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doi:10.1016/j.ijdevneu.2008.01.004 
Copyright © 2008 ISDN Published by Elsevier Ltd.
Oligomeric β-amyloid(1-42) induces the expression of Alzheimer disease-relevant proteins in cholinergic SN56.B5.G4 cells as revealed by proteomic analysis
Received 7 December 2007;
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Abstract
Alzheimer's disease (AD) is characterized by cholinergic dysfunction and progressive basal forebrain cell loss which has been hypothesized to be associated with extensive accumulation of β-amyloid (Aβ). To reveal whether oligomeric Aβ displays a particular toxicity for cholinergic neurons, the cholinergic cell line SN56.B5.G4 (SN56) was used as a model. Recently performed microarray analyses demonstrated that genes affected by exposure of SN56 cells with 50 μM oligomeric Aβ(1-42) for 24 h were involved in protein modification and degradation [Heinitz, K., Beck, M., Schliebs, R., Perez-Polo, J.R., 2006. Toxicity mediated by soluble oligomers of beta-amyloid(1-42) on cholinergic SN56.B5.G4 cells. J. Neurochem. 98, 1930–1945]. Using a proteomic approach, we compared the levels of proteins and specially of phosphorylated proteins in cytosolic fractions of cell lysates from cholinergic SN56 cells exposed to 50 μM Aβ(1-42) for 24 h to those in control incubations. We show here that the levels of calreticulin, and mitogen-activated protein kinase (MAPK) kinase 6c were up-regulated in cholinergic SN56 cells exposed to Aβ(1-42), while γ-actin appeared down-regulated. Aβ(1-42) exposure of cholinergic SN56 cells led to decreased phosphorylation of phosphoproteins, such as the Rho GDP dissociation inhibitor, the ubiquitin carboxyl terminal hydrolase-1, and the tubulin α-chain isotype Mα6, as compared to untreated control lysates. The proteins identified have also been reported to be affected in brains of AD patients, suggesting a potential role of Aβ in influencing the integrity and functioning of the proteome in AD.
Keywords: Cholinergic cell culture; Beta-amyloid toxicity; Alzheimer-relevant proteins; Mass spectrometry; Two-dimensional electrophoresis; Protein identification; Proteomics
Abbreviations: Aβ(1-42), β-amyloid(1-42) peptide; AD, Alzheimer's disease; SN56, SN56.B5.G4 cholinergic cell line; 2D PAGE, two-dimensional polyacrylamide gel electrophoresis; MALDI-TOF, matrix-assisted laser desorption/ionization time-of-flight; MAPK, mitogen-activated protein kinase; RuBPS, Ruthenium-II-Tris(Bathophenanthrolin disulphonate)
Article Outline
- 1. Introduction
- 2. Materials and methods
- 2.1. Cell culture
- 2.2. Treatment of cells and protein extraction
- 2.3. Two-dimensional gel electrophoresis
- 2.4. Image analysis
- 2.5. Sample preparation for mass spectrometry
- 2.6. Protein identification using MALDI-TOF-mass spectrometry and database searching
- 3. Results
- 3.1. Identification of proteins in cell lysates of cholinergic SN56 cells, affected by exposure to Aβ(1-42)
- 3.2. Identification of phosphorylated proteins in cell lysates of SN56 cells after Aβ(1-42) treatment
- 4. Discussion
- Acknowledgements
- References
