Virus Research 207 (2015) 38–46

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Parkinson’s disease as a member of Prion-like disorders Maria Eugenia Herva ∗ , Maria Grazia Spillantini The Clifford Allbutt Building, Department of Clinical Neurosciences, University of Cambridge, Cambridge CB2 0AH, UK

a r t i c l e

i n f o

Article history: Received 21 July 2014 Received in revised form 29 September 2014 Accepted 14 October 2014 Available online 4 November 2014

a b s t r a c t Parkinson’s disease is one of several neurodegenerative diseases associated with a misfolded, aggregated and pathological protein. In Parkinson’s disease this protein is alpha-synuclein and its neuronal deposits in the form of Lewy bodies are considered a hallmark of the disease. In this review we describe the clinical and experimental data that have led to think of alpha-synuclein as a prion-like protein and we summarize data from in vitro, cellular and animal models supporting this view. © 2014 Published by Elsevier B.V.

Keywords: Alpha-synuclein Parkinson Prion Spreading Misfolding

1. Parkinson’s disease and other alpha-synucleinopathies Parkinson’s disease (PD) is the second most common neurodegenerative disease and the most common movement disorder. It principally affects people over the age of 50 and its prevalence increases with age. The majority of PD cases are sporadic but a small percentage is familiar and in these cases the age of onset can be much earlier (Thenganatt and Jankovic, 2014). Patients with PD show characteristic motor symptoms such as postural instability, bradykinesia, rigidity and resting tremor. However, clinical studies have evidenced a wide range of other nonmotor symptoms, including anosmia, sleep problems, constipation and psychological alterations as depression with around 40% of the patients developing dementia (Khoo et al., 2013). PD belongs to a group of diseases named alpha-synucleinopathies due to the presence of alpha-synuclein aggregates. These diseases include among others Dementia with Lewy Bodies (DLB) and Multiple System Atrophy (MSA). DLB is clinically characterized by progressive dementia with visual hallucinations, and hyper-sensitivity to neuroleptic medications (McKeith et al., 1996). MSA is the third most common parkinsonism syndrome with a great variety of symptomatology comprising autonomic dysfunction parkinsonian movement disorders, cerebellar ataxia (Gilman et al., 1999).

∗ Corresponding author. Tel.: +44 1223331173. E-mail address: [email protected] (M.E. Herva). http://dx.doi.org/10.1016/j.virusres.2014.10.016 0168-1702/© 2014 Published by Elsevier B.V.

Neuropathological, biochemical and genetic evidences (Spillantini et al., 1998, 1997; Vekrellis et al., 2011) have supported the implication of alpha-synuclein as the cause of these diseases. More specifically, mutations, duplications and triplications of the alpha-synuclein gene (SNCA) locus are causes of familial forms of PD and DLB (Houlden and Singleton, 2012) and SNCA has also been identified as a risk factor in all the PD genome wide association studies (Singleton et al., 2013). Additionally, alpha-synuclein deposits can be found with other proteinaceous aggregates (Tau, A␤, prions, etc.) characteristics of other neurodegenerative diseases (Arima et al., 1998; Charles et al., 2000; Doherty et al., 2004; Forman et al., 2002; Haïk et al., 2002; Lippa et al., 1999; Spillantini et al., 1998; Takeda et al., 1998; Wakabayashi et al., 2000). 2. The alpha-synuclein protein Alpha-synuclein is a small protein of 140 amino acids highly expressed in the brain, with a pre-synaptic localization (Iwai et al., 1995; Jakes et al., 1994), but has been reported to be expressed in other organs including kidney, liver and heart (Baltic et al., 2004) besides been present in blood cells (Nakai et al., 2007). It is part of the synuclein family, which has two other members with different percentage of homology to alpha-synuclein, beta-synuclein with 63% and gamma-synuclein with 55% homology. The alphasynuclein gene, SNCA, resides in chromosome 4 and although three alternative spliced transcripts have been described, proteins have been identified only for the full length 1–140 protein (Tofaris and Spillantini, 2007). Structurally alpha-synuclein can be divided in

