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Belbaraka, Loubaba. "Growth, differentiation and cell-cell coupling in the human neuroblastoma cell line SH-SY5Y." Thesis, University of Ottawa (Canada), 2002. http://hdl.handle.net/10393/6112.
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Neuroblastoma is one of the most common paediatric solid tumours, frequently occurring in infancy with the primary lesion in the adrenal gland and sympathetic nervous system. It originates from primitive neural crest cells. In rare cases, this tumour regresses spontaneously to a more benign ganglioneuroma probably by neuronal differentiation or apoptosis. We investigated the role of induction of neuronal differentiation and apoptosis in vitro in SH-SY5Y neuroblastoma cells. A variety of agents that are known to induce neuronal differentiation including retinoic acid, nerve growth factor (NGF), protein kinase C (PKC) inhibitors and a cAMP-dependent protein kinase A (PKA) activator were tested solely or in combination for their capacity to induce terminal differentiation. The cells were characterised for markers of differentiation as well as their ability to withdraw from the cell cycle. We found that the combination of 8-Br-cAMP (PKA activator) with NGF in the presence of the cell cycle inhibitor aphidicolin was the best treatment to induce terminal differentiation in SH-SY5Y cells. Treated cells showed long neurites resembling those of neurones. They expressed markers characteristic of the cytoskeleton (NF200, NF68) and of neuronal function (tyrosine hydroxylase, choline acetyltransferase and the neurone-specific enolase) and showed an increase in the expression of TrkA, the receptor for NGF. Furthermore, the expression of the N-myc oncogene that is normally overexpressed in these cells decreased. Since neurones are dependent on NGF for survival, we tested the ability of terminally differentiated SH-SY5Y cells to survive in the absence of NGF. We found that the cells became NGF-survival dependent and that deprivation from this neurotrophic factor induced programmed cell death. We also tested the effect of PKC specific and non-specific inhibitors on cell proliferation, differentiation and apoptosis in SH-SY5Y cells. We found that only the non-specific PKC inhibitors staurosporine and H7 induced morphological and molecular differentiation as well as decreased N-myc amplification followed by apoptosis. The effect of differentiation induction on p53 sub-localisation was also investigated in undifferentiated and differentiated SH-SY5Y cells. p53 immunostaining showed that p53 was sequestered in the cytoplasm in undifferentiated SH-SY5Y cells and that the induction of differentiation resulted in the partial transfer of p53 to the nucleus where it probably became able to regulate the cell cycle and apoptosis since p53 is not mutated in neuroblastoma. Gap junction intercellular communication (GJIC) is known to be involved in the regulation of cell growth and homeostasis and has been shown to contribute to many diseases including cancer. We investigated the role of GJIC in neuroblastoma. We found that SH-SY5Y cells have altered GJIC due to an aberrant localisation in the perinuclear region of connexin 43 (Cx43), one of the proteins of GJIC normally present at the plasma membrane. GJ channel formation and gating is regulated by PKC, PKA and/or MAPK. We found that induction of differentiation using the PKA activator 8-Br-cAMP relocalised Cx43 to the plasma membrane region and restored GJIC. Furthermore, inhibition of p38 MAPK induced a block of cell proliferation associated with GJIC proficiency while inhibition of the subfamily of MAPK, Erk1/Erk2, likely promoted the degradation of Cx43.
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Song, Juxian, and 宋聚先. "Protective effects of chrysotoxine on Parkinsonian neurotoxins induceddopaminergic neuronal cell death in SH-SY5Y cells." Thesis, The University of Hong Kong (Pokfulam, Hong Kong), 2011. http://hub.hku.hk/bib/B47150129.
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Samms, Warren C. "Catecholamine disturbance and SH-SY5Y cell toxicity of halogenated 3-amino-2 phenylpropenes." Diss., Wichita State University, 2009. http://hdl.handle.net/10057/2378.
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Parkinson’s disease (PD) is a progressive neurodegenerative disease that
affects between 1% and 2% of the population over age 65. Despite decades of
research and the development of many molecular models of PD, there is far from a
consensus as to the etiology of this disease. Current molecular models, such as the
role of the quaternary ammonium ion MPP+ and its effect on cell death, in the
presence/absence of monoamine transporters as opposed to distinct intracellular
activity are still disputed.
The 3-amino-2-phenylpropene (APP) class of compounds has been previously
characterized as reversible inhibitors for the bovine adrenal chromaffin granule vesicular
monoamine transporter (VMAT) as well as potent irreversible dopamine-β-
monooxygenase (DβM) and monoamine oxidase (MAO) inhibitors. These effects result
in perturbation of catecholamine uptake, storage and/or metabolism, leading to the
potential for increased oxidative stress.
Herein, we report that halogen substitution on the 4'-position of the aromatic ring
gradually increases VMAT inhibition potency from 4'-F to 4'-I, parallel to the
hydrophobicity of the halogen. We show that these derivatives are taken up into both
neuronal and non-neuronal cells, and into resealed chromaffin granule ghosts efficiently
through passive diffusion. In addition, these derivatives are highly toxic to human
neuroblastoma SH-SY5Y cells, they are not toxic to several non-neuronal cell lines at
similar concentrations. These compounds drastically perturb DA uptake and
metabolism in SH-SY5Y cells under sub-lethal conditions, and are able to deplete both
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vesicular and cytosolic catecholamines similar to amphetamines. Additionally, (4'-iodo)
3-amino-2-phenylpropene (4'-IAPP) treatment significantly increases intracellular
reactive oxygen species (ROS) and decreases glutathione reduced form (GSH) levels in
SH-SY5Y cells, and cell death is significantly attenuated by the common antioxidants α-
tocopherol, N-acetyl-L-cysteine (NAC) and glutathione (GSH).
This suggests that ROS production is intricately involved with the mechanism of
cell death. Although DNA fragmentation analysis supports that apoptosis occurs, the
fact that a non-specific caspase inhibitor provided no significant protection suggests that
that cell death is likely due to a caspase-independent ROS-mediated apoptotic pathway.
Based on these and other findings, we propose that drastic perturbation of DA
metabolism in SH-SY5Y cells by 4'-halo APP derivatives causes increased oxidative
stress leading to apoptotic cell death. These compounds, which induce catecholaminespecific
neurotoxic effects following nonspecific cellular entry may be a unique resource
in the modeling of PD both in vitro and in vivo.Thesis (Ph.D.)--Wichita State University, College of Liberal Arts and Sciences, Dept. of Chemistry
4
Lin, Shu-Ting. "Effects of inclusion body formationand alpha-synuclein on apoptotic cell death in SH-SY5Y." Thesis, King's College London (University of London), 2005. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.417323.
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FERNANDEZ, ABASCAL JESUS. "Role of cytochrome P450 against toxic insult in neuroblastoma SH-SY5Y cells." Doctoral thesis, Università di Siena, 2018. http://hdl.handle.net/11365/1051261.
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Cytochrome P450 (CYP) is one of the main metabolic systems involved in xenobiotic clearance in live organisms. Its activity is mainly carried out at hepatic level, but in the last two decades, its function has been also reported to be important in other extrahepatic tissues, especially in the central nervous system. In brain, CYP isoenzymes vary their expression depending on the brain area and the cell type. However, the role of the different isoforms is not yet well characterized. Some of them have been involved in detoxification and/or toxic activation of xenobiotics; while other have been related with production of oxidative stress through the metabolism of endogenous compounds. Therefore, the presence and the function of the CYP in the brain has been related with neurodegenerative diseases such as Parkinson’s Disease (PD).
On the other hand, the study of the degeneration of dopaminergic neurons in vitro has been carried out by the use of several toxins that promote apoptosis and similar pathological features that the observed in PD. Among these xenobiotics, 1-methyl-4-phenylpyridinium (MPP+), rotenone, and paraquat are the most used because they are able to promote neurodegeneration of dopaminergic cells by directly targeting complex I of mitochondria.
Recently, it has been reported that some CYP isoforms, such as CYP 2D6, can be involved in the development of this neurodegenerative disease. To better characterize this role, we have studied the induction of some isoforms in an in vitro system. Undifferentiated SH-SY5Y cells were treated with well-known inducers of CYP for 48 hours, namely β-naphtoflavone (β-NF), ethanol (EtOH), and cyclophosphamide (CPA); and qRT-PCR, Western Blot (WB) and confocal microscopy analysis were performed. CPA increased the mRNA levels of CYP 1A1, 2D6 and 2E1, while the other inducers promoted a slight increase on these isoforms compared to CPA. WB analysis confirmed the induction promoted by CPA in CYP 1A1 and 2D6, and revealed that CYP 2D6 was also inducible by EtOH. Moreover, CYP 2E1 was increased by β-NF and EtOH treatments. In differentiated SH-SY5Y cells, a preliminary WB analysis of CYP 2D6 and 2E1 was also performed. The results suggested a change in the regulation of the expression of CYP 2D6 when treated with β-NF, and also of CYP 2E1 levels when treated with β-NF and CPA. Immunohistochemistry analysis confirmed the inducibility of CYPs and showed a co-localization of CYP 2D6 with mitochondria. These data indicate that CYP can be induced in both undifferentiated and differentiated neuroblastoma cells, and underline the possibility to use this in vitro system for studying the role of CYPs in neurodegeneration.
Moreover, as showed by our group and others, β-naphtoflavone and ethanol have the ability to induce the expression of some CYP isoforms. In order to study the possible role of CYP induction in neurodegeneration, we have use the same in vitro model than before, where undifferentiated neuroblastoma SH-SY5Y cells have been treated with these inducers separately previous and during exposure of MPP+. In both experimental conditions, the toxic effect of MPP+ was partially reverted. MTT assays showed an increase in cell viability of approximately 17% in β-NF treatment, while EtOH showed an increase between 13-15%. The analysis of apoptotic population by flow cytometry showed that both treatments were able to restore the populations to control values compared to the toxic effect of MPP+. The mitochondrial fission-fusion kinetics revealed that both treatments were able to avoid the impairment of this mitochondrial motility after exposure to the toxin. Finally, this neuroprotection was confirmed by a lowest effect of MPP+ upon complex I activity when cells were preincubated with the inducers. These results bring new insights about the possible role of CYPs, specially CYP 2D6, in neuroprotection and possible development of therapeutic drugs.
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Klein, Juliane. "Amyloid precursor protein (APP) and copper homeostasis in the human neuroblastoma cell line SH-SY5Y." Thesis, University of Newcastle upon Tyne, 2011. http://hdl.handle.net/10443/1201.
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Alzheimer’s disease (AD) is characterised by cerebellar accumulation and aggregation of amyloid beta (Aβ), a cleavage product of the transmembrane amyloid precursor protein (APP). APP contains a range of functional domains in its large extracellular portion, among which are two copper-binding motifs and one zinc-binding motif. The copper-binding motifs are present in the amino-terminal region of APP and within the Aβ region of the protein and readily reduce Cu(II) to Cu(I), thus APP and its cleavage products are linked to copper metabolism and have been hypothesised to participate in cellular copper homeostasis. In this project human neuroblastoma cell lines SH-SY5Y were utilised to determine the effect of expressing a familial AD mutation on intracellular copper concentrations and possible functional alterations or deficits of enzymes that require copper as a co-factor. The familial AD mutation first found in a Swedish population was previously shown to increase the total amount of released Aβ. Direct phenotypic comparison between SH-SY5Y APPWT cell lines expressing endogenous levels of APP and APPswe Coupling native two dimensional liquid chromatography with metal analysis, SDS-PAGE and Principal Component Analysis identified one major copper and zinc containing pool as copper-zinc superoxide dismutase (SOD1) in soluble whole cell protein extracts. Comparative analysis of metal content between APP cell lines overexpressing APP carrying the Swedish mutation was performed in standard culture and manipulated copper concentrations. WT and APPswe cultures indicated a difference in metallation of SOD1 with copper. APPswe cultures displayed reduced metallation of SOD1, whereas SOD1 metallation with zinc remained unaltered. Functional analysis of copper-binding enzymes, such as SOD1 and cytochrome c oxidase (CCO), using standard biochemical approaches, identified lower activities for both enzymes during standard cell culture in APPswe cells. Upon treatment of cultures with increasing concentrations of exogenous copper APPWT enzyme activities remained unaltered but enzyme activities in APPswe cultures increased in direct correlation with increasing copper concentrations. Combined with phenotypic analysis of growth, survival and intracellular metal content it appears that APPswe cultures are copper deficient, but this can be overcome by copper supplementation. Lower copper accumulation also enables greater survival of APP swe Overall, these data suggest that APP cells in elevated copper. swe overexpression in SH-SY5Y cells results in functional copper deficiency which can be rescued by supplementation of cultures with exogenous copper. APPswe further confers resistance to copper toxicity not only via increased Aβ release, but also via increased copper delivery to enzymatic target proteins improving cellular antioxidant response and energy metabolism. These data are consistent with a function of APP in copper efflux either in a regulatory capacity or directly contributing to copper egress.
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Lau, Kwok-wai. "Evaluation of using all-trans-retinoic acid to differentiate human neuroblastoma SH-SY5Y cells in neurodegeneration research." Click to view the E-thesis via HKUTO, 2007. http://sunzi.lib.hku.hk/HKUTO/record/B39557339.
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Lau, Kwok-wai, and 劉國威. "Evaluation of using all-trans-retinoic acid to differentiate human neuroblastoma SH-SY5Y cells in neurodegeneration research." Thesis, The University of Hong Kong (Pokfulam, Hong Kong), 2007. http://hub.hku.hk/bib/B39557339.
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Fjæraa, Alfredsson Christina. "Effects of ellagic acid in human neuroblastoma cells." Doctoral thesis, Karlstads universitet, Institutionen för hälsovetenskaper, 2013. http://urn.kb.se/resolve?urn=urn:nbn:se:kau:diva-29905.
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A diet rich in polyphenols has been proposed to have beneficial health effects and to reduce risk of disease. Ellagic acid, a polyphenol common in red berries and pomegranates, has potential anti-tumorigenic effects that make it interesting to further study in different cancer cell systems. Neuroblastoma is a childhood cancer that arises during development of the peripheral nervous system. Neuroblastoma, being an embryonal tumor, show loss of function of genes controlling differentiation and apoptosis. Neuroblastoma is a heterogenic tumor disease, and highly malignant neuroblastomas are difficult to treat despite different treatment modalities, identifying a need for new and combinatory treatments. A common model for human neuroblastoma is the SH-SY5Y cell line resembling immature neuroblasts that can be differentiated in vitro with several agents including the phorbol ester 12-O-tetradecanoylphorbol-13-acetate and the vitamin A-derivative all-trans retinoic acid. Here, the effect of ellagic acid on proliferation, cell detachment and apoptosis in non-differentiated and in vitro-differentiated SH-SY5Y cells were studied with the aim of identifying cellular target mechanisms and a possible therapeutic potential for ellagic acid. In non-differentiated cells, ellagic acid reduced cell number, inhibited cell cycle activity, and induced cell detachment and apoptosis. Apoptosis was partly mediated by the intrinsic pathway. 12-O-tetradecanoylphorbol-13-acetate and all-trans retinoic acid both induced morphological differentiation, while only the latter induced G0/G1-arrest. Single-cell analysis revealed that 12-O-tetradecanoylphorbol-13-acetate-treated cells continued cycling during neuritogenesis while these two read-outs were mutually exclusive in all-trans retinoic acid-treated cells. 12-O-tetradecanoylphorbol-13-acetate- and especially all-trans retinoic acid-differentiated cells showed lower sensitivity to ellagic acid-dependent cell detachment and apoptosis.Artikel 4 ("Altered sensitivity...") ingick som manuskript i avhandlingen, nu publicerad.
