A novel N18TG2 x mesencephalon cell hybrid expresses properties that suggest a dopaminergic cell line of substantia nigra origin

Crawford, G. D.; Le, Weidong; Smith, Roy G.; Xie, Wen; Stefani, Enrico; Appel, Stanley H.

doi:10.1523/jneurosci.12-09-03392.1992

Cited by 165 publications

(108 citation statements)

References 35 publications

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“…The cells were routinely propagated in Sato's N1 medium (87.5% (v/v) DMEM, glutamine (4 mM), NCS (2%, v/v), FBS (5%, v/v), penicillin/streptomycin (1%, v/v), 15 mM HEPES (pH 7.4), and 1x SATO (50x SATO: insulin, 0.25 mg/mL; human transferrin, 0.25 mg/mL; pyruvic acid, 2.43 mg/mL; putrescine, 0.2 mg/mL; sodium selenite, 0.25 μg/mL; progesterone, 0.315 μg/mL) as described [34]. Transfections were carried out by treating the cells with pcDNA3 or pcDNA3-MsrA in the presence of Lipofectamine 2000 (Invitrogen).…”

Section: Transfection Of Mes235 Cellsmentioning

confidence: 99%

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Methionine sulfoxide reductase A protects dopaminergic cells from Parkinson's disease-related insults

Liu

Hindupur

Nguyen

et al. 2008

Free Radical Biology and Medicine

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Parkinson's disease (PD) is a neurologic disorder characterized by dopaminergic cell death in the substantia nigra. PD pathogenesis involves mitochondrial dysfunction, proteasome impairment, and α-synuclein aggregation, insults that may be especially toxic to oxidatively stressed cells including dopaminergic neurons. The enzyme methionine sulfoxide reductase A (MsrA) plays a critical role in the antioxidant response by repairing methionine-oxidized proteins and by participating in cycles of methionine oxidation and reduction that have the net effect of consuming reactive oxygen species. Here, we show that MsrA suppresses dopaminergic cell death and protein aggregation induced by the complex I inhibitor rotenone or mutant α-synuclein, but not by the proteasome inhibitor MG132. By comparing the effects of MsrA and the small-molecule antioxidants N-acetyl-cysteine and vitamin E, we provide evidence that MsrA protects against PD-related stresses primarily via methionine sulfoxide repair rather than by scavenging reactive oxygen species. We also demonstrate that MsrA efficiently reduces oxidized methionine residues in recombinant α-synuclein. These findings suggest that enhancing MsrA function may be a reasonable therapeutic strategy in PD. Keywords aggresome; dopamine; glutathione; heat shock protein; methionine sulfoxide reductase; neurodegeneration; oxidative stress; Parkinson's disease; proteasome; protofibril; rotenone; synuclein Parkinson's disease (PD) is a neurologic disorder that involves a selective loss of dopaminergic neurons from the substantia nigra [1,2]. The postmortem brains of PD patients are characterized by reduced activity of mitochondrial complex I, an enzyme of the mitochondrial electron transport chain [3,4]. In turn, this defect may cause a 'leakage' of electrons from mitochondria, leading to the accumulation of reactive oxygen species (ROS) that damage *Corresponding author. Address: Jean-Christophe Rochet, Department of Medicinal Chemistry and Molecular Pharmacology, Purdue University, 575 Stadium Mall Drive, RHPH 410A, West Lafayette, Indiana, 47907-209147907- . Fax: 765-494-1414. E-mail: rochet@pharmacy.purdue.edu. Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. NIH Public Access Author ManuscriptFree Radic Biol Med. Author manuscript; available in PMC 2009 August 1. NIH-PA Author ManuscriptNIH-PA Author Manuscript NIH-PA Author Manuscript proteins, lipids, and nucleic acids [3,5]. The brains of PD patients also show evidence of impaired proteasomal function [6], a defect that results in increased oxidative stress and decreased elimination...

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Section: Transfection Of Mes235 Cellsmentioning

confidence: 99%

“…The cells were plated at a density of 50,000 to 100,000 cells per well in media supplemented with dibutyryl-cAMP (1 mM) [34]. After 24 h, the cells were treated with fresh media with rotenone (100 nM), MG132 (2 μM), or vehicle (0.0006-0.02% [v/v] DMSO, final concentration in media) for 48 h, unless otherwise specified.…”

Section: Induction Of Aggresome Formationmentioning

confidence: 99%

Methionine sulfoxide reductase A protects dopaminergic cells from Parkinson's disease-related insults

Liu

Hindupur

Nguyen

et al. 2008

Free Radical Biology and Medicine

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“…Somatic cell fusion is an alternative technique that permits the immortalization of postmitotic neuronal cells from brain regions which express specific neurotransmitter phenotypes (Hammond et al, 1986;Lee et al, 1990a,b;Choi et al, 1991;Crawford et al, 1992). Furthermore, cell lines produced in this manner may be used as models for the examination of the molecular mechanisms governing the differential expression of specific neurochemical phenotypes (Choi et al, 1992).…”