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Fig. 1. Schematic of alpha-synuclein featuring: sequence with regions of interest and most abundant mutations (A), tertiary structure of the protein (B) and post-translational modifications (C) where Ac is acetylation, Ub = ubiquitinilation, P = phosphorylation and scissors show some of the truncated epitopes.

three regions: the N-terminal part containing 7 imperfect repeats of 11 amino acids with a highly conserved KTKEGV motif and with propensity to form alpha-helical structures (Chandra et al., 2003; Eliezer et al., 2001) (Fig. 1B). The alpha-synuclein gene mutations A30P, E46K, A53T, H50Q and G51D that are associated with disease are concentrated in this N-terminal region (Appel-Cresswell et al., 2013; Kiely et al., 2013; Krüger et al., 1998; Lesage et al., 2013; Polymeropoulos, 1997; Proukakis et al., 2013; Zarranz et al., 2004). The second region is the NAC region, which stands for nonamyloid component and has this name because it was first described in amyloid plaques in Alzheimer’s disease brains (Uéda et al., 1993), location that was not confirmed later (Culvenor et al., 1999). This hydrophobic region of 35 amino acids is indispensable for the aggregation of alpha-synuclein (Crowther et al., 1998; Giasson et al., 2001) and it has a degree of sequence similarity with other amyloidogenic peptides such as ␤-amyloid (El-Agnaf and Irvine, 2002). The third region is the C-terminal part, a proline rich area, negatively charged and highly unstructured (Fig. 1A and B) where several proteins have been reported to bind (Burré et al., 2012; Dev et al., 2003). The alpha-synuclein protein undergoes a number of posttranslational modifications mostly pathology associated. These include N-acetylation and nitration in tyrosine 39 (Giasson et al., 2000; Trexler and Rhoades, 2012). There are several ubiquitinylated residues in lysines 12, 21 and 23, as well as fragments truncated at Asp-115, Asp-119, Glu-120, Asn-122, Tyr-133, and Asp-135 (Anderson et al., 2006; Baba et al., 1998; Hasegawa et al., 2002) (Fig. 1C) that have been detected. Also phosphorylation, mainly in Ser-129 and Ser-87 have been strongly associated with pathology (Fujiwara et al., 2002; Paleologou et al., 2010). The alpha-synuclein protein is considered to be a natively unfolded protein that can become misfolded and aggregates forming ␤-sheet structures (Fauvet et al., 2012; Weinreb et al., 1996). However it has also been reported that alpha-synuclein can be natively in a tetrameric form as suggested by data obtained using recombinant, cell or brain extracted alpha-synuclein (Bartels et al., 2011; Selkoe et al., 2014; Wang et al., 2011). It is known that alpha-synuclein structure is stabilized by its binding to lipid membranes (Davidson et al., 1998) and a recent report has shown that multimers are found only when alpha-synuclein is bound to cell membranes, not finding tetramers or other oligomers when the protein is in its cytosolic not-bound state (Burré et al., 2014). These discrepancies might be explained by different methods of producing or extracting the alpha-synuclein (Coelho-Cerqueira et al., 2013). The events that could lead to alpha-synuclein aggregation and gain of pathological function have been summarized (Anichtchik et al., 2013). Despite a debate is ongoing about the structural physiological characteristics of alpha-synuclein, in the pathological condition the protein becomes insoluble with amyloid