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Sadideen, Doraid, and Doraid Sadideen. "Exploring G-Protein-Coupled Receptors Regulation, Specificity and Controllability of Exosomes Release in the Neuronal Cell Line SH-SY5Y." Thesis, The University of Arizona, 2016. http://hdl.handle.net/10150/621166.
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Parkinson's disease is a neurodegenerative disease characterized by the buildup of aggregated and spread of misfolded alpha-synuclein. How the misfolded alpha-synuclein contributing to the toxicity and death of neuronal cells has been the focal point of research. The spread of alpha-synuclein has been attributed to many mechanisms, one of which is via cell-derived vesicles called exosomes. This project aims to examine the controllability of exosome release. SH-SY5Y, MCF-7 and CHO-K1 cells were transfected with dopamine receptor 3-green fluorescent protein, G-protein receptor 143 or green fluorescent protein and treated with either dopamine or L-DOPA. Medium was harvested and subjected to ultracentrifugation and a silver stain and western blot were performed. There was no significant difference in the total protein in the exosome fraction lanes between the treatment groups or within them. Another aim was to test the specificity of exosomes. Exosomes isolated from SH-SY5Y or MCF-7 were labeled with Exo-Red dye and introduced to wells containing SH-SY5Y, MCF-7 and CHO-K1 cells at room temperature and -4C. At room temperature, exosomes were observed intercellular in all of the cell lines, however, they did not deliver their content. At -4C exosome uptake was halted and they remained on the surface of the cells. Exo-Red labeled SH-SY5Y exosomes were treated with proteinase K and were introduced to CHO-K1 cells at -4C and room temperature. CHO-K1 did not take up exosomes, suggesting exosomes contain one or more necessary proteins needed to interact with the cellular membrane to initiate internalization. CHO-K1 cells were treated with versene to examine the involvement of integrin proteins. Exo-Red labeled SH-SY5Y exosomes were trapped on the surface of CHO-K1 after versene treatment. Lastly, Exo-Red labeled SH-SY5Y exosomes were biotinylated and magnetically captured then introduced to SH-SY5Y and MCF-7 cells and a silver stain and a biotinylated blot were performed. MCF-7 bound more Exo-Red labeled SH-SY5Y exosomes.
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Mateus, José Carlos Barreiro. "Optimization of a multielectrode array (MEA)-based approach to study the impact of Aβ on the SH-SY5Y cell line." Master's thesis, Universidade de Aveiro, 2015. http://hdl.handle.net/10773/15796.
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Mestrado em Biomedicina MolecularThe human brain stores, integrates, and transmits information recurring to millions of neurons, interconnected by countless synapses. Though neurons communicate through chemical signaling, information is coded and conducted in the form of electrical signals. Neuroelectrophysiology focus on the study of this type of signaling. Both intra and extracellular approaches are used in research, but none holds as much potential in high-throughput screening and drug discovery, as extracellular recordings using multielectrode arrays (MEAs). MEAs measure neuronal activity, both in vitro and in vivo. Their key advantage is the capability to record electrical activity at multiple sites simultaneously. Alzheimer’s disease (AD) is the most common neurodegenerative disease and one of the leading causes of death worldwide. It is characterized by neurofibrillar tangles and aggregates of amyloid-β (Aβ) peptides, which lead to the loss of synapses and ultimately neuronal death. Currently, there is no cure and the drugs available can only delay its progression. In vitro MEA assays enable rapid screening of neuroprotective and neuroharming compounds. Therefore, MEA recordings are of great use in both AD basic and clinical research. The main aim of this thesis was to optimize the formation of SH-SY5Y neuronal networks on MEAs. These can be extremely useful for facilities that do not have access to primary neuronal cultures, but can also save resources and facilitate obtaining faster high-throughput results to those that do. Adhesion-mediating compounds proved to impact cell morphology, viability and exhibition of spontaneous electrical activity. Moreover, SH-SY5Y cells were successfully differentiated and demonstrated acute effects on neuronal function after Aβ addition. This effect on electrical signaling was dependent on Aβ oligomers concentration. The results here presented allow us to conclude that the SH-SY5Y cell line can be successfully differentiated in properly coated MEAs and be used for assessing acute Aβ effects on neuronal signaling.
O cérebro humano armazena, integra e transmite informação recorrendo a milhões de neurónios, interconetados por inúmeras sinapses. Embora os neurónios comuniquem entre si através de sinais químicos, a informação é codificada e conduzida sob a forma de sinais elétricos. A neuroeletrofisiologia foca-se no estudo deste tipo de sinalização. Tanto abordagens intra, como abordagens extracelulares são usadas em investigação, mas nenhuma detém tanto potencial em screening de alto débito e na descoberta de fármacos, como medições extracelulares baseadas em matrizes de multi-elétrodos (MEA). MEAs medem a atividade neuronal, tanto em in vitro como em in vivo. A sua principal vantagem é a capacidade de medir atividade elétrica a partir de vários locais simultaneamente. A doença de Alzheimer (DA) é a doença neurodegenerativa mais comum e uma das principais causas de morte em todo o mundo. É caracterizada por emaranhados neurofibrilares e agregados de péptidos amilóides (Aβ), que conduzem à perda de sinapses e em última instância, à morte neuronal. Atualmente, não existe cura e os tratamentos disponíveis apenas retardam a sua progressão. Os ensaios in vitro com MEA permitem uma seleção rápida dos compostos neuroprotectores e neurotóxicos. Portanto, as medições com recurso a MEA são de grande utilidade na investigação básica e clínica da DA. O principal objetivo desta tese foi otimizar a formação de redes neuronais SH-SY5Y em MEAs. Estas podem ser extremamente úteis para instalações que não têm acesso a culturas neuronais primárias, mas também podem economizar recursos e facilitar a obtenção mais rápida de resultados para aquelas que têm acesso. Compostos mediadores de adesão provaram afetar a morfologia, viabilidade e a exibição espontânea de atividade elétrica das células. Além disso, as células SH-SY5Y foram diferenciadas com sucesso e demonstraram efeitos agudos sobre a função neuronal após a adição de Aβ. Este efeito sobre a sinalização elétrica foi dependente da concentração dos oligómeros de Aβ. Os resultados aqui apresentados permitem concluir que a linha celular SH-SY5Y pode ser diferenciada com sucesso em MEAs devidamente tratados e pode ser usada para avaliar os efeitos agudos do Aβ sobre a sinalização neuronal.
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Holmes, Sophie. "The effect of glucagon-like peptide-1 (GLP-1) receptor agonists on cell viability, ADAM10 maturation and the proteolysis of ADAM10 substrates in SH-SY5Y cells." Thesis, Lancaster University, 2018. http://eprints.lancs.ac.uk/129821/.
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Arguably the causative agent of Alzheimer's disease (AD), amyloid beta (Aβ)- peptides, are generated by sequential proteolysis of the amyloid precursor protein (APP) by β- and γ-secretase activities. Alternatively, APP can be processed nonamyloidogenically by an α-secretase activity that cleaves within the Aβ-domain, precluding the formation of intact Aβ-peptides and liberating a neuroprotective fragment, sAPPα. This latter proteolytic event is catalysed by a disintegrin and metalloproteinase (ADAM) 10. Recent research has suggested that glucagon-like peptide-1 (GLP-1) analogues; Lixisenatide, Dulaglutide, Liraglutide, and Exenatide (Exendin-4), currently used to treat diabetes, may also be beneficial in the treatment of neurodegenerative conditions such as AD, as they have been shown to exert neuroprotective properties. Research has also suggested that one of these compounds, exendin-4, can enhance the amount of membrane-associated ADAM10. The current study, therefore, aims to determine firstly; whether GLP-1 analogues can protect neuroblastoma, SH-SY5Y cells, against chemical stressors of relevance to AD and, secondly, whether these compounds alter ADAM10 maturation/activity and the proteolysis of the enzyme's substrates, APP, prion protein (PrP) and neuroligin1 (NLGN-1). Cells treated with hydrogen peroxide (as an oxidative stressor), cobalt chloride (as a hypoxic mimic), methylglyoxal (MG) or thapsigargin (TG) (as Endoplasmic Reticulum stressors) were co-treated with or without GLP-1 analogues and cell viability was subsequently monitored along with the expression and proteolysis of ADAM10 and its substrates. The results of the study showed that the GLP-1 analogue, liraglutide, had no major effect in terms of protecting SH-SY5Y cells against any of the afore-mentioned chemical stressors. However, a second compound, exendin-4, was protective against TG-induced cytotoxicity. Thapsigargin impaired the proteolytic maturation of ADAM10 suggesting a decrease in the activity of the enzyme but exendin-4 was unable to reverse this effect. Nonetheless, exendin-4 was able to partly reverse the inhibitory effect of TG on the expression of endogenous APP, and shedding of over-expressed NLGN1, and sAPPα in cells over-expressing APP695. Interestingly, TG caused an increase in expression of APP695, and an intracellular accumulation of PrP and, subsequently, did not alter the cell surface ADAM-mediated shedding of this protein. Collectively, these data indicate that liraglutide was ineffective in the protection of SH-SY5Y cells against the chemical stressors employed. However, exendin-4, is mildly protective against thapsigargin-mediated oxidative stress and is able to partly restore the TG-induced decrease in expression of endogenous APP, and shedding of APP695 and NLGN1. Unusually though, this latter effect was not associated with restored proteolytic maturation of ADAM10.
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Santiago, Fernando Enrique. "BAG2 is repressed by NF- kB signaling, and its overexpression is sufficient to shift AB1-42 from neurotrophic to neurotoxic in undifferentiated SH-SY5Y neuroblastoma." reponame:Repositório Institucional da UFABC, 2015.
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Orientador: Prof. Dr. Daniel Carneiro CarrettieroTese (doutorado) - Universidade Federal do ABC, Programa de Pós-Graduação em Neurociência e Cognição, 2015.
A doenca de Alzheimer (AD) e a mais diagnosticada de todas as demencias e afeta aproximadamente 12,5% das pessoas com idade superior a 65 anos. A doenca piora progressivamente, causando um alarmante declinio na funcao cognitiva e eventualmente a morte. Duas caracteristicas histopatologicas da doenca sao presenca das placas do peptideo beta-amiloide (AB) e emaranhados neurofibrilares da proteina tau hyperphosphorilada. A hipotese amiloide da AD propoe que o beta-amiloide (AB) induz a fosforilacao da tau, resultando na formacao de agregados toxicos, perda da funcao neuronal e morte neuronal. Curiosamente, em neuronios nao-diferenciados AB e neuroprotetora em vez de neurotoxica. Em neuronios diferenciados, a neurotoxicidade de AB e dependente da expressao da proteina tau. A co-chaperona BAG2 foi identificada como capaz de estimular a degradacao da proteina tau fosforilada, sugerindo que pode reverter a eventual agregacao de tau hiperfosforilada citotoxica. A relevancia da BAG2 nos processos neurotoxicos induzidos pela AB, dentro de um contexto de maturac.o neuronal, nao foi anteriormente pesquisada e representa uma interessante hipotese no quanto possa ajudar a elucidar os mecanismos celular-moleculares responsaveis pela patologia da doenca de Alzheimer. O objetivo deste estudo foi caracterizar a funcao e regulacao de BAG2 no contexto da morte celular induzida por AB em neuronios nao-diferenciados e diferenciados. A expressao de BAG2 em celulas SH-SY5Y foi analisada sob condicoes de nao-diferenciacao e diferenciacao. O BAG2 foi superexpressada em celulas SH-SY5Y nao-diferenciadas, que foram depois tratadas 15 com o peptido AB. A morte celular foi avaliada por exclusao de azul de tripano. A regiao do promotor de BAG2 foi analisada para avaliar a frequencia e distribuicao de sitios-alvo de varios fatores de transcricao para identificar fatores que regulam a expressao do BAG2. Os niveis de expressao de BAG2 foram determinados utilizando RT-PCR. Nos mostramos que a expressao de BAG2 aumenta com a diferenciacao das celulas SH-SY5Y. AB e neurotrofico para as celulas SH-SY5Y nao-diferenciadas, porem e neurotoxica de celulas diferenciadas. A superexpressao da co-chaperona BAG2 foi suficiente para causar uma mudanca na sinalizacao de AB de neurotrofico para neurotoxico em celulas nao-diferenciadas. A superexpressao de BAG2, na ausencia de AB, nao teve efeito na morte celular ou no grau de diferenciacao em celulas SH-SY5Y nao-diferenciadas. Em seguida analisamos a regiao do promotor de BAG2 e descobrimos uma prevalencia de sitios do NF .kB, os quais co-localizavam com o sitio de iniciacao da transcricao do BAG2. O tratamento das celulas, tanto com ativadores e inibidor do NF-kB, revelou que o BAG2 e reprimido pela atividade do NF- kB. Em conjunto, estes dados sugerem que a ausencia de BAG2 em celulas nao-diferenciadas e suficiente para lhes conferir resistencia a morte induzida por AB. Assim, sugerimos que a inducao da expressao de BAG2 atraves da interrupcao ou alteracao de sinalizacao de NF-kB pode criar um ambiente celular em neuronios maduros que e sensivel, em vez de resistente, a morte celular induzida por AB.
Alzheimer¿s disease (AD) remains the most-diagnosed of all dementias, affecting approximately 12.5% of persons over 65 years of age. The disease progressively worsens causing an alarming decline in cognitive function, often culminating in death. Two histopathological hallmarks of Alzheimer¿s disease are beta-amyloid (AB) peptide plaques and neurofibrillary tangles of hyperphosphorylated tau protein. According to the amyloid hypothesis of AD, AB induces tau phosphorylation which results in the formation of toxic protein aggregates, loss of neuron function, and neuronal death. Interestingly, AB has been shown to be neurotrophic rather than neurotoxic to neural precursors in animal and cell culture models, and the neurotoxicity of AB in differentiated neurons has been found to be dependent upon tau protein expression. The co-chaperone BAG2 has recently been shown to stimulate the degradation of accumulated phosphorylated tau protein, suggesting that it may abrogate aggregation of tau into cytotoxic neurofibrillary tangles. The relevance of BAG2 to AB-induced neurotoxicity within the context of neuron maturation is not presently understood, and may help to further elucidate the cell-molecular mechanisms responsible for Alzheimer¿s disease pathology. The aim of this study was to characterize the function and regulation of BAG2 within AB-induced cell death in undifferentiated and differentiated neurons. BAG2 expression in SH-SY5Y cells was analyzed under undifferentiated and differentiated conditions. BAG2 was overexpressed in undifferentiated SH-SY5Y cells, which were then treated with AB peptide. 13 Cell death was evaluated by Trypan blue exclusion. The BAG2 promoter region was analyzed for the frequency and distribution of various transcription factor regulatory motifs to identify BAG2-regulating factors. BAG2 expression levels were determined using RT-PCR. We show that BAG2 expression increases on differentiation of SH-SY5Y cells. AB is neurotrophic to undifferentiated SH-SY5Y cells, while it is neurotoxic to differentiated cells. Overexpression of the co-chaperone BAG2 was sufficient to cause a switch in AB signaling from neurotrophic to neurotoxic in undifferentiated cells. BAG2 overexpression, in the absence of AB, had no effect on cell death or degree of differentiation in undifferentiated SH-SY5Y cells. We next analyzed to putative BAG2 promoter region and discovered a preponderance of NF-kB binding motifs that co-localized to the BAG2 transcription start site. Treatment of cells with NF-kB activators and inhibitor reagents revealed that BAG2 is repressed by NF-kB signaling. Together, these data suggest that the absence of BAG2 in undifferentiated cells is sufficient to render them refractive to AB-induced death. We thus suggest that the expression of BAG2 expression via the disruption or modification of NF-kB signaling may create a cellular environment in mature neurons that is sensitive rather than resistant to AB-induced cell death.
