Lysophosphatidylcholine increases the neurotoxicity of Alzheimer's amyloid β1-42 peptide: role of oligomer formation.
Oligomer formation is considered as a critical process for the neurotoxic effects of Alzheimer's amyloid β (Aβ) peptide. Previously we have demonstrated that lysophosphatidylcholine (LPC) increases the oligomer formation of Aβ1-42, the major Aβ peptide found Alzheimer's disease (AD) lesions. In this study, we have investigated whether LPC affects the neurotoxic effects of Aβ1-42 in a neuronal cell line (A1) culture. Dimethyl thiazolyl diphenyl tetrazolium (MTT) assay revealed that up to 10μM concentration, LPC did not affect A1 cell viability. Aβ1-42 decreased the cell viability, and such effect was dose dependently enhanced by LPC. However, neither LPC nor Aβ1-42, alone or in combination increased lactate dehydrogenase (LDH) release from A1 cells after 24-h treatment. Terminal deoxynucleotidyl transferase dUTP-biotin nick-end-labeling (TUNEL) assay showed that LPC increased Aβ1-42-induced apoptotic cell number. To determine the underlying mechanisms, the proteins implicated in apoptosis pathways including Bcl-2- and caspase-family were analyzed by Western blotting. The results demonstrated that Aβ1-42 decreased Bcl-2 in A1 cells at 24h, whereas LPC had no effect at any time point. Both LPC and Aβ1-42 increased Bax level at 24h, and their combined stimulation showed a synergistic effect. Similar synergistic effect of LPC and Aβ1-42 on caspase9 activation was observed. Dot blot immunoassay and Western blotting showed that LPC augmented Aβ1-42 oligomer formation in cell culture medium. Removing LPC-induced early-formed Aβ1-42 oligomer from the culture medium by immunoprecipitation decreased active caspase9 level and neurotoxicity, as revealed by Western blotting and MTT assay. Furthermore, dihydroethidium (DHE) assay showed that Aβ1-42 increased reactive oxygen species level in A1 cells, such effect was further enhanced by LPC. Thus, our results demonstrated that LPC increased the oligomer formation process of Aβ1-42 peptide in culture condition, and consequently increased apoptotic neuronal death. Such process might be important for the pathogenesis of AD, and inhibition of LPC generation could be a therapeutic target for the disease.
Department of Laboratory Medicine, Shimane University School of Medicine, 89-1 Enya Cho, Izumo 693-8501, Japan.
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Sheikh
Department of Biochemistry, Graduate School of Medical Sciences, Nagoya City University, Nagoya, Japan.
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Michikawa
Department of Neurology, UBC Hospital, University of British Columbia, Vancouver, Canada; Medical Research Institute, Chung-Ang University College of Medicine, Seoul, Republic of Korea.
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Kim
Department of Laboratory Medicine, Shimane University School of Medicine, 89-1 Enya Cho, Izumo 693-8501, Japan. Electronic address: anagai@med.shimane-u.ac.jp.
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Nagai
D016229:Amyloid beta-Peptides / Q000378:metabolism*; D017209:Apoptosis / Q000502:physiology*; D015153:Blotting, Western; D053453:Caspase 9 / Q000378:metabolism; D045744:Cell Line, Tumor; D002470:Cell Survival / Q000502:physiology; D006801:Humans; D015151:Immunoblotting; D047468:Immunoprecipitation; D020287:In Situ Nick-End Labeling; D007770:L-Lactate Dehydrogenase / Q000378:metabolism; D008244:Lysophosphatidylcholines / Q000378:metabolism*; D009474:Neurons / Q000502:physiology*; D010446:Peptide Fragments / Q000378:metabolism*; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000378:metabolism; D017382:Reactive Oxygen Species / Q000378:metabolism; D051028:bcl-2-Associated X Protein / Q000378:metabolism
D016229:Amyloid beta-Peptides; C494826:BAX protein, human; C000595707:BCL2 protein, human; D008244:Lysophosphatidylcholines; D010446:Peptide Fragments; D019253:Proto-Oncogene Proteins c-bcl-2; D017382:Reactive Oxygen Species; C075222:amyloid beta-protein (1-42); D051028:bcl-2-Associated X Protein; D007770:L-Lactate Dehydrogenase; C506433:CASP9 protein, human; D053453:Caspase 9
Alzheimer’s disease; amyloid β; lysophosphatidylcholine; neuronal apoptosis; oligomer