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Neurobiol Dis. 2015 May;77:266-75. doi: 10.1016/j.nbd.2014.07.003. Epub 2014 Jul 16.

A cell culture model for monitoring α-synuclein cell-to-cell transfer.

Author information

1
Neuronal Survival Unit, Department of Experimental Medical Science, Wallenberg Neuroscience Center, Lund University, Sweden.
2
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.
3
Laboratory for Translational Parkinson's Disease Research, Center for Neurodegenerative Science, Van Andel Research Institute, Grand Rapids, MI, USA.
4
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.
5
MRC Centre for Regenerative Medicine, Institute for Stem Cell Research, University of Edinburgh, Edinburgh EH9 3JQ, UK.
6
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.

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.

KEYWORDS:

Dynamin; Dynasore; Flow cytometry; HCA; Heparin; Parkinson's disease; Prion-like; Synucleinopathy; iPS cells

PMID:
25046995
DOI:
10.1016/j.nbd.2014.07.003
[Indexed for MEDLINE]

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