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confidence: 99%

“…Somatic cell fusion has been used for the generation of adrenergic cell lines from the peripheral nervous system (Greene et al, 1975) as well as those of central nervous system lineage including septal, hippocampal (Hammond et al, 1986(Hammond et al, , 1990Lee et al, 1990a,b), and ventral spinal cord (Cashman, 1991) cell lines. In addition, this technique has been used to establish dopaminergic cell lines derived from embryonic murine mesencephalic neurons (Choi et al,199 1) as well as a rat mesencephalic cell line (Crawford et al, 1992). As an approach to developing model systems to provide a stable, homogenous model of primary corpus striatum neurons, we have used the somatic cell fusion technique to generate monoclonal cell lines of neuronal origin from the corpus striatum.…”

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confidence: 99%

Immortalized murine striatal neuronal cell lines expressing dopamine receptors and cholinergic properties

et al. 1995

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Immortalizedhybrid ceils were generated by somatic cell fusion of 18-d-old embryonic corpus striatum of the mouse strain C57BL/6J with the N18TG2 neuroblastoma.One of the cell populations obtained was treated with a combination of 1 mM n-butyric acid and 10 PM SKF 38393 (a specific D, agonist), and a surviving cell population (ElX) was subcloned. Twenty-seven monoclonal cell lines were obtained and screened for the expression of striatal-specific characteristics including 7-aminobutyric acid (GABA), choline acetyltransferase (ChAT), acetylcholine (ACh), mRNA for specific dopamine receptors, and dopamine-and adenosine negatively coupled to adenylate cyclase activity. These findings suggest that the immortalized monoclonal hybrid cell lines are of neuronal origin and have incorporated elements of the medium spiny and cholinergic neurons of the developing striatum.

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“…In the present study, we aim to verify the hypothesis that increased DMT1 expression caused the iron accumulation. We chose MES23.5 cells as the experimental neuronal model, a dopaminergic cell line hybridized from murine neuroblastoma-glioma N18TG2 cells with rat mesencephalic neurons exhibiting several properties similar to the primary neurons originated in the SN (Crawford et al, 1992). We employed a gene reconstruction technique to construct recombinant adenovirus expression vector encoding human DMT1 + IRE (AdDMT1 + IRE).…”

Section: Introductionmentioning

confidence: 99%

Over-expressed human divalent metal transporter 1 is involved in iron accumulation in MES23.5 cells

Jiang

Wang

et al. 2008

Neurochemistry International

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Elevated iron accumulation has been reported in brain regions in some neurodegenerative disorders. However, the mechanism for this is largely unknown. Divalent metal transporter 1 (DMT1) is an important divalent cation transporter. The aim of the present study is to construct recombinant adenovirus encoding human DMT1 with iron responsive element (DMT1 + IRE) and infect MES23.5 dopaminergic cells in order to investigate the relationship between increased DMT1 + IRE expression and iron accumulation. The human DMT1 gene was obtained by RT-PCR from tissues of human duodenum. AdDMT1 + IRE was successfully constructed and identified by PCR, restriction endonuclease analyses and DNA sequencing, respectively. It was able to efficiently infect MES23.5 cells, which was confirmed by RT-PCR and Western blots. When incubated with 100 mM ferrous iron for 6 h, the intracellular iron levels dramatically increased in AdDMT1 + IRE infected MES23.5 cells compared to the solely adenovirus infected cells. Meanwhile, the levels of hydroxyl free radicals and malondialdehyde (MDA) in these cells increased. This led to the activation of caspase-3. The apoptosis in AdDMT1 + IRE infected cells was shown with hypercondensed nuclei using Hoechst staining. Analysis of DNA extracted from these cells showed the typical ''ladder pattern'', indicating the formation of mono-and oligonucleosomes. These results suggested that increased DMT1 + IRE expression in MES23.5 cells caused the increased intracellular iron accumulation. This resulted in the increased oxidative stress leading to ultimate cell apoptosis. #

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A novel N18TG2 x mesencephalon cell hybrid expresses properties that suggest a dopaminergic cell line of substantia nigra origin

Cited by 165 publications

References 35 publications

Methionine sulfoxide reductase A protects dopaminergic cells from Parkinson's disease-related insults

Methionine sulfoxide reductase A protects dopaminergic cells from Parkinson's disease-related insults

Immortalized murine striatal neuronal cell lines expressing dopamine receptors and cholinergic properties

Over-expressed human divalent metal transporter 1 is involved in iron accumulation in MES23.5 cells

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