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Lopes, Fernanda Martins. "Caracterização da diferenciação neural induzida por ácido retinóico da linhagem de neuroblastoma humano SH-SY5Y e seu uso como ferramenta para pesquisa em neurociências." reponame:Biblioteca Digital de Teses e Dissertações da UFRGS, 2012. http://hdl.handle.net/10183/49007.
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Os mecanismos moleculares que levam ao dano da via nigroestriatal durante a progressão da Doença de Parkinson (DP) ainda não estão totalmente elucidados. Dessa forma, existe a necessidade de desenvolver modelos experimentais adequados para o estudo desse distúrbio neurodegenerativo. A linhagem de neuroblastoma humano SH-SY5Y tratada com neurotoxinas indutoras deste distúrbio (ex.: 6-hidroxidopamina - 6-OHDA) é amplamente utilizada como modelo in vitro da DP. Muitos estudos mostram que esta linhagem pode ser diferenciada em células dopaminérgicas através da combinação da diminuição do soro fetal bovino (SFB) em meio de cultura e da adição de neurotrofinas como o ácido retinóico (AR). No entanto, há poucos estudos mostrando as diferenças entre células proliferativas e diferenciadas da linhagem de neuroblastoma SH-SY5Y, além do efeito do tratamento com 6-OHDA. Ainda, não há um consenso nos protocolos de diferenciação. Dessa forma, o objetivo deste estudo foi estabelecer um protocolo de diferenciação dopaminérgica da linhagem de neuroblastoma humano SH-SY5Y, bem como avaliar a potencialidade do modelo como plataforma para o screening de neurotoxicidade/neuroproteção de compostos e a possibilidade de manipulação gênica. As células proliferativas SH-SY5Y foram mantidas em meio de cultura DMEM/F12 (1:1) suplementado com 10% de SFB. A diferenciação foi induzida pela combinação de 10 μM de AR e meio de cultura com 1% de SFB durante 4, 7 e 10 dias. Foram avaliados parâmetros morfológicos (presença de neuritos) e neuroquímicos, através marcadores de diferenciação neuronal (DAT- transportador de dopamina; TH – tirosina hidroxilase; ENS – enolase neurônio específica; NeuN – proteína nuclear de neurônio; Nestina). Ainda, avaliamos parâmetros de estresse oxidativo através da atividade de enzimas antioxidantes e dos níveis de tióis reduzidos. Nossos dados mostraram que as células SH-SY5Y diferenciadas por 7 dias apresentaram mudanças morfológicas e o aumento do imunoconteúdo de todos os marcadores neuronais testados, e a concomitantemente diminuição do imunoconteúdo de nestina (marcador de células indiferenciadas). Além disso, o fenótipo neuronal apresentou uma maior atividade de alguns sistemas antioxidantes. Também foi avaliada a citotoxicidade frente ao H2O2 e à 6-OHDA nos dois fenótipos. As células diferenciadas se mostraram mais resistentes ao dano causado pelo H2O2 e mais sensíveis à 6-OHDA. Dessa forma, a citotoxicidade da 6-OHDA pode estar relacionada com o aumento do imunoconteúdo do DAT, visto que a neurotoxina entra na célula dopaminérgica através deste transportador. Interessantemente, as células diferenciadas apresentaram aumento dos níveis da proteína neuroprotetora DJ-1, que está relacionada a uma forma prematura de Parkinsonismo em humanos. Após a caracterização do modelo, nós utilizamos o fenótipo diferenciado como plataforma experimental para o screening de compostos neuroprotetores como os organocalcogênios. Nós determinamos a citotoxidade destes compostos em células diferenciadas da linhagem de neuroblastoma SH-SY5Y. A partir destes dados, foram selecionados compostos com baixa citotoxicidade e avaliamos a morfologia celular (densidade de neuritos). Nós verificamos que antes da perda de viabilidade, ocorre a perda de neuritos, sendo que este parâmetro é outra vantagem do modelo de célula diferenciada para avaliação da neurototoxicidade. Ainda, verificamos que estes compostos são capazes de prevenir o dano celular causado pela 6-OHDA. Além disso, nós caracterizamos a capacidade do modelo de ser manipulado geneticamente através da transfecção e superexpressão de plasmídeo contendo a proteína verde fluorescente, onde verificamos que a expressão é mantida durante a diferenciação. Dessa forma, nossos dados mostraram a eficácia da padronização da diferenciação induzida por AR da linhagem de neuroblastoma humano SH-SY5Y, pois estas células apresentam características morfológicas e neuroquímicas adequadas de neurônio dopaminérgico bem como pode ser aplicado não só para avaliação de neurototoxicidade/neuroproteção, mas também pode ser manipulado geneticamente.The molecular mechanisms underlying the massive cellular loss found in the nigrostriatal pathway during the progression of Parkinson’s disease (PD) are not completely understood. Therefore, it is important to develop more suitable experimental models to study the molecular mechanisms of this neurodegenerative disorder. Proliferative human neuroblastoma cell line SH-SY5Y challenged with neurotoxins (e.g.: 6-hydroxydopamine – 6-OHDA) has been widely used as an in vitro model for PD. Many lines of evidence showed that this cell line differentiates with the combination of lower fetal bovine serum (FBS) and retinoic acid (RA) to dopaminergic-like neural cell. However, there are few studies addressing the differences between proliferative and RA-differentiated SH-SY5Y cells as well as their responses to 6-OHDA cytotoxicity. Moreover, there is no consensus in differentiation protocols. Hence, the objective of this study was to establish a RAinduced dopaminergic differentiation protocol and also evaluate its capabilities for drug screening of neurotoxicity/neuroprotection and genetic manipulation. Exponentially growing SH-SY5Y cells were maintained with DMEM/F12 (1:1) medium plus 10% FBS. Differentiation was triggered by the combination of 10 μM of RA plus medium with 1% of FBS during 4, 7 and 10 days. We evaluated the cell morphology (neurites) and the neuronal markers (Dopamine Transporter- DAT, Tyrosine Hydroxylase-TH, Neuron-Specific Enolase-NSE, Neuronal Nuclei Protein- NeuN, and Nestin immunocontent). Furthermore, we verify the activity of antioxidant enzymes and the reduced thiol levels. Our data demonstrated that SH-SY5Y cells differentiated for 7 days expresses all neuronal markers tested with concomitant decrease in nondifferentiated marker (nestin). Besides, they showed a higher activity of some antioxidant systems. We also evaluated the cytotoxicity of H2O2 and 6-OHDA in both phenotypes. Differentiated cells are more resistant to H2O2 and more sensitive to 6- OHDA. Hence, the damage caused by 6-OHDA could be related with the increase of DAT immuncontent, because this neurotoxin enters into the dopaminergic cell through this transporter. Interestingly, the differentiated cells have more levels of neuroprotective DJ-1 protein, which is related with a juvenile Parkinsonism. After establish the conditions of differentiation, we used the neuronal phenotype to perform a drug screening with organoselenide compounds. We verify the cytotoxicity of these compounds in differentiated cells. From these data, we selected compounds with low toxicity and evaluated the cell morphology (neurites density). We verify that before the loss of viability, there is a loss of neurites. This parameter is another advantage of the differentiated cells model to neurotoxicity evaluation. Moreover, these compounds were able to prevent neuronal cell death caused by 6-OHDA. We also characterized the ability of the model to be manipulated genetically through transfection and overexpression of a green fluorescent protein (GFP) plasmid. We verify that the expression of GFP is maintained during the differentiation. Hence, our data showed the efficacy of the RA-induced differentiation protocol of the neuroblastoma cell line SH-SY5Y, because these cells have morphological and neurochemical characteristics of dopaminergic neurons. Furthermore, the neuronal phenotype can be applyed not only to evaluate neurocytotoxicity/neuroprotection but also can be manipulated genetically.
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Jesus, João André Fernandes de. "Aß effects in protein phosphatase 1 complexes." Master's thesis, Universidade de Aveiro, 2017. http://hdl.handle.net/10773/21535.
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Mestrado em Biomedicina MolecularThe brain is a complex structure, which is comprised by neurons capable of communication through synapses. These usually occur between an axon terminal and a dendritic spine of different neurons. The dendritic spines are dynamic structures that allow for a rapid adaptation to different stimuli. PP1 is a phosphatase protein that catalyses the majority of dephosphorylation reactions in the human body. It has different functions that vary, from glycogen metabolism to synaptic regulation. Neurabins (1 and 2) are two PP1 regulator subunits that are structurally and functionally similar to each other. They are highly enriched in the dendritic spines, interacting with several proteins, including PP1, and targeting them to receptors, cytoskeleton or other cellular compartments. Thus, regulating neuronal morphology and synaptic transmission and plasticity. Phactr3 belongs to the actin regulatory protein family, which comprises four members. Phactr3 is involved in cell migration and regulates cytoskeleton dynamics. It interacts with PP1 forming a complex that is controlled by changes in cytoplasmic G-actin concentration, thus, regulating actin cytoskeleton dynamics. Neurodegenerative diseases are usually characterised by the loss of synapses, neural death, gradual loss of cognitive functions and memory. It is believed that Aβ is the major culprit in these changes. Aβ results from an abnormal cleavage of APP, via the amyloidogenic pathway, resulting in the overproduction of toxic peptides. The main aim of this thesis was to evaluate the effects of Aβ on Neurabins and Phactr3 expression, and in PP1 complexes. The results show a slight decrease in neurabins’ expression, and a slight increase in Phactr3 expression. The results also show a decrease in interaction between PP1 and Neurabin-1, possibly due to direct effect of Aβ on Neurabin-1 or the imbalance of phosphatases and kinases, and between PP1 and Phactr3, possibly due to variations of G-actin levels which competes with PP1 to binding with Phactr3. The same changes were not observed in Neurabin-2/PP1 complexes. A possible explanation is that both are differently regulated by several kinases. The results allow us to conclude that Aβ interferes in the expression of all three proteins and in the interaction of Neurabin-1/PP1. However, additional studies are required in order to better understand the physiological relevance of these complexes.
O cérebro é uma estrutura complexa, que é composta por neurónios capazes de comunicar através de sinapses. Estas normalmente ocorrem entre um terminal axónico e espinha dendritica de neurónios diferentes. As espinhas dendriticas são estruturas dinâmicas que permitem uma rápida adaptação a diferentes estímulos. A PP1 é uma proteína fosfatase que catalisa a maioria das desfosforilações que ocorrem no corpo humano. Tem diversas funções, que variam desde metabolismo de glicogénio a regulação sináptica. As neurorabinas (1 e 2) são duas subunidades reguladores da PP1 que são estruturalmente e funcionalmente idênticas entre si. São muito enriquecidas nas espinhas dendriticas e interagem com diversas proteínas, incluindo a PP1, e guiam as proteínas para receptores, citoesqueleto ou outros compartimentos celulares, regulando assim a morfologia neuronal e transmissão e plasticidade sináptica. A Phactr3 pertence à família de proteínas reguladoras de actina, á qual pertencem quatro membros. A Phactr3 está envolvida na migração celular e regula a dinâmica do citoesqueleto. Interage com a PP1, formando um complexo que é controlado por alterações na concentração de G-actina citoplasmática, regulando assim a dinâmica do citoesqueleto. Doenças neurodegenerativas, são normalmente caracterizadas pela perda de sinapses, morte neuronal, e perda gradual de funções cognitivas e memória. Acredita-se que o principal fator responsável por essas anomalias seja o péptido Aβ. Este resulta de uma clivagem anormal de APP, pela via amiloidogénica, resultando numa sobreprodução do péptido tóxico. O objectivo desta tese era avaliar os efeitos de Aβ nos níveis de expressão das neurorabinas e Phactr3, assim como os complexos formados com a PP1. Os resultados mostram uma ligeira diminuição nos níveis de expressão das neurorabinas, e num ligeiro aumento na expressão de Phactr3. Os resultados também mostram uma diminuição na interação do complexo Neurorabina-1/PP1, provavelmente devido a um efeito direto de Aβ sobre Neurorabina-1 ou um desequilíbrio nas fosfatases e cinases, e Phactr3/PP1, provavelmente devido a variação nos níveis de G-actina, que compete com a PP1 para interagir com Phactr3. As mesmas alterações não foram verificadas no complexo Neurorabina-2/PP1. Uma explicação possível é que ambas as proteínas são reguladas de forma diferente por diversas cinases. Estes resultados permitem concluir que Aβ interfere na expressão das três proteínas e nos níveis de interação de Neurabina-1/PP1 e Phactr3/PP1. No entanto são necessários mais estudos para obter uma melhor compreensão da relevância fisiológica destes complexos.
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Gawinecka, Joanna Verfasser], Mathias [Akademischer Betreuer] [Bähr, Inga [Akademischer Betreuer] Zerr, Thomas [Akademischer Betreuer] Bayer, and Mikael [Akademischer Betreuer] Simons. "Prion protein-induced proteome alterations in sporadic Creutzfeldt-Jakob disease and in SH-SY5Y cell culture model / Joanna Gawinecka. Gutachter: Inga Zerr ; Thomas Bayer ; Mikael Simons. Betreuer: Mathias Bähr." Göttingen : Niedersächsische Staats- und Universitätsbibliothek Göttingen, 2011. http://d-nb.info/1042639671/34.
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Popova, Dina. "In vitro cellular models for neurotoxicity studies : neurons derived from P19 cells." Doctoral thesis, Umeå universitet, Institutionen för farmakologi och klinisk neurovetenskap, 2017. http://urn.kb.se/resolve?urn=urn:nbn:se:umu:diva-133030.
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Humans are exposed to a variety of chemicals including environmental pollutants, cosmetics, food preservatives and drugs. Some of these substances might be harmful to the human body. Traditional toxicological and behavioural investigations performed in animal models are not suitable for the screening of a large number of compounds for potential toxic effects. There is a need for simple and robust in vitro cellular models that allow high-throughput toxicity testing of chemicals, as well as investigation of specific mechanisms of cytotoxicity. The overall aim of the thesis has been to evaluate neuronally differentiated mouse embryonal carcinoma P19 cells (P19 neurons) as a model for such testing. The model has been compared to other cellular models used for neurotoxicity assessment: retinoic acid-differentiated human neuroblastoma SH-SY5Y cells and nerve growth factor-treated rat pheochromocytoma PC12 cells. The chemicals assessed in the studies included the neurotoxicants methylmercury, okadaic acid and acrylamide, the drug of abuse MDMA (“ecstasy”) and a group of piperazine derivatives known as “party pills”. Effects of the chemicals on cell survival, neurite outgrowth and mitochondrial function have been assessed. In Paper I, we describe a fluorescence-based microplate method to detect chemical-induced effects on neurite outgrowth in P19 neurons immunostained against the neuron-specific cytoskeletal protein βIII-tubulin. In Paper II, we show that P19 neurons are more sensitive than differentiated SH-SY5Y and PC12 cells for detection of cytotoxic effects of methylmercury, okadaic acid and acrylamide. Additionally, in P19 neurons and differentiated SH-SY5Y cells, we could demonstrate that toxicity of methylmercury was attenuated by the antioxidant glutathione. In Paper III, we show a time- and temperature-dependent toxicity produced by MDMA in P19 neurons. The mechanisms of MDMA toxicity did not involve inhibition of the serotonin re-uptake transporter or monoamine oxidase, stimulation of 5-HT2A receptors, oxidative stress or loss of mitochondrial membrane potential. In Paper IV, the piperazine derivatives are evaluated for cytotoxicity in P19 neurons and differentiated SH-SY5Y cells. The most toxic compound in both cell models was TFMPP. In P19 neurons, the mechanism of action of TFMPP included loss of mitochondrial membrane potential. In conclusion, P19 neurons are a robust cellular model that may be useful in conjunction with other models for the assessment of chemical-induced neurotoxicity.
