David Pozo1* Abstract Glial Innate Immunity Generated by Non-Aggregated Alpha-Synuclein in Mouse: Differences between Wild-type and Parkinson’s Disease-Linked Mutants Cintia Roodveldt1, Adahir Labrador-Garrido1, Elena Gonzalez-Rey2, Rafael Fernandez-Montesinos1, Marta Caro2, Christian C. Lachaud1, Christopher A. Waudby3,4, Mario Delgado2, Christopher M. Dobson 3 , 1 CABIMER-Andalusian Center for Molecular Biology and Regenerative Medicine, Consejo Superior de Investigaciones Cient´ıficos, University of Seville-UPO-Junta de Andalucia, Seville, Spain, 2 Institute of Parasitology and Biomedicine Lopez-Neyra, Consejo Superior de Investigaciones Cient´ıficos, Granada, Spain, 3 Department of Chemistry, University of Cambridge, Cambridge, United Kingdom, 4 Department of Structural and Molecular Biology, University College, London, United Kingdom Background: Parkinson’s disease (PD) is a progressive neurodegenerative disorder characterized pathologically by the presence in the brain of intracellular protein inclusions highly enriched in aggregated alpha-synuclein (a-Syn). Although it has been established that progression of the disease is accompanied by sustained activation of microglia, the underlying molecules and factors involved in these immune-triggered mechanisms remain largely unexplored. Lately, accumulating evidence has shown the presence of extracellular a-Syn both in its aggregated and monomeric forms in cerebrospinal fluid and blood plasma. However, the effect of extracellular a-Syn on cellular activation and immune mediators, as well as the impact of familial PD-linked a-Syn mutants on this stimulation, are still largely unknown. Methods and Findings: In this work, we have compared the activation profiles of non-aggregated, extracellular wild-type and PD-linked mutant a-Syn variants on primary glial and microglial cell cultures. After stimulation of cells with a-Syn, we measured the release of Th1- and Th2- type cytokines as well as IP-10/CXCL10, RANTES/CCL5, MCP-1/CCL2 and MIP-1a/CCL3 chemokines. Contrary to what had been observed using cell lines or for the case of aggregated a-Syn, we found strong differences in the immune response generated by wild-type a-Syn and the familial PD mutants (A30P, E46K and A53T). Conclusions: These findings might contribute to explain the differences in the onset and progression of this highly debilitating disease, which could be of value in the development of rational approaches towards effective control of immune responses that are associated with PD. Citation: Roodveldt C, Labrador-Garrido A, Gonzalez-Rey E, Fernandez-Montesinos R, Caro M, et al. (2010) Glial Innate Immunity Generated by Non-Aggregated Alpha-Synuclein in Mouse: Differences between Wild-type and Parkinson’s Disease-Linked Mutants. PLoS ONE 5(10): e13481. doi:10.1371/journal.pone.0013481 Editor: Joseph Najbauer, City of Hope National Medical Center, United States of America Received February 24, 2010; Accepted September 24, 2010; Published October 26, 2010 Copyright: 2010 Roodveldt et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: CR held a Federation of the Societies of Biochemistry and Molecular Biology Long-Term Fellowship during part of this work, and currently holds a postdoctoral fellowship by the Spanish Ministry of Science and Innovation (Juan de la Cierva Programme). RFM holds an FPI fellowship at the University of Seville Department of Normal and Pathological Cytology and Histology. This work was supported by Instituto de Salud Carlos III - Fund for Health of Spain (to MD and DP); Junta de Andalucia BIO-323 (DP) and CTS-541 (MD); Alicia Kloplowitz Foundation (to MD and DP); and Wellcome Trust and Leverhulme Trust grants (CMD). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: david.pozo@cabimer.es Introduction Parkinson’s disease (PD) is the second most common neurode- generative disorder, after Alzheimer’s disease. It is characterized pathologically by the presence of deposits of aggregated a- synuclein (a-Syn) in intracellular inclusions, known as Lewy bodies, in the substantia nigra pars compacta (SN) of the brain [1,2], and by the loss of dopaminergic neurons [3,4]. There is considerable evidence indicating a role of a-Syn in the etiology of PD, in which the conversion of a-Syn from soluble monomers to aggregated amyloid-like insoluble forms is a key event in PD pathogenesis [5]. However, the cellular and molecular mecha- nisms underlying the pathological actions of a-Syn are still not completely understood. Traditionally, a-Syn has been viewed as an exclusively intracellular, cytoplasmic protein which is highly expressed in dopaminergic neuronal cells. Lately, accumulating evidence showing the uptake of extracellular a-Syn by glia and neurons via endocytosis [6,7], the release and exocytosis of a-Syn to the medium [8,9], and the presence of a-Syn in cerebrospinal fluid [10,11] and blood [11] both in its aggregated and non- aggregated forms has pointed at the importance of studying the effects of extracellular a-Syn on surrounding cells in the brain. Alpha-Syn is a 140-amino acid protein that is highly enriched in presynaptic neuronal terminals, in particular in the neocortex, hippocampus, and SN [12], as well as within astrocytes and oligodendroglia [13,14]. The physiological role of a-Syn is still being established, but its interaction with pre-synaptic membranes suggests that one function may be the regulation of synaptic vesicle pools, including control of dopamine levels [15]. Alpha-Syn belongs to the group of proteins described as natively unfolded PLoS ONE | www.plosone.org 1 October 2010 | Volume 5 | Issue 10 | e13481