Ser129D mutant alpha-synuclein induces earlier motor dysfunction while S129A results in distinctive pathology in a rat model of Parkinson's disease Fabia Febbraro a , Gurdal Sahin b , Aina Farran a , Sofia Soares a , Poul H. Jensen a , Deniz Kirik b , Marina Romero-Ramos a, ⁎ a Department of Biomedicine, Aarhus University, DK-8000 Aarhus C, Denmark b Brain Repair and Imaging in Neural Systems (B.R.A.I.N.S.) Unit, BMC D11, Lund University, 22184 Lund, Sweden abstract article info Article history: Received 24 June 2012 Revised 19 March 2013 Accepted 26 March 2013 Available online 5 April 2013 Keywords: Alpha-synuclein Serine phosphorylation Adeno-associated virus Striatum Alpha-synuclein phosphorylated at serine 129 (S129) is highly elevated in Parkinson's disease patients where it mainly accumulates in the Lewy bodies. Several groups have studied the role of phosphorylation at the S129 in α-synuclein in a rat model for Parkinson's disease using recombinant adeno-associated viral (rAAV) vectors. The results obtained are inconsistent and accordingly the role of S129 phosphorylation in α-synuclein toxicity remains unclear. This prompted us to re-examine the neuropathological and behavioral effects of the S129 modified α-synuclein species in vivo. For this purpose, we used two mutated forms of human α-synuclein in which the S129 was replaced either with an alanine (S129A), to block phosphoryla- tion, or with an aspartate (S129D), to mimic phosphorylation, and compared them with the wild type α-synuclein. This approach was similar in design to previous studies, however our investigation of dopami- nergic degeneration also included performing a detailed study of the α-synuclein induced pathology in the striatum and the analysis of motor deficits. Our results showed that overexpressing S129D or wild type α-synuclein resulted in an accelerated dopaminergic fiber loss as compared with S129A α-synuclein. Fur- thermore, the motor deficit seen in the group treated with the mutant S129D α-synuclein appeared earlier than the other two forms of α-synuclein. Conversely, S129A α-synuclein showed significantly larger patho- logical α-synuclein-positive inclusions, and slower dopaminergic fiber loss, when compared to the other two forms of α-synuclein, suggesting a neuroprotective effect of the mutation. When examined at long-term, all three α-synuclein forms resulted in pathological accumulations of α-synuclein in striatal fibers and dopami- nergic cell death in the substantia nigra. Our data show that changes in the S129 residue of α-synuclein influ- ence the rate of pathology and neurodegeneration, with an overall deleterious effect of exchanging S129 to a residue mimicking its phosphorylated state. © 2013 Elsevier Inc. All rights reserved. Introduction Studies during the last decade have established a central role for alpha-synuclein (α-syn) in the neurodegeneration seen in Parkinson's disease (PD) (Marti et al., 2003). Not only is α-syn the main component of Lewy bodies (LBs) and Lewy neurites (LNs), but its mutation or in- creased expression due to gene multiplications also leads to PD (Eriksen et al., 2005; Wakabayashi et al., 2007). The mechanism initiated and prob- ably sustained by abnormal handling of α-syn that ultimately leads to neurodegeneration and cell death is still unclear. Several studies have shown that post-translational modifications in the protein per se could in- fluence α-syn toxicity, not only by causing changes in α-syn solubility, but also by leading to alterations in α-syn function as well as by influenc- ing the interactions with other proteins (Gorbatyuk et al., 2008; Kuwahara et al., 2012; McFarland et al., 2009; Michell et al., 2007; Tofaris and Spillantini, 2007; Wakamatsu et al., 2008; Yu et al., 2010). LBs are mainly composed of α-syn filaments, although other compo- nents have also been found in these pathological protein accumulations (Wakabayashi et al., 2007). Numerous studies have been conducted fo- cusing on the aggregation and/or formation of α-syn insoluble fila- ments and on the factors affecting the aggregation process. It is now widely accepted that the formation of filaments is preceded by meta-stable soluble species known as oligomers, which have been ob- served in brain samples of synucleinopathy patients (Paleologou et al., 2009) and are proposed to be the toxic form of the protein (Volles and Lansbury, 2003). Studies in post-mortem human tissue have re- vealed that the α-syn species in the LBs have different forms of modifi- cation such as nitration, truncation and phosphorylation (Baba et al., 1998; Fujiwara et al., 2002; Giasson et al., 2000). These modifications Neurobiology of Disease 56 (2013) 47–58 ⁎ Corresponding author at: Ole Worms Alle 3, bldg. 1170 Aarhus University, Aarhus C DK-8000, Denmark. Fax: +45 86131160. E-mail address: mrr@biokemi.au.dk (M. Romero-Ramos). Available online on ScienceDirect (www.sciencedirect.com). 0969-9961/$ – see front matter © 2013 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.nbd.2013.03.014 Contents lists available at SciVerse ScienceDirect Neurobiology of Disease journal homepage: www.elsevier.com/locate/ynbdi