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Jordão, Nuno Ricardo de Oliveira. "Study of the cell surface proteome for the analysis of Parkinson’s disease associated DJ-1 mutations." Master's thesis, Universidade de Aveiro, 2015. http://hdl.handle.net/10773/15483.
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Mestrado em Biotecnologia - Biotecnologia MolecularParkinson’s disease (PD) is a neurodegenerative disease, characterized with selective neurodegeneration and dopamine depletion. Despite most cases appear to have sporadic origin, it has been associated various monogenic mutations to the onset of a parkinsonian phenotype. DJ-1 protein is of particular interest given its neuroprotective role against oxidative stress and mitochondria impairment, and the identification of several mutations correlated with early onset PD. For this study, it were then produced two pathological mutations of DJ-1, M26I and E163K. SDS-PAGE and LC-MS/MS analysis confirmed the adequate production and purification of the both mutant proteins, and SEC-HPLC secured the structural perseverance of the mutations as homodimers, a key feature of DJ-1 essential for its biological activity. On the other hand, SH-SY5Y viability assays indicated that despite the native form protective role against oxidative stress, M26I and E163K mutations showed a compromised neuroprotective capacity. To better understand the reasons for this biological impairment, it was developed a protocol for cell surface proteins labelling with Sulfo-NHSLC-biotin and avidin pull-down for enrichment and downstream MS analysis. Assays such as western blotting, LC-MS/MS and confocal microscopy confirmed the adequacy of the proposed procedure. When applied for the analysis of proteome variations related to oxidative stress, in enriched fractions from SH-SY5Y biotinylation and avidin pull-down of crude membrane sub cellular part, it allowed the identification of several proteins of interest, namely four proteins with significant difference caused by oxidative stress induction, and of other proteins of interest. It was also performed the direct pull-down of whole protein extract, offering inconclusive results regarding the preferential use of ultracentrifugation before pull-down. Nevertheless, it was the first time that SH-SY5Y cell surface was analysed in a PD context, and it could be used in the future to study cell surface proteome alterations modulated by oxidative stress and extracellular presence of native or mutant DJ-1, providing new insights regarding its intake and signalling modulation in pathological conditions, and hence contributing for a new perspective over preventive or eliciting mechanisms associated to the onset of Parkinson’s disease.
A doença de Parkinson é uma doença neurodegenerativa caracterizada por uma neurodegeneração selectiva e depleção de dopamina, e apesar de grande parte dos casos terem origem esporádica diversas mutações monogénicas têm sido associadas ao desenvolvimento de um fenótipo parkinsoniano. A proteína DJ-1 é de particular interesse, dado o seu papel neuroprotector contra stress oxidativo e disfunção mitocondrial, e a identificação de mutações correlacionadas a doença de Parkinson precoce. Neste estudo, foram produzidas duas mutações patológicas da proteína DJ-1, M26I e E163K. Uma análise SDS-PAGE e LC-MS/MS comprovou uma produção e purificação adequada das mutações, e SEC-HPLC assegurou a preservação estrutural das mutações de DJ-1 como homodímeros, uma característica chave de DJ-1 fundamental para a sua actividade biológica. Por outro lado, estudos de viabilidade de SH-SY5Y indicaram que, apesar do papel protector da forma nativa contra stress oxidativo, as mutações M26I e E163K demonstraram uma reduzida capacidade neuroprotectiva. Para melhor compreender os motivos desta disfunção biológica, foi desenvolvido um protocolo para marcação das proteínas de superfície celular com Sulfo-NHS-LC-biotina e pull-down com avidina para enriquecimento e subsequente análise MS. Vários ensaios como western blotting, LC-MS/MS e microscopia confocal confirmaram a adequação do protocolo sugerido. Quando aplicado para uma análise de variações proteómicas relacionadas com stress oxidativo, em fracções enriquecidas provenientes da biotinilação de SH-SY5Y e pull-down da parte membranar do extracto celular, permitiu a identificação de várias proteínas de interesse, nomeadamente quatro proteínas com diferença significativa resultante da indução de stress oxidativo. Também foi realizado um pull-down com a totalidade extracto celular, que resultou em dados não conclusivos relativamente ao uso de ultracentrifugação antes do pull-down. Não obstante, este estudo correspondeu à primeira análise da superfície celular de SH-SY5Y realizada num contexto da doença de Parkinson, que poderá ser usada no futuro para estudar alterações no proteoma de superfície celular em ambiente de stress oxidativo e de adição da proteína DJ-1, na forma nativa e mutante, de forma a fornecer novas pistas referentes ao seu intake e modulação da sinalização em ambiente oxidativo, e em suma contribuindo para uma nova perspectica sobre os mecanismos protectores ou despoletadores da doença de Parkinson.
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Olsson, Anna-Karin. "Ras-MAPK signaling in differentiating SH-SY5Y human neuroblastoma cells." Doctoral thesis, Uppsala University, Department of Genetics and Pathology, 2000. http://urn.kb.se/resolve?urn=urn:nbn:se:uu:diva-1251.
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Neuroblastoma is a malignant childhood cancer, originating from sympathetic neuroblasts of the peripheral nervous system. Neuroblastoma is a heterogenous group of tumours, while some are highly malignant others can spontaneosly mature into a more benign form or regress. Less than half of the patients survive and this statistics has improved only modestly over the past 20 years.
SH-SY5Y is a human neuroblastoma cell line established from a highly malignant tumour. The cells have retained a capacity to differentiate in vitro in response to low concentrations of the phorbolester 12-O-tetradecanoylphorbol-13-acetate (TPA) in the presence of serum or defined growth factors. Differentiated cells are characterised by neurite formation and upregulation of neuronal marker genes. SH-SY5Y are unresponsive to nerve growth factor (NGF), but when transfected to express the NGF-receptor TrkA, they differentiate in response to NGF. Protein kinase C (PKC) is pivotal for the differentiation response to take place.
We have investigated the role of signaling through the Ras-MAPK pathway in differentiating SH-SY5Y, with respect to neurite formation, expression of neuronal marker genes and growth control. Our results show that differentiation-promoting treatment induced a sustained activation and nuclear accumulation of the MAPK ERK in SH-SY5Y. The nuclear accumulation of ERK was PKC-dependent. However, nuclear accumulation of ERK was not sufficient for a differentiation response to take place in these cells, but ERK activity was needed for the characteristic upregulation of NPY and GAP-43 induced by TPA. ERK activity did not induce neurite formation, neither was it necessary for TPA-induced neurite formation. Instead, stimulation of a pathway distinct from MEK/ERK, but downstream of Ras, was needed for morphological differentiation. We could also show that differentiated cells still entered S-phase and that there was no correlation between expression of the CKI p21cip1 (an ERK target), BrdU-incorporation or neurite formation.
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Al-Jarari, Nouh Mohamed Hamed. "Regulation of adenosine receptors in human SH-SY5Y neuroblastoma cells." Thesis, University of Nottingham, 2006. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.438430.
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Riddoch, Fiona C. "Ca²⺠signaling in human neuroblastoma SH-SY5Y cells." Thesis, University of Newcastle Upon Tyne, 2005. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.416990.
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Yamakawa, Kentaro. "Dopamine facilitates α-synuclein oligomerization in human neuroblastoma SH-SY5Y cells." Kyoto University, 2010. http://hdl.handle.net/2433/120568.
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Hernandez-Martinez, Juan-Manuel. "Role of kynurenines and oxidative stress in the differentiation of SH-SY5Y cells." Thesis, University of Glasgow, 2015. http://theses.gla.ac.uk/6133/.
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Neuroblastoma is the most common solid extracranial tumour in children. The neuroblastoma SH-SY5Y cell line is a third successive subclone established from a metastatic bone tumour biopsy. It can be induced to differentiate (regress) into a neuronal phenotype when treated with any of several molecules including retinoic acid (RA). This characteristic has been exploited in several studies that use the SH-SY5Y cell line as a neuronal model. These studies have had far- reaching implications in shaping our understanding of certain key aspects of neurotoxicity and neurodevelopment yet their genuine relevance becomes evident when approached from an oncological point of view, as they provide information about the process underlying tumour regression which in turn can lead to the development of better therapies for the clinical management of this malignancy. It has been shown both in vitro and in vivo that several tumours constitutively catabolize the essential amino acid tryptophan (Trp) promoting cancer- associated inflammation, immune response suppression, immune escape and tumour outgrowth. The main degradation pathway of Trp is the kynurenine pathway: it involves its transformation into several bioactive compounds such as kynurenic acid (KA) and quinolinic acid (QA). QA has been implicated in several neurodegenerative diseases where it is believed to induce excitotoxic neuronal death through the activation of the N-methyl-D-aspartate (NMDA) receptor, a type of ionotropic glutamate receptor, as well as by causing oxidative stress and energy metabolism disruption. Conversely, KA acts as an NMDA receptor antagonist and exerts neuroprotection. Similarly, glutamate signaling and its dysregulation has been implicated in the development and progression of cancer. Furthermore, several glutamate receptor antagonists, including kynurenic acid, have been shown to inhibit the proliferation and migration of neoplastic cells. Conversely, it has recently been reported that QA increases the proliferation of SK-N-SH cells and protects gliomas against oxidative stress by acting as a precursor of NAD+. In view of all that has been mentioned thus far, the SH-SY5Y cell line was used as a model to investigate the effect of certain Trp metabolites such as QA, KAand 3-hydroxyanthranilic acid (3-HAA) on cellular morphology, viability and neurite extension. An important part of this study was to determine whether the available methods could reliably be employed to investigate these parameters in the SH-SY5Y cell line. It was confirmed that the acquired SH-SY5Y cell line retains its ability to differentiate and to die, and that both processes can be accurately quantified. Additionally, the optimal culturing conditions for the SH- SY5Y cell line were determined. Treatment with RA (10 μM) was used as a positive control of differentiated SH- SY5Y cells. Overall, the morphology adopted by cells after QA (50 μM) treatment was similar to the one that follows RA-induced differentiation. It was demonstrated that QA caused an increase in the neurite/soma ratio in SH-SY5Y cells, which was confirmed by Western blot analysis as evidenced by an increase in the total cellular content of β3-tubulin. These results were also confirmed by a neurite outgrowth assay that selectively quantified the neuritic mass present in cultures. However, unlike RA, QA did not decrease the levels of the neuronal proliferation marker doublecortin; the term neuritogenesis is therefore more appropriately used to refer to the series of morphological and molecular changes induced by QA in SH-SY5Y cells. The morphological changes induced by QA were not reproduced by application of NMDA, nor were they inhibited by blockade of the NMDA receptor with MK-801. Furthermore, SH-SY5Y cells were not susceptible to NMDA excitotoxic death. In view of this, the expression of GluN1 protein was determined by Western blot. GluN1 could not be detected in either undifferentiated or differentiated SH-SY5Y cells, confirming that QA-induced neuritogenesis occurs through a mechanism independent of NMDAR activation. The results herein contained suggest that the SH-SY5Y cell line does not have functional NMDARs, nonetheless it is recognized that a more exhaustive study would be necessary to fully establish which glutamate receptor subtypes are found in the SH-SY5Y cell line. The effect of QA on the production of reactive oxygen species (ROS) was also investigated. QA caused an increase in the intracellular levels of ROS as evidenced by an increase in the fluorescence of oxidised ethidium. Additionally QA-treatment caused an increase in the expression of NRF2, a transcriptionfactor that responds to oxidative stress and which has been implicated in ROS- induced differentiation in SH-SY5Y cells. In contrast, superoxide dismutase (SOD; 300 U/ml) significantly reduced the levels of ROS induced by QA treatment, which in turn caused an increase in cell proliferation and a reduction in the number of neurites. Similarly, diphenylene iodonium (DPI; an inhibitor of NADPH oxidase) also inhibited QA-induced neuritogenesis. These results suggest that the action mechanism of QA is mainly via the production of ROS, most likely superoxide (O2•-) through NADPH-oxidase. Interestingly, nicotinamide (1 nM-1mM; another precursor of NAD+) caused a dose dependent increase in the number of neurites and in the expression of β3- tubulin, which suggests that the action mechanism of QA may be mediated by metabolites of the nicotinate and Nam pathway, including NAD+ either before or after the induction of ROS. Cells were treated with 3-hydroxyanthranilic acid (3-HAA) in order to ascertain whether other pro-oxidant molecules could induce neuritogenesis as well. Single and repeated application of 3-HAA (100 μM) induced cell death in SH-SY5Y cells. Furthermore, when 3-HAA was delivered in combination with SOD, there was a shift in the IC50 values indicating that toxicity was potentiated by SOD. Catalase (CAT; 100 U/ml) afforded complete protection from the exacerbated damage induced by the single co-application of 3-HAA + SOD. However, when repeated treatments were performed, CAT no longer afforded any protection. Interestingly, the serum concentration in the medium did not affect the IC50 of 3-HAA but it did modulate the response to CAT, indicating that the specific ROS produced after 3-HAA treatment depend on the medium in which 3-HAA is delivered. At sublethal doses, 3-HAA interfered with the expression of NeuN (neuronal marker) through a mechanism that involves high production of ROS. The ability of some kynurenines to induce differentiation and cell death in SH- SY5Y cells may open new and exciting avenues of research. If these results can be confirmed in vivo they could impact the way in which certain neuroblastomas are treated.
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Graça, Júlio César Gomes. "Avaliação dos efeitos de acetato em células de neuroblastoma SH-SY5Y e células-tronco humanas de dente decíduo esfoliado cultivadas na presença de glutamato." Universidade Federal de Juiz de Fora (UFJF), 2017. https://repositorio.ufjf.br/jspui/handle/ufjf/5829.
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O glutamato, aminoácido não-essencial, é um neurotransmissor excitatório do sistema nervoso central (SNC), sendo liberado durante o impulso nervoso. Em situações de patologia cerebral, o acúmulo de glutamato ocorre no espaço extracelular, causando dano neuronal e, eventualmente, apoptose. Muitos trabalhos relataram que a citotoxicidade do glutamato está associada a várias doenças neurológicas. Neste contexto, o acetato, um ácido graxo de cadeia curta, pode beneficiar o SNC de forma energética e estrutural. A acetil-coenzima A, forma metabolicamente ativa de acetato, é utilizada como substrato em vias bioquímicas envolvidas no metabolismo de carboidratos, lipídios e proteínas, além de aumentar a acetilação das histonas, alterando a expressão de genes inflamatórios. A linhagem celular de neuroblastoma humano SH-SY5Y é comumente usada em estudos relacionados a doenças neurodegenerativas, com uma capacidade de expansão em larga escala antes da diferenciação, enquanto que a linhagem de células-tronco de polpa de dente de leite decíduo esfoliado (SHED) é comumente utilizada em modelos de estudo do comportamento celular. O objetivo desta pesquisa foi avaliar os efeitos do acetato mediante a citotoxicidade causada pelo glutamato em células SH-SY5Y e SHED. Células SH-SY5Y e SHED foram cultivadas, respectivamente, em meio DMEM/F12 suplementado com 10% (v/v) de soro fetal bovino (SFB) e 1% (v/v) de penicilina-estreptomicina, e meio alfa-MEM, suplementado com 10% (v/v) de SFB, 1% (v/v), 1% (v/v) de penicilina-estreptomicina, 0,01 mM de aminoácidos não essenciais e 2 mM L-glutamina. A viabilidade celular em diferentes concentrações de acetato (5 a 75 mM) e glutamato (25 a 150mM) foi medida pelo ensaio MTT. A diferenciação foi realizada em SH-SY5Y pela suplementação do meio com 10 μM de ácido retinóico (AR) e redução de SFB para 1% (v/v) durante 4, 7 e 10 dias em cultura, e em SHED pela substituição do meio de cultivo por DMEM Low-Glicose, suplementado com 10% (v/v) de SFB, 1% (v/v) de penicilina-estreptomicina, 10-7 M de dexametasona, 50 μM de 2-fosfato ácido ascórbico e 2 mM de β-glicerolfosfato, a fim de verificar como essas células respondem à mistura de acetato/glutamato. A análise estatística foi realizada teste ANOVA de uma ou duas vias, bem como pelo teste de Kruskal-Wallis, quando apropriado, com p < 0,05 considerado estatisticamente significativo. Após 7 dias de incubação, as concentrações de 5 e 25 mM de acetato apresentaram menor influência sobre a viabilidade de células SH-SY5Y e SHED, enquanto que o IC50% de glutamato ficou em torno de 75mM e 50mM para estas linhagens, respectivamente. Ao submeter as células ao tratamento combinado de acetato e glutamato, observou-se que o acetato não exerceu citoproteção mediante exposição celular ao glutamato. Após análise qualitativa da diferenciação osteogênica em SHEDs, foi observado maior mineralização nas células tratadas com AR e acetato, em comparação com as células controle. Estudos subsequentes, que permitam identificar como tais células respondem ao acetato em nível molecular, considerando a expressão de ciclinas, compactação da cromatina e a presença de marcadores bioquímicos característicos durante a diferenciação de cada linhagem, por exemplo, poderão fornecer um entendimento mais completo de como esse composto atua na dinâmica metabólica e bioenergética celular.
