressed by silencing of Nrf2 with siRNA. Therefore, PB activated the Erk1/2–Nrf2 signaling pathway resulting in mitochondrial protection in SH-SY5Y cells exposed to MG. Our work shows that PB is a strong candidate to figure among mitochondria-focusing agents with pharmacological potential.     

Construction of SH-EP1-α4β2-hAPP695 Cell Line and Effects of Nicotinic Agonists on β-amyloid in the Cells

(1) Nicotinic acetylcholine receptors in central nervous system are thought to be new targets for Alzheimer’s disease. However, the most involved nicotinic receptor subtype in Alzheimer’s disease is unclear. α4β2 receptor is the most widely spread subtype in brain, involving in several important aspects of cognitive and other functions. We constructed cell line by transfecting human amyloid precursor protein (695) gene into SH-EP1 cells which have been transfected with human nicotinic receptor α4 subunit and β2 subunit gene, to observe effects of α4β2 receptors activation on β-amyloid, expecting to provide a new cell line for drug screening and research purpose. (2) Liposome transfection was used to express human amyloid precursor protein (695) gene in SH-EP1-α4β2 cells. Function of the transfected α4β2 receptors was tested by patch clamp. Effects of nicotine and epibatidine (selective α4β2 nicotinic receptor agonist) on β-amyloid were detected by Western blot and ELISA. Effects of nicotine and epibatidine on amyloid precursor protein (695) mRNA level were measured using real-time PCR. (3) Human amyloid precursor protein (695) gene was stably expressed in SH-EP1-α4β2 cells; Nicotine (1 μM) and epibatidine (0.1 μM) decreased intracellular and secreted β-amyloid in the cells; and activation of α4β2 receptors did not affect amyloid precursor protein (695) mRNA level. (4) These results suggest that the constructed cell line, expressing both amyloid precursor protein (695) gene and human nicotinic receptor α4 subunit and β2 subunit gene, might be useful for screening specific nicotinic receptor agonists against Alzheimer’s disease. Alteration of Aβ level induced by activation of α4β2 nAChR in our study might occur at a post-translational level.     

Neuroprotective effects of Mycoplasma hyorhinis against amyloid-β-peptide toxicity in SH-SY5Y human neuroblastoma cells are mediated by calpastatin upregulation in the mycoplasma-infected cells

Mycoplasmas are frequent contaminants of cell cultures. Contamination leads to altered synthetic and metabolic pathways. We have found that contamination of neuroblastoma SH-SY5Y cells by a strain of Mycoplasma hyorhinis derived from SH-SY5Y cell culture (NDMh) leads to increased levels of calpastatin (the endogenous inhibitor of the Ca(2+)-dependent protease, calpain) in NDMh-infected cells. We have now examined effects of amyloid-β-peptide (Aβ) (central to the pathogenesis of Alzheimer's disease) on uncontaminated (clean) and NDMh-infected SH-SY5Y cells. Aβ was toxic to clean cells, resulting in necrotic cell damage. Aβ treatment led to activation of calpain and enhanced proteolysis, cell swelling, cell membrane permeability to propidium iodide (PI) (without nuclear apoptotic changes), and diminished mitochondrial enzyme activity (XTT reduction). Aβ-toxicity was attenuated in the high calpastatin-containing NDMh-infected cells, as shown by inhibition of calpain activation and activity, no membrane permeability, normal cell morphology, and maintenance of mitochondrial enzyme activity (similar to attenuation of Aβ-toxicity in non-infected cells overexpressing calpastatin following calpastatin-plasmid introduction into the cells). By contrast, staurosporine affected both clean and infected cells, causing apoptotic damage (cell shrinkage, nuclear apoptotic alterations, caspase-3 activation and caspase-promoted proteolysis, without PI permeability, and without effect on XTT reduction). The results indicate that mycoplasma protects the cells against certain types of insults involving calpain. The ratio of calpastatin to calpain is an important factor in the control of calpain activity. Exogenous pharmacological means, including calpastatin-based inhibitors, have been considered for therapy of various diseases in which calpain is implicated. Mycoplasmas provide the first naturally occurring biological system that upregulates the endogenous calpain inhibitor, and thus may be of interest in devising treatments for some disorders, such as neurodegenerative diseases.     

Investigation of the Effects of 2.1 GHz Microwave Radiation on Mitochondrial Membrane Potential (ΔΨ m), Apoptotic Activity and Cell Viability in Human Breast Fibroblast Cells

In the present study we aimed to investigate the effects of 2.1 GHz Wideband Code Division Multiple Access (W-CDMA) modulated Microwave (MW) Radiation on cell survival and apoptotic activity of human breast fibroblast cells. The cell cultures were exposed to W-CDMA modulated MW at 2.1 GHz at a SAR level of 0.607 W/kg for 4 and 24 h. The cell viability was assessed by MTT [3-(4, 5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] method. The percentage of apoptotic cells was analyzed by Annexin V-FITC and PI staining. 5,5′,6,6′-Tetrachloro-1,1′,3,3′- tetraethylbenzimidazolcarbocyanine iodide (JC-1) was used to measure Mitochondrial Membrane Potential (ΔΨ _m). sFasL and Fas/APO-1 protein levels were determined by ELISA method. 2.1 GHz MW radiation was shown to be able to inhibit cell proliferation and induce apoptosis in human breast fibroblast cells. The cell viability of MW-exposed cells was decreased significantly. The percentages of Annexin V-FITC positive cells were higher in MW groups. ΔΨ _m was decreased significantly due to MW radiation exposure. However, neither sFas nor FasL level was significantly changed in MW-exposed fibroblast cells. The results of this study showed that 2.1 GHz W-CDMA modulated MW radiation-induced apoptotic cell death via the mitochondrial pathway.     

The influence of inhibiting or stimulating the expression of the α3 subunit of the nicotinic receptor in SH-SY5Y cells on levels of amyloid-β peptide and β-secretase

To examine the effects of the α3 subunit of the nicotinic acetylcholine receptor (nAChR) on the expression of β-secretase and the concomitant level of amyloid-β (Aβ), SH-SY5Y neuroblastoma cells were either transfected with small interference RNAs (siRNAs) specifically targeting this subunit or exposed to nicotine. The levels of α3 nAChR mRNA and protein, as well as the corresponding levels of BACE1 (which cleaves the β-site of APP) and BACE2 (cleaving in the Aβ domain) were determined by real-time PCR and Western blotting, respectively. The levels of Aβ_1–42 in culture media were determined by an Elisa procedure.     

Mapping of the human gene encoding the mutual signal-transducing subunit (β-chain) of granulocyte-macrophage colony-stimulating factor (GM-CSF), interleukin-3 (IL-3), and interleukin-5 (IL-5) receptor complexes to chromosome 22q13.1

The human gene encoding the mutual signal-transducing subunit (-chain) of granulocyte-macrophage colony-stimulating factor, interleukin-3, and interleukin-5 receptor complexes has been mapped to chromosome 22q13.1 by the fluorescence in situ hybridization method.     

Formation of Triple Helices at Irregular Poly (R·Y) Sites Located in Critical Positions in the Human BCR Promoter

Abstract

Two polypurine sequences interrupted respectively by one and two adjacent pyrimidines have been identified in the promoter of the human bcr gene. Although these targets are irregular they are recognised and tightly bound by AG and GT motif triplex-forming oligonucleotides. Thermodynamic and kinetic data are presented.