A new model to study cell-to-cell transfer of aSynuclein in vivo Gabriela Mercado a, b, c ,N  elida L  opez a, b, c , Alexis Martínez a, b, c , Sergio P. Sardi d , Claudio Hetz a, b, c, e, f, * a Biomedical Neuroscience Institute (BNI), Faculty of Medicine, University of Chile, Independencia 1027, Santiago, 8380453, Chile b Program of Cellular and Molecular Biology, Institute of Biomedical Sciences, Faculty of Medicine, University of Chile, Independencia 1027, Santiago, 8380453, Chile c FONDAP Center for Geroscience, Brain Health and Metabolism (GERO), Independencia 1027, Santiago, 8380453, Chile d Neuroscience Therapeutic Area, Sanofi, 49 New York Avenue, Framingham, MA, 01701, USA e Buck Institute for Research on Aging, Novato, CA, 94945, USA f Department of Immunology and Infectious Diseases, Harvard School of Public Health, Boston, 02115, MA, USA article info Article history: Received 5 July 2018 Accepted 10 July 2018 Available online xxx Keywords: Parkinson's disease aSynuclein Cell-to-cell transfer Sub-cellular compartments Endoplasmic reticulum Dopaminergic neuron abstract Parkinson's disease (PD) compromises motor control due to the loss of dopaminergic neurons in the substantia nigra pars compacta. At the histopathological level, PD is characterized by the accumulation of Lewy bodies, large protein inclusions containing aggregated aSynuclein (aSyn). The progression of PD involves the spreading of aSyn misfolding through the brain mediated by a prion-like mechanism, where the protein is transferred between cells. Here we report that aSyn internalization is a dynamic process, where the protein transits through different sub-cellular compartments. Importantly, cells incorporating aSyn develop larger protein-like inclusions when compared to aSyn producing cells. We developed a new tool to monitor cell-to-cell transfer of aSyn in vivo using an adeno-associated viral (AAV) vector expressing aSyn fused to a red fluorescent protein in addition to soluble EGFP to label donor cells. Intra- nigral delivery of this reporter AAV construct allowed the visualization of aSyn incorporation into sur- rounding neurons. This work provides a new tool to study aSyn cell-to-cell transfer in vivo and may open new opportunities to study PD pathogenesis. © 2018 Elsevier Inc. All rights reserved. 1. Introduction Parkinson's disease (PD) is the second most common neurode- generative disease and the predominant cause of movement problems [1]. PD is recognized mostly by the appearance of motor symptoms such as bradykinesia, resting tremor and postural ri- gidity. The impairment of motor control in PD is the result of pro- gressive degeneration of dopaminergic neurons in the substantia nigra pars compacta (SNpc). PD is histopathologically characterized by the accumulation of intracellular protein inclusions known as Lewy Bodies (LBs). Fibrillar aggregates of misfolded aSynuclein (aSyn) constitute a major component of these intracellular in- clusions [2], which are also found in other neurological disorders including dementia with LBs and multiple system atrophy (reviewed in Ref. [3]). The majority of PD cases are idiopathic with no clear genetic components. Point mutations in aSyn, as well as duplication and triplication of the SNCA locus, result in the development of PD, involving the pathogenic aggregation of the protein and neuro- degeneration [4]. Fibrillization of aSyn occurs through a two-step polymerization process where monomers are converted into sol- uble oligomeric intermediates, which then serve as seeds for sub- sequent elongation into fibrillar structures that can give rise to large protein inclusions [5,6]. Accumulating evidence suggests that pre-fibrillar soluble oligomers are the major toxic species in PD [7]. It has long been debated whether PD results from acute damage that leads to progressive neurodegeneration or whether it is an Abbreviations: aSyn, aSynuclein; aSyn-CGFP, aSynuclein fused to carboxy-ter- minal; AAV, adeno-associated virus; BiFC, Bi-molecular Fluorescence Complemen- tation; CM, conditioned media; CSF, cerebrospinal fluid; ER, endoplasmic reticulum; GFAP, glial fibrillary acidic protein; GFP, green fluorescent protein; iPSC, induced pluripotent stem cell; LBs, Lewy Bodies; NGFP-aSyn, aSynuclein fused to amino-terminal; PD, Parkinson's disease; SNpc, Substantia Nigra pars compacta; YFP, yellow fluorescent protein. * Corresponding author. Institute of Biomedical Sciences, University of Chile, Independencia 1027, Santiago, 8380453, Chile. E-mail address: chetz@hsph.harvard.edu (C. Hetz). URL: http://www.hetzlab.cl Contents lists available at ScienceDirect Biochemical and Biophysical Research Communications journal homepage: www.elsevier.com/locate/ybbrc https://doi.org/10.1016/j.bbrc.2018.07.053 0006-291X/© 2018 Elsevier Inc. All rights reserved. Biochemical and Biophysical Research Communications xxx (2018) 1e9 Please cite this article in press as: G. Mercado, et al., A new model to study cell-to-cell transfer of aSynuclein in vivo, Biochemical and Biophysical Research Communications (2018), https://doi.org/10.1016/j.bbrc.2018.07.053