Glutamate, a non-essential amino acid, is an excitatory neurotransmitter of the central nervous system (CNS), being released during the nerve impulse. In situations of cerebral pathology, the accumulation of glutamate occurs in the extracellular space, causing neuronal damage and, eventually, apoptosis. Many studies have reported that glutamate cytotoxicity is associated with various neurological diseases. In this context, acetate, a short chain fatty acid, can benefit the CNS energetically and structurally. Acetyl-coenzyme A, a metabolically active form of acetate, is used as a substrate in biochemical pathways involved in the metabolism of carbohydrates, lipids and proteins, in addition to increasing the acetylation of histones, altering the expression of inflammatory genes. The SH-SY5Y human neuroblastoma cell line is commonly used in studies related to neurodegenerative diseases, with a large scale expansion capacity prior to differentiation, whereas the stem cell line of exfoliated deciduous teeth (SHED) is commonly used in models of cellular behavior. The objective of this research was evaluate the effects of acetate by glutamate-induced cytotoxicity on SH-SY5Y and SHED cells. SH-SY5Y and SHED cells were cultured respectively in DMEM / F12 medium supplemented with 10% (v/v) fetal bovine serum (FBS) and 1% (v/v) penicillin-streptomycin, and alpha-MEM medium, supplemented with 10% (v/v) FBS, 1% (v/v), 1% (v/v) penicillin-streptomycin, 0.01 mM non-essential amino acids and 2 mM L-glutamine. Cell viability at different concentrations of acetate (5 to 75 mM) and glutamate (25 to 150 mM) was measured by the MTT assay. Differentiation was performed on SH-SY5Y by supplementing the medium with 10 μM retinoic acid (RA) and reducing FBS to 1% (v/v) for 4, 7 and 10 days in culture, and in SHED by replacing the culture medium with DMEM Low-Glucose, supplemented with 10% (v/v) FBS, 1% (v/v) penicillin-streptomycin, 10-7 M dexamethasone, 50 μM ascorbic acid 2-phosphate and 2 mM β- Glycerol phosphate in order to verify how these cells respond to the acetate/glutamate mixture. Statistical analysis was performed one-way or two-way ANOVA, as well as Kruskal-Wallis test, when appropriate, with p < 0.05 considered statistically significant. After 7 days of incubation, concentrations of 5 and 25 mM acetate had less influence on the viability of SH-SY5Y and SHED cells, whereas the IC50% of glutamate was around 75mM and 50mM for these lines, respectively. By subjecting the cells to the combined treatment of acetate and glutamate, it was observed that acetate did not exert cytoprotection through cellular exposure to glutamate. After qualitative analysis of the osteogenic differentiation in SHEDs, greater mineralization was observed in the cells treated with RA and acetate, in comparison with the control cells. Subsequent studies to identify how these cells respond to acetate at the molecular level, considering the expression of cyclins, chromatin compaction, and the presence of characteristic biochemical markers during differentiation of each lineage, for example, may provide a more complete understanding of how this component acts on the metabolic dynamics and cellular bioenergetics.
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Balaraman, Priyadharshini. "An investigation of the mechanism of cisplatinum-induced apoptosis in SH-SY5Y neuroblastoma cells." Thesis, University College London (University of London), 2005. http://discovery.ucl.ac.uk/1444506/.
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Neuroblastomas are common paediatric tumours derived from the sympathoadrenal lineage. Neuroblastoma cells may arise from neuroblasts, which failed to differentiate or which were not eliminated by apoptosis at an appropriate stage of development. The aim of this thesis was to identify the signalling pathway by which cis-diamminedichloroplatinum (II) (CDDP) a chemotherapeutic agent, triggers caspase activation and apoptosis in the SH-SY5Y neuroblastoma cell line. An understanding of this may prove to be useful for developing better therapeutic agents for treating neuroblastoma and for understanding mechanisms of drug resistance. CDDP was found to induce apoptosis in SH-SY5Y cells via the mitochondrial death pathway, with cytochrome c release and activation of caspases-9 and -3. CDDP, a DNA damaging agent, activates p53 in SH-SY5Y cells and p53 is known to induce apoptosis via the mitochondrial pathway. Bcl-2 family members play a central role in the regulation of the mitochondrial death pathway and may have pro- or antiapoptotic activity. The pattern of expression of members of the Bcl-2 family following CDDP treatment was investigated to determine their regulatory role. PUMA (p53 upregulated modulator of apoptosis), a proapoptotic BH3-only protein and a direct transcriptional target of p53, was found to be upregulated at both the mRNA and protein levels during CDDP-induced apoptosis of SH-SY5Y cells. PUMA has three transcripts that encode PUMA- a, P and 5. PUMA-a and PUMA-(3 are proapoptotic and contain the BH3 domain. PUMA-a was identified as the transcript that increased during CDDP treatment in SH-SY5Y cells. Overexpression of PUMA-a in SH-SY5Y cells was sufficient to induce apoptosis. To identify other genes regulated by CDDP, we performed oligonucleotide microarray analysis using Affymetrix human genome-U133A microarrays and RNA extracted from SH-SY5Y cells treated with DMSO, transplatinum (an isomer of CDDP which does not induce cell death) and CDDP. The results provide a detailed picture of the changes in gene expression that occur following CDDP treatment.
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Sharma, Mohit Kumar. "Analysis of the effects of incretin-based peptides on human neuroblastoma SH-SY5Y cells." Thesis, Ulster University, 2013. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.673817.
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Incretins are growth factors that have demonstrated neuroprotective properties in a range of studies. Here, we analyse the neuroprotective properties of the Glucagon like peptide-l (GLP-l) analogues in human neuroblastoma SH-SY5Y cells against methyl glyoxal (MG) stress. The results demonstrate a range of growth factor-related cytoprotective processes induced by liraglutide (Victoza®), currently used to treat type 2 diabetes. FUl1her, we study the comparative long-term effects of three different GLP-l analogues on cell viability, proliferation and cytotoxicity and demonstrate the long-term exposure of liraglutide and lixisenatide (Lyxumia®) to be more protective when compared to the exendin-4 (Byetta~ . We also report the absence of any additive effect on cell viability and proliferation, when using GLP-l analogues in combination, but a 3-fold increase in cytoprotection observed when comparing 100 nM doses of liraglutide and lixisenatide to exendin-4 (p<0.001). In addition, we demonstrate that lixisenatide (10 and 50 nM) is neuroprotective when compared to 50 nM liraglutide (p<0.05) and 50 and 100 nM exendin-4 (p<0.001) against MG post stress. Further, the most effective doses for native Glucose dependent insulinotropic peptide (GIP), (D-Ala2)GIP and (Pro3)GIP were 200, 100 and 1 nM exhibiting a 43 ± 3% decrease in LDH levels (p<0.001). Lastly, we show a novel mechanism wherein blockade ofMEK1I2 leads to the activation of AktIPKB (p<0.001). A decrease in the survival (p<0.05) and an increase in the cytotoxicity (p<0.05) during inhibition of MEK1I2 followed by liraglutide treatment, suggest a role ofMEK1I2 in protecting the cells. Our studies show that the liraglutide pre-treatment confers neuroprotection by increasing the cell survival and decreasing cytotoxicity but inhibition of MEK1I2 withdraws this neuroprotective effect, suggesting the involvement of MAPKIERK pathway in the neuroprotection conferred by liraglutide. Overall, incretins confer protection and have potential to be developed as possible drugs to treat neurodegenerative disorders.
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Chiaino, Elda. "CYP450 expression and regulation in SH-SY5Y cells. Possible role in neurotoxin-mediated injury." Doctoral thesis, Università di Siena, 2022. http://hdl.handle.net/11365/1211396.
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The Cytochrome P450 isozymes involved in xenobiotic metabolism are ubiquitous enzymes, predominantly expressed in the liver in comparison to the extrahepatic tissues. In the brain, CYP expression is approximately 0.5-2% of that in liver microsomes, and most of the isoforms appears to be very low for playing a role in overall total body clearance. Brain basal expression and up-regulation can however significantly affect local disposition of xenobiotic or endogenous compounds. Several reports, in fact, indicate that environmental toxins may play a role in the pathogenesis of neurodegenerative disorders by directly damaging neurons or by CYPs-mediated their bioactivation into toxic compounds. Among the different isoforms, CYP2D6 is particularly involved in the metabolism of exogenous drugs, neurotoxins such as 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP, which selectively target nigrostriatal dopaminergic pathways), as well as endogenous compounds, including dopamine. Moreover, CYP2D6 extended polymorphism is significantly associated with an increased PD risk, owing to a lower capability in the metabolism of neurotoxic compounds such as pesticides. On the contrary, other studies did not support the association between PD and the poor CYP2D6-dependent metabolism, thus suggesting that PD is most likely the result of interactions between multiple genetic and environmental factors.
The aim of the present study was to increase the knowledge on the role of neuronal CYP-dependent oxidative metabolism and to clarify whether it might affect xenobiotic-promoted neurodegeneration by using an in vitro model based on human neuroblastoma-derived SH-SY5Y cells.
The first step was to promote the differentiation of neuroblastoma cells into mature human dopaminergic neurons phenotype. Two different protocols based on retinoic acid and phorbol ester or retinoic acid and brain derived neurotrophic factor were set up. The switch into dopaminergic phenotype was assessed by studying the cell morphology and the expression of the most important proteins recognized as neuronal biomarkers. Results showed that after differentiation, SH-SY5Y cells demonstrated extensive and elongated neuritic projections, a significant increased content of NeurineN, Synaptophysin, and β-tubulin III, as well as dopamine transporter.
Two well know inducers such as β-naphtoflavone (βNF) and ethanol (EtOH) were then used for promoting CYPs induction in both undifferentiated (UD) and differentiated SH-SY5Y cells. qRT-PCR analysis showed that both compounds significantly increased the mRNA expression of CYP2E1 and CYP2D6 isoforms in all SH-SY5Y cells population, while 1A1 and 3A4 were substantially unaffected.
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To explore the role of CYP in the protection toward neurotoxicity, SH-SY5Y cells possessing increased CYPs content were treated with 1-methyl-4-phenylpyridinium (MPP+), the MAO-B-dependent toxic metabolite of MPTP, or rotenone, two neurotoxins widely used to reproduce PD models in vivo and in vitro.
The treatment with EtOH and βNF protected UD cells against MPP+ toxicity, whereas only βNF partially reverted the cytotoxic insult promoted by Rotenone. Moreover, the differentiated SH-SY5Y cells resulted to be less sensitive to the cytotoxic effects caused by the neurotoxins and consequently less responsive to the protection promoted by the CYPs induction. Furthermore, both βNF and EtOH treatments partially reverted the loss in mitochondria membrane potential, complex I impairment, as well as the increased ROS formation when UD were exposed to MPP+ or rotenone.
Taken together, these results support the possible role of CYP isoforms in the neuroprotection against xenobiotic insult, especially as far as concerned the metabolic activity of CYP2D6 and CYP2E1. However, it would be interesting to investigate the induction dynamics of the CYPs here analysed and to understand the pathways triggered by βNF and EtOH leading to CYP induction. In conclusion, the impact that these isoforms have in the metabolism of other drugs, together with the present results, bring new insights on the role of brain CYPs in the effects of drugs and can drive future therapeutic approaches for ameliorating the therapy of neurological diseases.
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Góes, Lizandra Moreira. "Caracterização dos efeitos tóxicos do 1,2-dihidroxibenzeno em células do sistema nervoso central: investigação do efeito protetor de derivados de plantas." reponame:Repositório Institucional da FIOCRUZ, 2013. https://www.arca.fiocruz.br/handle/icict/7303.
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Fundação Oswaldo Cruz. Centro de Pesquisa Gonçalo Moniz. Salvador, BA, Brasil / Universidade Federal da Bahia. Faculdade de Medicina. Salvador, BA, Brasil
Catecóis são derivados do benzeno, podendo apresentar citotoxicidade, que pode constituir um modelo experimental útil para o desenvolvimento de novos fármacos. No bioma brasileiro inúmeras plantas produzem metabólitos com atividades diversas, como antioxidantes, ou inibidores do crescimento celular. No Brasil, as neoplasias são a segunda causa de óbito, especialmente aquelas derivadas do sistema nervoso, aumentando o interesse por novos antineoplásicos e agentes neuroprotetores. Este trabalho caracteriza efeitos citotóxicos do 1,2-dihidroxibenzeno (CAT) e discretamina (DSC) em células do sistema nervoso in vitro. Determinou-se a EC50 de CAT e DSC usando brometo de 3-(4,5-dimetiltiazol-2-il)-2,5-difeniltetrazolium (MTT), investigou-se sua auto-oxidação por espectrofotometria, avaliou-se mudanças morfológicas e condensação/fragmentação nuclear por microscopia. Avaliou-se a proteção de DSC e 8-metoxipsoraleno (8-MOP) contra a citotoxicidade do CAT. O padrão de morte celular foi analisado por citometria de fluxo. A espoliação de glutation reduzido (GSH) foi analisada usando monoclorobimano. A toxicidade do CAT para células SH-SY5Y e C6 depende da dose e associa-se à formação de quinonas. Houve mudanças morfológicas, condensação/fragmentação da cromatina e morte apoptótica, não relacionada à espoliação de GSH. DSC não foi tóxica para células SH-SY5Y, porém protegeu contra os efeitos do CAT em baixas concentrações. DSC mostrou-se citotóxica para células de glioma (GL-15 e C6) e potencializou o CAT. Pré-tratamento por 30 minutos com DSC protegeu contra a ação do CAT após 72 horas. 8-MOP potencializou os efeitos do CAT, não revertendo seus efeitos na viabilidade celular, morfologia celular, condensação/fragmentação nuclear, e espoliação de GSH. Esses resultados caracterizam um modelo de citotoxicidade que pode ser aplicado no desenvolvimento de novos agentes farmacológicos. Estudos complementares são necessários para elucidar a proteção da DSC.
