title: A cell culture model for monitoring α-synuclein cell-to-cell transfer.
abstract: The transfer of α-synuclein (α-syn) between cells has been proposed to be the primary mechanism of disease spreading in Parkinson's disease. Several cellular models exist that monitor the uptake of recombinant α-syn from the culture medium. Here we established a more physiologically relevant model system in which α-syn is produced and transferred between mammalian neurons. We generated cell lines expressing either α-syn tagged with fluorescent proteins or fluorescent tags alone then we co-cultured these cell lines to measure protein uptake. We used live-cell imaging to demonstrate intercellular α-syn transfer and used flow cytometry and high content analysis to quantify the transfer. We then successfully inhibited intercellular protein transfer genetically by down-regulating dynamin or pharmacologically using dynasore or heparin. In addition, we differentiated human induced pluripotent stem cells carrying a triplication of the α-syn gene into dopaminergic neurons. These cells secreted high levels of α-syn, which was taken up by neighboring neurons. Collectively, our co-culture systems provide simple but physiologically relevant tools for the identification of genetic modifiers or small molecules that inhibit α-syn cell-to-cell transfer.
authors.affiliation: Neuronal Survival Unit, Department of Experimental Medical Science, Wallenberg Neuroscience Center, Lund University, Sweden.
authors.forename: Juan F
authors.initials: JF
authors.lastname: Reyes
authors.affiliation: Neuronal Survival Unit, Department of Experimental Medical Science, Wallenberg Neuroscience Center, Lund University, Sweden; Laboratory for Translational Parkinson's Disease Research, Center for Neurodegenerative Science, Van Andel Research Institute, Grand Rapids, MI, USA.
authors.forename: Tomas T
authors.initials: TT
authors.lastname: Olsson
authors.affiliation: Laboratory for Translational Parkinson's Disease Research, Center for Neurodegenerative Science, Van Andel Research Institute, Grand Rapids, MI, USA.
authors.forename: Jennifer T
authors.initials: JT
authors.lastname: Lamberts
authors.affiliation: MRC Centre for Regenerative Medicine, Institute for Stem Cell Research, University of Edinburgh, Edinburgh EH9 3JQ, UK; Department of Molecular Neuroscience, UCL Institute of Neurology, Queen Square, London WC1N 3BG, UK.
authors.forename: Michael J
authors.initials: MJ
authors.lastname: Devine
authors.affiliation: MRC Centre for Regenerative Medicine, Institute for Stem Cell Research, University of Edinburgh, Edinburgh EH9 3JQ, UK.
authors.forename: Tilo
authors.initials: T
authors.lastname: Kunath
authors.affiliation: Neuronal Survival Unit, Department of Experimental Medical Science, Wallenberg Neuroscience Center, Lund University, Sweden; Laboratory for Translational Parkinson's Disease Research, Center for Neurodegenerative Science, Van Andel Research Institute, Grand Rapids, MI, USA. Electronic address: Patrik.Brundin@vai.org.
authors.forename: Patrik
authors.initials: P
authors.lastname: Brundin
mesh_terms: D000818:Animals; D002454:Cell Differentiation / Q000502:physiology; D045744:Cell Line, Tumor; D018920:Coculture Techniques; D015536:Down-Regulation / Q000187:drug effects / Q000235:genetics*; D034281:Dynamins / Q000378:metabolism; D004797:Enzyme-Linked Immunosorbent Assay; D005434:Flow Cytometry; D006493:Heparin / Q000031:analogs & derivatives / Q000494:pharmacology; D008164:Luminescent Proteins / Q000235:genetics / Q000378:metabolism; D051379:Mice; D018613:Microscopy, Confocal; D009447:Neuroblastoma / Q000473:pathology; D039904:Pluripotent Stem Cells / Q000502:physiology; D021381:Protein Transport / Q000502:physiology; D011509:Proteoglycans / Q000494:pharmacology; D034741:RNA, Small Interfering / Q000494:pharmacology; D013997:Time Factors; D014162:Transfection; D051844:alpha-Synuclein / Q000235:genetics* / Q000378:metabolism*
chemical_list: D008164:Luminescent Proteins; D011509:Proteoglycans; D034741:RNA, Small Interfering; D051844:alpha-Synuclein; C036090:heparin proteoglycan; D006493:Heparin; D034281:Dynamins
keywords: Dynamin; Dynasore; Flow cytometry; HCA; Heparin; Parkinson's disease; Prion-like; Synucleinopathy; iPS cells