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

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

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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Cell Differentiation / physiology
  • Cell Line, Tumor
  • Coculture Techniques
  • Down-Regulation / drug effects
  • Down-Regulation / genetics*
  • Dynamins / metabolism
  • Enzyme-Linked Immunosorbent Assay
  • Flow Cytometry
  • Heparin / analogs & derivatives
  • Heparin / pharmacology
  • Luminescent Proteins / genetics
  • Luminescent Proteins / metabolism
  • Mice
  • Microscopy, Confocal
  • Neuroblastoma / pathology
  • Pluripotent Stem Cells / physiology
  • Protein Transport / physiology
  • Proteoglycans / pharmacology
  • RNA, Small Interfering / pharmacology
  • Time Factors
  • Transfection
  • alpha-Synuclein / genetics*
  • alpha-Synuclein / metabolism*

Substances

  • Luminescent Proteins
  • Proteoglycans
  • RNA, Small Interfering
  • alpha-Synuclein
  • heparin proteoglycan
  • Heparin
  • Dynamins