Catechols are benzene derivatives, which may exhibit cytotoxic activity that can be employed to develop new drugs. Plants are important sources of metabolites with pharmacological activities such as antioxidants, or cell growth inhibitors. In Brazil, cancer is the second leading cause of death, especially those derived from the nervous system, which increase the interest for new antineoplastic and neuroprotective drugs. The cytotoxic effects promoted by 1,2-dihydroxybenzene (CAT) and discretamine (DSC) in nervous system cells were characterized in vitro. The protective effects of DSC and 8-methoxypsoralen (8-MOP) against CAT-induced cytotoxicity were also evaluated. CAT and DSC EC50 was determined by using 3-(4,5-dimethylthiazol-2-yl) 2,5-diphenyltetrazolium bromide (MTT). CAT auto-oxidation was investigated by spectrophotometry. Morphological changes and nuclear condensation/ fragmentation were evaluated by microscopy. The pattern of cell death was obtained by flow cytometry. Reduced glutathione (GSH) depletion was analyzed by using monochlorobimane. CAT induced a dose-dependent toxicity to SH-SY5Y and C6 cells, associated with reactive quinones formation. It also induced morphological changes, nuclear condensation/fragmentation, and apoptotic death not caused by GSH depletion. DSC was not toxic to SH-SY5Y cells, but protected against CAT effects at low concentrations. DSC was be cytotoxic to glioma cells (GL-15 and C6) and potentiated CAT effects. However, pretreatment for 30 minutes with DSC protected them against CAT after 72 hours. 8-MOP also potentiated CAT effects instead to protect cells. These results characterize an experimental model useful for studies searching new pharmacological agents. However, further studies are needed to elucidate the DSC protective effects.
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Song, Xiaoou. "A study of the neurotoxicity of MPTP and analogs in human neuroblastoma SH-SY5Y cells." Diss., This resource online, 1996. http://scholar.lib.vt.edu/theses/available/etd-08082007-120224/.
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Manning, Alicia. "Profiling biochemical changes associated with retinoic acid-induced differentiation of SH-SY5Y human neuroblastoma cells." Thesis, Manning, Alicia (2015) Profiling biochemical changes associated with retinoic acid-induced differentiation of SH-SY5Y human neuroblastoma cells. Honours thesis, Murdoch University, 2015. https://researchrepository.murdoch.edu.au/id/eprint/29631/.
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SH-SY5Y neuroblastoma cells have been widely used in neuroscience research to model neurotoxic processes. It is unclear how well, in their undifferentiated, immortalised state, the cells model the original tissue type. SH-SY5Y cells can be differentiated, with compounds such as retinoic acid (RA), to become more ‘tissue-like’, and potentially better reflect the biochemistry of a mature neuronal cell. Studies that have investigated the RA-induced differentiation process have focused on upstream changes associated with a small number of genes or proteins. This study proposed to use a novel approach in untargeted gas chromatography-mass spectrometry (GC-MS) metabolomic analysis to investigate the downstream biochemical changes in the metabolome, which are closer to the phenotype associated with an RA-differentiated cell. Metabolomics was utilised to profile a wide range of biochemical changes in SH-SY5Y cells exposed to RA and how these processes were affected by exposure to malaoxon, a toxic metabolite of the pesticide malathion, to assess whether differentiated cells are more appropriate in future toxicology research.
Cultured human SH-SY5Y neuroblastoma cells were exposed to 10 μM RA for 120 hours and 1 μM malaoxon for 24 hours. The samples were quenched, harvested, extracted and derivatised before metabolomic analysis was carried out using untargeted GC-MS. The metabolites that contributed most to the variance were identified using principal component analysis. This indicated that RA-differentiated cells, and undifferentiated and differentiated cells exposed to malaoxon, were grouped in relation to metabolite profile. The important metabolites were subsequently analysed to determine changes due to RA and malaoxon exposure, with the aim to produce a profile of RA-differentiation cells and of the differential toxicity between undifferentiated and differentiated cells.
Changes were profiled in a range of metabolites including amino acids, carbohydrates, fatty acids and unknowns, indicating that exposure to RA caused a biochemical response in relation to neurite outgrowth, survival and apoptosis, decreased energy demand for cell proliferation and increased synthesis and activation of proteins involved in cell signalling. Data suggest that exposure to malaoxon induced a glutamate-mediated excitotoxic response causing oxidative stress, mitochondrial dysfunction, energy depletion and damage to the cell membrane. In the differentiated state, induction of survival and apoptotic responses may have facilitated a neuroprotective response that decreased the toxic effects of malaoxon.
This preliminary study has demonstrated that GC-MS-based metabolomic analysis can be used to detect changes in metabolite profile after RA and malaoxon exposure, illustrating the efficacy of this technique. The data indicates that there were a range of biochemical changes associated with an RA differentiated phenotype and that using such cells to study the effects of neurotoxins should be done with caution due to the potential neuroprotective response. The outcomes of this study have implications for research using RA-differentiated SH-SY5Y cells, with the decision to use differentiated cells resting on the particular neurotoxic model being investigated.
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Seoposengwe, K. M. (Keabetswe Millicent). "The effect of selected medicinal plants on rotenone-induced toxicity in SH-SY5Y neuroblastoma cells." Diss., University of Pretoria, 2013. http://hdl.handle.net/2263/33342.
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Parkinson's disease (PD) is the second most common chronic neurodegenerative disease characterized by dopamine decrease in the substantia nigra. Currently, there is no promising cure for PD and this has resulted in extensive research into alternative medicines. The aim of this study was to investigate the effect of methanol and ethyl acetate extracts of Lannea schweinfurthii (Engl. Engl) (Anacardiaceae), Zanthoxylum capense (Thunb. Harv) (Rutaceae), Scadoxus puniceus ((L.) Friis & Nordal) (Amaryllidaceae) and Crinum bulbispermum (Burm. f.) Milne-Redh. & Schweick) (Amaryllidaceae) on rotenone-induced toxicity in SH-SY5Y neuroblastoma cells. The latter which mimics PD symptoms in vitro.
Cytotoxicity of the plant extracts was assessed using sulforhodamine B (SRB) assay. Intracellular reactive oxygen species (ROS) were measured fluorometrically with the use of the fluorescent dye 2‟,7‟-dichlorodihydrofluorescein diacetate (H2DCF-DA). Intracellular glutathione content was measured fluorometrically after staining with monochlorobimane (MCB). Fluorescent dye 5,5‟ ,6,6‟ -tetrachloro-1,1‟ ,3,3‟ -tetraethylbenzimidazolcarbocyanine iodide (JC-1) was used to assess the mitochondrial membrane potential (MMP) status of cells. Apoptosis was assessed by determining caspase-3 activity through detection of 7-amino-4-methylcoumarin (AMC) which is a product of caspace-3 substrate, acetyl-Asp-Glu-Val-Asp 7-amino-4-methylcoumarin (Ac-DEVD-AMC), cleaved by the caspase-3 enzyme.
Rotenone was used as an in vitro model to induce PD-like symptoms. Cytotoxicity studies for methanol extract of Zanthoxylum capense revealed the highest IC50 value of 121.3 μg/mL, indicating low toxicity. The ethyl acetate extract of Crinum bulbispermum was observed to have no effect on the normal proliferation of the SH-SY5Y cells and produced an IC50 value >100 μg/mL. The calculated IC50 value obtained from rotenone cytotoxicity studies was 112
iv
nM. Zanthoxylum capense and Scadoxus puniceus plant extracts were observed to be neuroprotective against rotenone-induced toxicity.
A decrease in intracellular glutathione content as well as MMP was also observed in cells exposed to rotenone alone (50 nM). There was no intracellular ROS generation observed in cells exposed to rotenone alone (50 nM) after 24 h and 72 h. However, apoptotic cell death was observed in cells treated with rotenone (50 nM).
Intracellular ROS production was observed to be elevated by methanol and ethyl acetate extracts of C. bulbispermum. Methanol extracts of Z. capense was observed to increase intracellular glutathione content. MMP was increased effectively following treatment with ethyl acetate extract of C. bulbispermum. Moreover, both methanol and ethyl acetate plant extracts were found to decrease caspase-3 activity significantly (p<0.05), in cells exposed to 50 nM rotenone. Z. capense methanol extract reduced caspase-3 activity the most effectively.
Treatment with plant extracts was protective and decreased cell death. Furthermore, L. schweinfurthii, Z. capense, S. puniceus and C. bulbispermum, demonstrated strong antioxidant and anti-apoptotic effects against rotenone-toxicity, making them potential agents in developing therapies for treating PD.Dissertation (MSc)--University of Pretoria, 2013.
gm2014
Pharmacology
unrestricted
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Lorenzeti, Fabio Medici. "O papel do aminoácido leucina na modulação da atividade do peptídeo beta amiloide em células SH-SY5Y." Universidade de São Paulo, 2014. http://www.teses.usp.br/teses/disponiveis/39/39132/tde-20022015-101423/.
Full textAbstract:
Estudos demonstram que a indução do estresse oxidativo pelo peptídeo beta amiloide (A?) exerce um importante papel no desencadeamento da excitotoxicidade neuronal o que pode resultar no desenvolvimento de doenças neurodegenerativas. A formação do peptídeo A? se deve a alterações na proteína precursora de amiloide (APP) que é clivada para a formação do peptídeo A?. Por sua vez, os mecanismos de ação do A? no S.N.C. ocorrem através da sinalização do receptor NMDA (N-metil D-aspartato) receptor este que quando ativado pelo glutamato exerce importante papel fisiológico no S.N.C., visto que apresenta atividade ionotrópica que permite o influxo de Na+ e Ca2+ para as células neuronais, auxiliando nos processos de formação da memória e aprendizagem. Entretanto, apesar do seu papel fisiológico, a ativação excessiva do receptor NMDA é fortemente correlacionada com lesões no S.N.C. decorrente da excessiva permeabilidade do íon Ca2+ para o citosol das células neuronais. Com isso as concentrações de glutamato na fenda sináptica são estritamente controladas para que não haja ativação excessiva dos receptores com atividade glutamatérgica, como o receptor NMDA. Estudos indicam que o transporte de glutamina/glutamato através da barreira hematoencefálica é menor do que de outros aminoácidos, sendo que cerca de 25% a 30% do transporte de aminoácidos dos vasos sanguíneos para o cérebro através da barreira hematoencefálica é ocupado pelo aminoácido leucina, sendo este um grande responsável pela síntese de glutamato/glutamina no S.N.C. Com isso, estudos tem demonstrado que dietas enriquecidas com aminoácidos de cadeia ramificada, dentre eles a leucina, é responsável por alterar o metabolismo do glutamato e aumentar a susceptibilidade à excitotoxicidade de células neurais. A fim de testar esta hipótese utilizamos um modelo de cultura de células de neuroblastoma humano e realizamos o tratamento com diferentes concentrações de aminoácido leucina associado com o tratamento de peptídeo beta-amilóide. Realizamos as analises de citotoxicidade (LDH), viabilidade celular (MTT) e apoptose celular por citometria de fluxo (marcação com PE Anexina V e 7-AAD). Nossos resultados indicam que houve diferenças apenas entre o controle em relação aos demais grupos de tratamentoStudies demonstrate that induction of oxidative stress by beta amyloid peptide (A?) plays an important role in triggering neuronal excitotoxicity which can result in the development of neurodegenerative diseases. The formation of A? peptide are due to changes in the amyloid precursor protein (APP) which is cleaved to form the peptide A?. On the other hand, the mechanisms of action of A? in the C.N.S. occur through signaling of the NMDA (N-methyl-D-aspartate) receptor that when activated by glutamate plays an important physiological role in the C.N.S., as has inotropic activity that allows the influx of Na+ and Ca2+ into the neuronal cells, assisting in procedures of memory formation and learning. However, despite its physiological role, the excessive activation of the NMDA receptor is strongly correlated with C.N.S. lesions due to excess permeability of Ca2+ ions into the cytosol of neuronal cells. Thus the concentrations of glutamate in the synaptic cleft are strictly controlled so that there is excessive activation of receptors with glutamatergic activity, as the NMDA receptor. Studies indicate that the transport of glutamine/glutamate across the blood brain barrier is lower than that of other amino acids, of which about 25% to 30% of the amino acid transport blood vessels to the brain through the blood brain barrier is occupied by leucine this being one largely responsible for the synthesis of glutamate/glutamine in the C.N.S. Thus, studies have shown that diets enriched in branched chain amino acids, including leucine, are responsible for altering the metabolism of glutamate and excitotoxic increase susceptibility to neural cells. To test this hypothesis we used a cell culture model of human neuroblastoma and carry out the treatment with different concentrations of leucine associated with the processing of amyloid-beta peptide. We performed analysis of cytotoxicity (LDH), cell viability (MTT assay) and apoptosis using flow cytometry (Annexin V staining with PE and 7-AAD). Our results indicate that there were differences only between the control compared to the other treatment groups
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Yang, Wonsuk. "The bipyridyl herbicide paraquat-induced toxicity in human neuroblastoma SH-SY5Y cells: relevance to dopaminergic pathogenesis." Texas A&M University, 2005. http://hdl.handle.net/1969.1/4384.
Full textAbstract:
Paraquat (PQ) is a cationic non-selective bipyridyl herbicide widely used in
agriculture to control weeds and grasses. Epidemiologic studies indicate that exposure to
pesticides can be a risk factor in the incidence of Parkinson`s disease (PD). A strong
correlation has been reported between exposure to paraquat and PD incidence in Canada,
Taiwan, and United States. This correlation is supported by animal studies showing that
paraquat produces toxicity in dopaminergic neurons of the rat and mouse brain. However,
it is unclear how paraquat triggers toxicity in dopaminergic neurons. Based on the
previous reports, it was hypothesized that paraquat may induce oxidative stress and
proteasomal dysfunction-mediated toxicity in dopaminergic neurons. To explore this
possibility, dopaminergic SH-SY5Y human neuroblastoma cells were treated with
paraquat, and several biomarkers of oxidative stress or proteasomal dysfunction were
investigated. First, a specific dopamine transporter inhibitor GBR12909 significantly
protected SY5Y cells against the toxicity of paraquat, indicating that paraquat exerts its
toxicity by a mechanism involving the dopamine transporter (DAT). Second, paraquat increased the levels of reactive oxygen species (ROS) in SY5Y cells, but decreased the
levels of glutathione. Third, paraquat inhibited glutathione peroxidase activity, but did
not affect glutathione reductase activity. On the other hand, paraquat increased GST
activity by 24 hr, after which GST activity returned to the control value at 48 hr. Fourth,
paraquat decreased mitochondrial transmembrane potential (MTP). Fifth, paraquat
produced the increases in malondialdehyde (MDA) and protein carbonyls, as well as
DNA fragmentation, indicating oxidative damage to major cellular components. Sixth,
paraquat decreased proteasomal activity, the activities of mitochondrial complex I and V,
and intracellular ATP levels, but increased the activities of caspase 3 and 9, indicating
that proteasomal inhibition is linked to mitochondrial dysfunction accompanied by the
activation of apoptotic signaling pathway. Seventh, paraquat increased the protein levels
of heme oxygenase-1 (HO-1), p53, Bax, ñ-synuclein and ubiquitinated proteins. Eighth,
paraquat induced nuclear condensation. Taken together, these findings support the
hypothesis that paraquat produces oxidative stress and proteasomal dysfunctionmediated
toxicity in SY5Y cells. Thus, current findings suggest that paraquat may
induce the pathogenesis of dopaminergic neurons through oxidative stress and
proteasomal dysfunction.
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Jonsson, Karl. "Are nAChRs and NMDA receptors involved in low dose ethanol-nicotine toxicity in SH-SY5Y cells?" Thesis, Uppsala universitet, Institutionen för farmaceutisk biovetenskap, 2013. http://urn.kb.se/resolve?urn=urn:nbn:se:uu:diva-193048.
Full textAbstract:
Consumption of alcohol and tobacco is common all around the world and these drugs are frequently consumed concomitantly. It has been estimated that 70-80 % of alcoholics are smokers and non-alcoholic drinkers are more often smokers than teetotallers. Alcohol and tobacco may affect the risk of developing neurological diseases and might influence this risk differently when combined compared to when only one of these compounds is consumed. Some in vitro-research have shown that non-toxic concentrations of ethanol and nicotine, in combination, can exert toxicity, and might do so in a synergistic way. In this work, investigations were made to see if the neuronal nicotinic acetylcholine receptors (nAChRs) and NMDA receptors are involved in this interactive behaviour between ethanol and nicotine. A human neuroblastoma SH-SY5Y cell line was treated with ethanol and nicotine at different concentrations and cell viability was measured through an MTT-assay. A significant reduction in cell viability was induced by chronic treatment with a low-dose combination of ethanol and nicotine. The cell viability reduction was completely inhibited by pretreatment with the non-specific nAChR antagonist mecamylamine. This suggests that nAChRs are involved in low-dose ethanol-nicotine interactions. The NMDA receptor antagonist memantine did not affect the ethanol-nicotine effect, which implies that NMDA receptors are not involved in low-dose ethanol-nicotine interactions in SH-SY5Y cells. However, it is unclear if the SH-SY5Y cell line expresses fully functional NMDA receptors. The expression of NMDA receptors might vary with cell passage number. Further research has to be done to uncover the contribution of specific nAChR subtypes to the ethanol-nicotine interaction. There also remains to be revealed if human neuroblastoma SH-SY5Y cells express fully functional NMDA receptors and how cell passage number affects the expression of these receptors.
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Caneda-Ferrón, B. "The effects of Parkinson's disease mimetics on the proteasomal and neurofilament systems in SH-SY5Y cells." Thesis, Nottingham Trent University, 2006. http://irep.ntu.ac.uk/id/eprint/105/.
Full textAbstract:
Mitochondrial impairment, glutathione depletion and oxidative stress have been implicated in the pathogenesis of Parkinson's disease, linked recently to proteasomal dysfunction. This study analyses how these factors influence the various activities of the proteasome in SH-SY5Y human neuroblastoma cells treated with the PD mimetics MPP+ (a complex I inhibitor) or dopamine. Treatment with these toxins led to dose and time dependent reductions in ATP and glutathione levels and also chymotrypsin-like and postacidic-like activities; however, trypsin-like activity was unaffected. Antioxidants blocked the effects of dopamine but not MPP+, suggesting that oxidative stress was more important in the dopamine-mediated effects. With MPP+, ATP depletion was a pre-requisite for loss of proteasomal function. This study also shows that addition of MPP+ or dopamine to purified samples of the human 20S proteasome also reduced proteasomal activities; with dopamine being most damaging. As was the case with toxin-treated cells chymotrypsin-like activity was the most sensitive and trypsin-like activity, the least sensitive. The direct effect of both compounds on proteasomal activity was, at least, partly due to oxidative damage to the proteasome, since the antioxidant vitamin C could partially alleviate the proteasomal impairment. Indeed, Western blot analyses showed that some of the ?- and ?-subunits of the proteasome were modified by dopamine treatment. One of the hallmarks of Parkinson's disease is the appearance of Lewy bodies, which are protein inclusions containing ?-synuclein, neurofilament proteins and ubiquitinated proteins. A growing body of evidence suggests that the UPS might be involved in the formation of these aggregates. This thesis, reports that neurofilaments can undergo proteasomal degradation and that MPP+ and dopamine alter the expression/phosphorylation and distribution of these cytoskeletal proteins in SH-SY5Y cells. Therefore aberrant changes in both neurofilament profiles and proteasomal degradation may influence inclusion formation in dopaminergic neurons.
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Mustafa, Mohamed. "Protective capabilities of allopregnanolone against induced toxicity in SH-SY5Y cells relative to Alzheimer´s disease." Thesis, Uppsala universitet, Institutionen för farmaceutisk biovetenskap, 2020. http://urn.kb.se/resolve?urn=urn:nbn:se:uu:diva-416643.
Full textAbstract:
When the brain is exposed to a traumatic injury, the brain produces high amounts of neurosteroids like allopregnanolone and progesterone which show protective and neurogenic capacities. Alzheimer’s disease patients also have lower amounts of these neurosteroids in brain tissue. Neurosteroids act on GABAA receptors and cholesterol receptors which is interesting since both the cholesterol transporter ApoE and excitotoxicity seems to be issues plaguing the patients. To study if there is a relationship between Alzheimer’s disease and neurosteroids, there are ongoing phase one studies but neurobiological studies are equally important in order to understand the mechanism. In this work protective capabilities of allopregnanolone on induced toxicity was investigated in human neuroblastoma SH-SY5Y cells. Protection and induced toxicity were assessed by studying cell viability with MTT assay. Toxins used were the oxidative stress inducing agent t-BHP, excitotoxic glutamate and amyloid β25-35. Previous studies have found allopregnanolone to induce neurogenesis, decrease ROS levels, inhibit apoptosis and to have immunoregulatory capabilities. The present study did see an increase in cell viability when treated to 1x10-8 M allopregnanolone but this effect was not observed when the concentration was increased further to 1x10-7 M and 1x10-6 M. When the SH-SY5Y cells were treated with toxins after pretreatment of allopregnanolone, additional decrease was seen when compared to cells only treated with toxins. The present study discovered the influence of components like cell density and cell generation which is of value for researchers planning future neurobiological studies. These neurobiological studies give insight of the correct mechanisms in the brain, opening up opportunities for new efficient drugs to be developed.
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Hynds, DiAnna Lynn. "Molecular mechanisms of ganglioside inhibition of proliferation and neurite outgrowth in sh-sy5y human neuroblastoma cells /." The Ohio State University, 1997. http://rave.ohiolink.edu/etdc/view?acc_num=osu1487948158628685.
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Rabelo, Thallita Kelly. "Caracterização redox-ativa do ácido úsnico e seu efeito citotóxico em células SH-SY5Y." Universidade Federal de Sergipe, 2013. https://ri.ufs.br/handle/riufs/3890.
Full textAbstract:
Conselho Nacional de Desenvolvimento Científico e TecnológicoUsnic acid (UA) is the most common and abundant lichenic secondary metabolite with potential therapeutic application. Anti-inflammatory and antitumour properties have already been reported and UA-enriched extracts are widely used to treat several diseases in the folk medicine. On the other hand, a growing body of evidence has suggested that UA present pro-oxidant properties, which might induce cellular damage mediated by reactive species. Based on this data, first we performed in silico evaluation of UA interactions with genes/proteins and important compounds for cellular redox balance. Then, we assessed UA redox properties against different reactive species (RS) generated in vitro, and evaluated its action on SH-SY5Y neuronal-like cells subjected to hydrogen peroxide (H2O2) treatment, since no in vitro neurotoxicological data has been reported so far. Total reactive antioxidant potential index (TRAP) showed a significant antioxidant capacity of UA at 20 μg/mL; UA was also effective against hydroxyl radicals and reduced nitric oxid formation. However, in vitro lipoperoxidation was enhanced by UA, and cell viability was decreased along 24 hours of treatment, according to MTT assay (3-[4,5-dimethythiazol-2-yl]-2,5-diphenyl tetrazolium bromide) and morphological analysis. Moreover, UA did not display protective effects against H2O2-induced cell death in any case. The DCFH- DA (2,7-dichlorfluorescein-diacetate) based assay indicated that UA enhanced basal reactive species production at 20 μg/mL for 1 hour and from 2 ng/mL to 20 μg/mL for 4 and 24 hours. In addition, UA appears to potentiate H2O2-induced reactive species production. Our results suggest that UA displays variable redox-active properties, acting either as antioxidant or pro-oxidant agent according to different system conditions and/or cellular environment. These pro-oxidant properties in SH-SY5Y might be responsible by potential neurotoxic effects of UA
O ácido úsnico (AU) é um dos mais comuns e abundantes metabólitos secundários liquênicos com potencial aplicação terapêutica. As propriedades anti-inflamatória e antitumoral já foram relatadas e extratos de liquens enriquecidos com ácido úsnico, são amplamente utilizados para tratar diversas doenças na medicina popular. Entretanto, um crescente número de estudos tem sugerido que o AU apresenta propriedades pró-oxidantes em sistemas biológicos, as quais podem induzir dano celular mediado por espécies reativas. Baseado nesses dados, primeiro foi realizado a avaliação in silico das interações do AU com genes / proteínas e compostos importantes para o equilíbrio celular redox. Além disso, analisamos as propriedades redox-ativa do AU contra diferentes espécies reativas (SR) geradas in vitro, e seu efeito em células neuronais SH-SY5Y na presença de peróxido de hidrogênio (H2O2), já que nenhum dado neurotoxicológico in vitro tem sido relatado até agora. O índice de potencial antioxidante reativo total (TRAP) mostrou uma capacidade antioxidante significativa do AU a 20 μg/mL; AU também foi eficaz contra os radicais hidroxila e reduziu a formação do óxido nítrico. No entanto, a lipoperoxidação in vitro foi induzida pelo AU, e a viabilidade celular foi diminuída ao longo de 24 horas de tratamento, de acordo com ensaio de MTT (brometo de 3-[4,5-dimetil-tiazol-2-il]-2,5-difeniltetrazólio) e análise morfológica. Além disso, o AU não protegeu a célula contra a morte celular induzida pelo H2O2. O ensaio DCFH-DA (2 7 diacetato de diclorofluoresceína) mostrou que o tratamento de AU (20 μg/mL) por 1 hora e (2 ng/mL a 20 μg/mL) durante 4 e 24 horas, aumentou a produção basal de espécies reativas e quando as células foram co-tratadas com o H2O2, o AU parece potencializar o H2O2 induzindo a produção de espécies reativas. Nossos resultados sugerem que o AU exibe variáveis propriedades redox-ativas, atuando como um agente antioxidante e pró-oxidante, de acordo com as diferentes condições do sistema e / ou ambiente celular. Estas propriedades pró-oxidantes em células SH-SY5Y podem ser responsáveis por possíveis efeitos neurotóxicos do AU.
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Tieu, Kim. "Involvement of bcl-2 and p53 genes in the survival of human catecholaminergic neuroblastoma SH-SY5Y cells." Thesis, National Library of Canada = Bibliothèque nationale du Canada, 2001. http://www.collectionscanada.ca/obj/s4/f2/dsk3/ftp05/NQ63933.pdf.
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Hewawitharana, Inoka Samanthi. "Part I: Characterization of Zn reconstituted Cytochrome B561 Part II: Studies of Catecholamine metabolism in SH-SY5Y and MN9D cells." Diss., Wichita State University, 2009. http://hdl.handle.net/10057/2370.
Full textAbstract:
Cytochrome b561, a transmembrane heme protein present in neurotransmitter
storage vesicles, shuttles electrons from the cytosolic Asc to the intragranullar matrix to
regenerate Asc. Although Cyt b561 has been purified, cloned and sequenced from
various sources, the physical structure is yet unresolved. Previous studies have shown
that one of the hemes in the protein is pH labile under mild alkaline conditions. In the
present study, Zn (II) protoporphyrin IX (ZnP) was reconstituted using the altered
protein to further study the mechanism of the transmembrane electron transfer reaction
of Cyt b561. The ZnP reconstituted protein was found to quench 90% of the fluorescence
compared to the same concentration of free ZnP in the solution. The Significance of
these findings with respect to the physiological role of Cyt b561 is discussed.
In part II, the research has focused on two commonly using CNS dopaminergic
models, SH-SY5Y and MN9D cells, which are poorly characterized with respect to their
catecholamine metabolism. Differentiated and undifferentiated cells of both cell lines
were used to detect the baseline levels and uptake studies. After performing the kinetic
experiments for low, medium and high passage cells, we concluded that SH-SY5Y
could not be considered as a fully functional CNS dopaminergic model and medium and
high passage cells may be characterized as noradrenergic. MN9D cells store high
baseline levels of DA but both the differentiated and undifferentiated forms show poor
catecholamine uptake characteristics, probably due to the high intracellular DA levels.
Therefore, MN9D may be a better CNS dopaminergic model with some deficiencies in
DA uptake and release.Thesis (Ph.D.)--Wichita State University, College of Liberal Arts and Sciences, Dept. of Chemistry
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Tsang, Yuen Ting. "Expression of brain-derived neurotrophic factor in reactive astrocytes provides neuroprotection to SH-SY5Y cells against six-hydroxydopamine toxicity invitro." HKBU Institutional Repository, 2012. https://repository.hkbu.edu.hk/etd_ra/1392.
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Rabie, Soheyla Mohd Souza. "Avaliação das propriedades redox-ativas e citotóxicas ou citoprotetoras do carvacrol em cultura de células de neuroblastoma humano SH-SY5Y." reponame:Biblioteca Digital de Teses e Dissertações da UFRGS, 2013. http://hdl.handle.net/10183/77086.
Full textAbstract:
Espécies reativas de oxigênio (ERO) são produzidas através da respiração aeróbica e durante processos inflamatórios. Além disso, agressões externas como radiações, poluição, estresse, alcoolismo e tabagismo aumentam a sua produção. Altos níveis de ERO podem ocasionar dano oxidativo à lipídios, proteínas e DNA, comprometendo a função normal da célula, podendo estar envolvidos na patogênese e agravamento de diversas doenças. Há evidências que sugerem que antioxidantes naturais presentes em alimentos conferem benefícios adicionais à saúde, atuando como anticarcinogênicos, antiinflamatórios ou agentes antimutagênicos. O orégano (Oreganum sp) é uma especiaria mediterrânea usada como condimento na alimentação e pela medicina popular para diversos tipos de moléstias. O óleo possui forte ação antimicrobiana, devido ao elevado conteúdo de monoterpenos, sendo os principais o carvacrol, o timol e o para-cimeno. O carvacrol (5-isopropil-2metilfenol) é um fenol monoterpênico, com sabor picante e odor característico e tem sido amplamente usado na indústria de alimentos como aditivo seguro para aumentar a vida útil dos alimentos, como aromatizante em produtos assados, doces, bebidas e gomas de mascar, e/ou agente antimicrobiano com atividades contra bactérias, fungos e leveduras. Estudos têm relatado efeito antidepressivo e ansiolítico do carvacrol em camundongos, assim como proteção contra a radiação UVB diminuindo a peroxidação lipídica, estresse oxidativo e danos no DNA em células linfocitárias humanas e atividade antioxidante em diferentes sistemas de lipídios. Nós avaliamos a viabilidade celular e parâmetros de citotoxicidade do carvacrol em células de neuroblastoma humano SH-SY5Y. Este parece modificar levemente a morfologia das células, sem modificar significativamente a biomassa celular, parecendo ser tóxico na concentração de 100 μg/mL. Nas demais concentrações (1 a 50 μg/mL) não houve citotoxicidade. No ensaio de DCFH-DA o carvacrol reduziu a produção de ERO intracelular e diminui significativamente a produção de radicais peroxil no ensaio TRAP. Esses dados reforçam a ideia do carvacrol ser um potencial antioxidante, sendo necessários mais estudos para avaliar o mecanismo de ação deste composto.Reactive oxygen species (ROS) are produced through aerobic respiration and during inflammation. Besides, external aggressions such as radiation, pollution, stress, alcoholism and smoking increase their production. Elevated levels of ROS can cause oxidative damage to lipids, proteins and DNA, compromising the normal cell function, and may be involved in the pathogenesis and progression of several diseases. There is suggesting that natural antioxidants found in foods provide additional health benefits, acting as anticarcinogenic, anti-inflammatory agents or antimutagenic. Oregano (Oreganum sp) is a Mediterranean spice used in food as a condiment and in popular medicine to treat several types of diseases. The essential oil has strong antimicrobial activity, due to the high content of monoterpenes, the main ones being carvacrol, thymol and para-cymene. Carvacrol (5-isopropyl-2metilfenol) is a phenol monoterpene with spicy taste and odor and has been widely used in food industry as additive to preserve foods, as flavoring agent in baked goods, candy, drinks and chewing gums, and/or antimicrobial agent with activity against bacteria, fungi and yeasts. Studies have reported antidepressant and anxiolytic effects of carvacrol in mice, as well as protection against UVB radiation, decreased lipid peroxidation, oxidative stress and DNA damage in human lymphocyte cells, and antioxidant activity in different lipid systems. We evaluated cell viability and cytotoxicity parameters of carvacrol in human neuroblastoma cells SH-SY5Y. Carvacrol induced morphology changes in cells without significant modification of the cellular biomass content and it was toxic at the concentration of 100 μg/mL. In other concentrations (1-50 μg/mL) it showed no cytotoxicity. In DCFH-DA assay carvacrol reduced the intracellular ROS production and significantly decreased the production of peroxyl radicals in the TRAP assay. These data reinforce the idea of carvacrol as a potential antioxidant and more research is needed to evaluate the mechanism of action of this compound.
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Lin, Yu-Mei, and 林于媺. "The Study of Thioredoxin Intake Mechanism into SH-SY5Y Cell." Thesis, 2014. http://ndltd.ncl.edu.tw/handle/30519097085135032072.
Full textAbstract:
碩士國立臺灣大學
生化科技學系
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1.Backgrounds: The recent results of our research laboratory (Hu, 2013) has confirmed the early findings of an NIMH research team headed by Prof Chiueh (Andoh, Chock and Chiueh, 2002) that the exogenously administered human redox protein Trx (MW: 11.7 kDa; < 1 μM) can enter SH-SY5Y cells to produce its anti-apoptotic and cyto-protective effects against oxidative stress evoked by serum deprivation, neurotoxins and chemotherapeutics through a thiol-sensitive redox mechanism. The possible uptake and/or transport mechanisms of Trx are not fully understood at the present. 2.Objective: The present research aim is to investigate possible transport mechanisms for the human redox protein Trx to enter the SH-SY5Y cells for producing biological functions. The primary research goal is to create point mutations on His-tag Trx for searching by which mechanism of Trx to enter the cell membrane through a specific transport system. 3.Methods: We searched the functional sequence associated with the transportation of Trx by utilizing the bioinformatics analysis of Trx peptide sequence and synthesized His-tag Trx mutants for studying interactions between Trx and plasma membrane proteins. The end point was to detect the entry of exogenously administered His-tag Trx in SH-SY5Y cells by the western blotting, the immunocytochemistry and the confocal imaging. 4.Results: The present results show that (i) the extracellularly administered Trx (< 1μM) suppressed the etoposide (83 μM)-induced apoptosis in SH-SY5Y cells. We synthesized recombinant protein His-tag Trx to distinguish from the endogenous Trx in SH-SY5Y cells and thus verified that (ii) both the extracellularly administered Trx in oxidized and reduced form entered the cells in a dose-dependent manner and the entry of oxidized Trx were more than the reduced Trx. (iii) The extracellularly administered Trx localized in cell membrane, cytosol and cell nucleus. (iv) The mutations on the tyrosine-based sorting signal (Tyr49-X-X-Val52) blocked the entry of His-tag Trx. The modification of peroxisome membrane protein docking site (Phe27-X-X-X-Trp31) did not alter the entry of His-tag Trx from extracellular space to the cytosol. 5.Conclusions: The study utilized the bioinformatic analysis and point mutation technique to search the transport-related peptide on Trx protein, including (i) the tyrosine-based sorting signal (Tyr49-X-X-Val52) interacts with adaptor proteins in endocytosis and (ii) the peroxisome membrane docking site (Phe27-X-X-X-Trp31) for the translocation of cytosol proteins. The present results show that Trx entered the cells through endocytosis and subsequently entered nucleus to mediate the anti-oxidative cyto-protection. These results infer that the levels of Trx in cancer cells may also play a role in drug resistance.
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Ni, Chun-Lun, and 倪群倫. "Proteomic Analysis on Neuroblastoma Cell Line SH-SY5Y Treated with β–Amyloid." Thesis, 2006. http://ndltd.ncl.edu.tw/handle/09711792237771502579.
Full textAbstract:
碩士國立臺灣大學
生化科學研究所
94
Amyloid is associated with debilitating human ailments including prominently Alzheimer''s disease (AD), Huntington''s disease, Prion-related diseases and possibly cataract formation. A central event in this kind of aging-related neurological diseases such as Alzheimer''s disease is the conformational change from normally circulating soluble amyloid beta peptides (Aβ) into amyloid fibrils, in the form of senile plaques and neurofibrillary tangles respectively. Some neurofibrillary tangles were also shown to consist of hyperphosphorylated tau proteins. Extracellular Aβ accumulation and intracellular tau tangling would stimulate a series of abnormal signaling and response such as disruption of intracellular ion homeostasis, irregular signal transduction and long-term inflammatory response, all progressively leading to neuronal cell death of neurons. Because brain dissections are often available only after patients'' death, sporadic samples obtained usually belong to the diseased state of a very late stage. Generally it is difficult to observe cellular changes in the early stage, which may play a critical role to unravel the mechanism underlying this group of neurological disorders. In this study we have therefore resorted to using the neuroblastoma cell line SH-SY5Y as a model system to study the effects of extracellular Aβ on the protein expression profiles of neuronal cells by a proteomic approach. We treated neuroblastoma cells with Aβ and found some changes in protein and gene expressions as judged by 2D-PAGE and RT-PCR, respectively. Additionally, some proteins modified by phosphorylation were detected by immunoblotting with antibodies against phosphotyrosine. We also found cells transfected for temporary expression of human αB-crystallin or its mutant, αB-crystallin R120G, showed different tolerance to the toxic effects of Aβ. Since AD is concerned with protein misfolding and αB-crystallin is a small heat-shock protein with chaperone activity, this protein might be involved in signal transduction and cell skeleton regulation. Detailed study of αB-crystallin and its role in the pathogenesis of amyloid formation and Alzheimer''s disease is warranted for future studies.
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Wu, Chi-Wei, and 吳志瑋. "Effects of Methamphetamine on Mitochondrial Function and Cell Growth in Human Neuroblastoma SH-SY5Y Cells." Thesis, 2005. http://ndltd.ncl.edu.tw/handle/18088895861325086847.
Full textAbstract:
碩士國立陽明大學
藥理學研究所
93
In Taiwan, methamphetamine (METH) is a wildly abused drug, which was originally used as a psychostimulant. Several studies have demonstrated that METH would cause neurotoxicity and damage neuronal cells in rat brain. In addition, METH would lead to neurodegeneration and cause a loss of neuronal function. Up to now, the cellular and molecular mechanisms of METH-induced damage still remain unclear, but the generally-accepted concept is that reactive oxygen species (ROS) play an important role in the process of METH-induced neurodegeneration. Mitochondria are the major source of energy in cells and they produce ATP to maintain normal cellular function through oxidative phosphorylation. Moreover, mitochondria are also the major source of ROS in cells and they are the major targets of ROS. On the other hand, mitochondria are also critical in the process of apoptosis (programmed cell death). According to the role of mitochondria in cell survival, the aim of this thesis is to study the effects of METH on mitochondria and cell growth in human neuroblastoma, SH-SY5Y cells. First, after treatment of the cells with 25~250 μg/ml METH, it was found that the viability of SH-SY5Y was decreased in a time- and dose-dependent manner. Secondly, 250 μg/ml METH caused a decrease in the protein level of cyclin D and also inhibited the phosphorylation of retinoblastoma (Rb), which then led to the cell cycle arrest at G0/G1 phase. After a 48 hr treatment of METH, apoptosis was found in 20% of total cell population. Thirdly, after exposure of METH to SH-SY5Y, it was found that mitochondrial membrane potential was decreased, but mitochondrial mass was increased. It was found that mitochondrial DNA (mt DNA) copy number was decreased. In addition, the mtDNA-encoded protein COX I and COX II were decreased after METH treatment. Moverover, METH treatment led to an increase of intracellular O2 consumption rate suggesting that the rate of electron transport in mitochondrial respiratory chain was elevated. It was also found that intracellular H2O2 production was increased after exposure of METH. Interestingly, pretreatment of coenzyme Q10 could attenuate the neurotoxicity induced by METH, which emphasized the importance of mitochondria in METH-induced neurotoxic process. In conclusion, these studies suggest that METH could result in the increase of intracellular reactive oxygen species (ROS) by impairing mitochondrial function. These effects may lead to more ROS production and causes neuronal cell death.
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Johnson, Jeffreys N. "INVESTIGATING EFFECTS OF THE ANTIBIOTIC CIPROFLOXACIN ON THE DOPAMINERGIC SH-SY5Y CELL LINE." 2015. https://scholarworks.umass.edu/masters_theses_2/275.
Full textAbstract:
Ciprofloxacin is a widely prescribed antibiotic which causes idiopathic sensory adverse effects and is known to induce oxidative stress. Dopaminergic cells are known to have intrinsic sensitivity to oxidative stress. To investigate whether ciprofloxacin potentiates cy- totoxicity of dopamine, effects of combined drug treatments on cell viability were assessed by resazurin reduction, and effects on mitochondrial health were assessed by morphology. The cell viability assays suggest that ciprofloxacin significantly potentiates dopamine cytotoxicity at clinically relevant doses, although dopamine possibly interferes with the viability assay. Effects of drug treatments on mitochondrial morphology were inconclusive.
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Hsu, Wen-Ling, and 許雯菱. "Investigation of subcellular proteomic changes that associate with SH-SY5Y neuroblastoma cell differentiation." Thesis, 2019. http://ndltd.ncl.edu.tw/handle/2p9d75.
Full textAbstract:
碩士國立陽明大學
生化暨分子生物研究所
107
Neuroblastoma is an aggressive embryonal malignancy arising from neural crest tissues of sympathetic nervous system. Until now, several gene mutations have correlations with congenital neuroblastoma, but the molecular changes underlying this tumor are mostly unknown. Although neuroblastoma is severely malignant in general, surprisingly, there is a considerably proportion of neuroblastoma that can self-differentiate into benign tumors or even vanish completely. For example, stage 4S is designated to a group of neuroblastoma tumors with spontaneous regression, and the long-term outcome has been excellent. Due to poor prediction of the prognosis, markers predicting self-differentiation can help clinicians to avoid unnecessarily aggressive treatment. Given that proteomics only found minimal changes in ATRA-induced differentiation, we would like to examine whether protein interaction changes are better predictors of neuroblastoma differentiation. In Chapter II, we used a modified in situ fractionation procedure as a protein interactomics tool to uncover proteins with all-or-none changes in a set of subcellular fractions during differentiation. When adapting this approach, we surprisingly found that detergent-permeabilized SH-SY5Y cells stably attached to the culture plate only under acidic pH like pH 4.2. Using two-dimensional differential gel electrophoresis, we found color changes in 37, 23, 18, 33 and 4 spots for the Triton X-100, DNase I, RNase A, high-salt and cell-architecture fractions, respectively. There were four proteins showing >10-fold increase in the Triton X-100 fraction from ATRA-treated cells, while other fractions still had only modest or minimal changes. These include GRP78, ERP29, NCS1 and CRABP2 proteins. Intriguingly, all four proteins are from ER-Golgi system, suggestive of an unnoticed role of this system in regulation of cell differentiation. Some cytoskeletal proteins with high expression were identified in certain subcellular fractions, implicating their participation in morphological changes like neurite formation. Ingenuity pathway analyses of proteins with significant changes imply that carbohydrate metabolism is involved in cell differentiation. We will further characterize whether these all-or-none protein interactions can be seen in vivo and how they can be used in choosing the strategy for neuroblastoma treatment.
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Hsiao, Yung-Hsuan, and 蕭詠瑄. "Palmitic acid induced neuron cell cycle arrest and apoptosis through protein palmitoylation in SH-SY5Y cells." Thesis, 2013. http://ndltd.ncl.edu.tw/handle/96697523778027325810.
Full textAbstract:
碩士臺北醫學大學
保健營養學研究所
101
Epidemiological studies suggested that high fat diet is one of the notable risk factors of neurodegenerative disease (NDs), especially Alzheimer’s disease (AD). The degree of fatty acid saturation is also critical point in AD development. Dietary saturated fatty acids (SFAs) stimulate brain to uptake SFAs from plasma through the blood brain barrier. The elevation of SFAs, especially palmitic acid (PA), may exacerbate cellular damages directly in the brain. It is also noteworthy that type 2 diabetes is a risk factor of AD development, which is characterized by an elevation of PA in blood. In the present study, we examined the effects of chronic palmitic acid exposure on the cell cycle of SH-SY5Y human neuroblastoma cells. We found that chronic palmitic acid exposure induced significant G2/M arrest in the neuronal cells through endoplasmic reticulum (ER) stress. Furthermore, the G2/M arrest and ER stress were inhibited by the palmitoylation inhibitor. These findings suggested that palmitic acid induced ER tress, G2/M arrest and neuronal cell apoptosis through palmitoylation.
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Chou, Chia-Hua, and 周佳樺. "GSKIP negatively regulates GSK3β to repress the differentiation in neuroblastoma SH-SY5Y cell line." Thesis, 2007. http://ndltd.ncl.edu.tw/handle/40580505077620615094.
Full textAbstract:
碩士高雄醫學大學
生物化學研究所碩士班
95
Glycogen synthase kinase 3β(GSK3β) is an essential protein kinase that regulates numerous functions within the cell. It is regulated by both phosphorylation and protein–protein interactions. Emerging evidence has shown that GSK3β plays a pivotal role in regulating the specification of axons and dendrites. The outgrowth of neurites from a neuronal cell and the differentiation into an axon and several dendrites depends on microtubule associated proteins such as tau protein. Tau is one of the critically important substrates of GSK3β. Phosphorylation of tau by GSK3β, decreases the ability of tau to bind and stabilize microtubules. In our previous study, we found a novel GSK3β interaction protein (GSKIP) that is homologous with the GSK3β interaction domain of Axin and is able to negatively regulate GSK3β. To examine the involvements of GSKIP in neuronal differentiation, a human neuroblastoma SH-SY5Y cell line that treatment with retinoic acid (RA) differentiates to neuron-like cells was used in this study. Using an indirect immuofluorescence assay, we found that transient expression of GSKIP in SH-SY5Y cell inhibited neurites outgrowth, which was the similar result as in the presence of a direct inhibitor of GSK3β (lithium or SB415286), suggesting that GSKIP maybe involved in neuronal differentiation via negatively regulating GSK3β. Moreover, we demonstrated that GSKIP could inhibit the site-specific phosphorylation of tau by GSK3β, but it has no effects on specific site that phosphorylated by CDK5. Furthermore, GSKIP could increase the amount of β-catenin and cyclin D1, and it also raised the proliferation rate of overexpressing GSKIP cells by MTT assay. Altogether, these data provide the first evidence and strongly indicate that overexpression of GSKIP is able to inhibit neurites outgrowth in differentiated SH-SY5Y cell via negatively affecting the phosphorylation of tau by GSK3β and promote cell proliferation.
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Desai, Anu. "Human neuroblastoma (SH-SY5Y) cell culture and differentiation in 3-D collagen hydrogels for cell-based biosensing." 2004. http://purl.galileo.usg.edu/uga%5Fetd/desai%5Fanu%5Fa%5F200412%5Fms.
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