characteristics and with, features similar to prions as summarized in Table 1. Misfolded alpha-synuclein has been described to have different conformations or strains and to be able to transmit the pathology from cell to cell explaining the progression of PD in affected individuals. However, although fidelity of transmission of strain characteristics has been suggested (Watts et al., 2013) more evidences are needed to definitely prove it (Table 1). 3. The prion protein Prions, (from proteinaceous infectious particles) are the causal agent of the Transmissible Spongiform Encephalopathies (TSEs). They are misfolded isoforms from an ubiquitiously expressed protein, PrPC (Prusiner et al., 1998). Prions occur as distinct strains, which share the sequence of the underlying PrPC but exhibit diverse phenotypes, including incubation period, clinical symptoms as well as biochemical and neuropathological features. It is generally accepted that prion strains differ in regard to the conformation of their PrPSc , which is propagated with high fidelity by seeded conversion (Weissmann, 2009). Biochemically, prions are characterized by a high content in ␤-sheet as compared with the fundamentally helical conformation of the PrPC , and high but not total resistance to proteinase K digestion. Purified prions are insoluble even in mild detergents and they form oligomers and fibrils named prion rods that can be observed using electron microscopy (Riesner, 2003). In addition to been self-propagating inside cells, prions are transmitted from cell to cell within individuals and between individuals. In cells several mechanisms of transmission have been reported, including release of exosomes and uptake by recipient cells and tunnelling nanotubules connecting donor and recipient cells (Fevrier et al., 2004; Gousset et al., 2009; Vella et al., Table 1 Comparison of prion and alpha-synuclein biochemical and transmission properties. *Although in vitro generation of alpha-synuclein fibril “strains” has been reported (Guo et al., 2013) their serial transmission in a prion-like manner has not been demonstrated. Prions

Alpha synuclein

Biochemical characteristics Oligomer and fibril formation High content in ␤-sheet Insolubility in mild detergents Partial resistance to PK Conformation diversity

Yes Yes Yes Yes Yes

Yes Yes Yes Yes Yes

Transmission properties Spread cell to cell Spread in an organism Transmission among individuals Spread of strains

Yes Yes Yes Yes

Yes Yes No No

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2007). As for transmission from one animal to another besides the well-known scrapie in sheep (Kimberlin, 1990), a good example is probably Chronic Wasting Disease in deer where there is around 50% prevalence of infection in free range animals (Gilch et al., 2011). Additionally, transmission of virtually every TSE to rodent models has been achieved by intracranial injection (Watts et al., 2014) and although in some TSEs experiment an initial transmission barrier requires long incubation times, soon adaptation occurs to the new host and next passages are accelerated (Torres et al., 2014).

4. In vitro evidences of alpha-synuclein prion behaviour There are many players in vivo that can influence the folding of a protein, such as chaperones, proteases and the efficiency of the cell-clearance systems (Leissring and Turner, 2013; Metcalf et al., 2012; Muchowski, 2002). However, in vitro misfolding of a protein involves structural identities. Lack of secondary structure conformation in proteins have been proposed to be a major force driving misfolding and aggregation (Kallberg et al., 2001; Villegas et al., 2000) and alpha-synuclein is a highly unfolded protein, therefore this could be the reason for its propensity to aggregate. The Cterminal region of the alpha-synuclein sequence seems to play a crucial role in maintaining the normal disordered conformation of the protein by interacting with the N-terminal region and possibly the NAC region and stabilizing the disordered structure of alphasynuclein (Hong et al., 2011; Hoyer et al., 2004). In fact, deletion of the last 20 amino acids in the C-terminal region promotes faster aggregation of alpha-synuclein (Crowther et al., 1998). In vitro aggregation of recombinant alpha-synuclein has been used for many years as a way to study the misfolding of the

protein, the significance of the point mutations in the misfolding rate, as well as to investigate compounds that can enhance and inhibit its aggregation (Giasson et al., 1999; Masuda et al., 2006; Narhi et al., 1999; Uversky and Eliezer, 2009). For these studies recombinant wild type or mutated alpha-synuclein expressed in E. coli and purified using size exclusion and anion exchange chromatography has been used. The recombinant protein in certain controlled conditions of temperature, high protein concentration, long incubation times and agitation forms aggregates of misfolded protein that can be detected by Thioflavin T, a dye that shows fluorescence when intercalated between ␤-sheet structures. The use of Thioflavin T has been widely used for detecting alpha-synuclein aggregates. However, small variations in the reaction conditions, pH, temperature, molecular crowding, etc. can profoundly alter the efficiency of the outcome, leading also to structurally different alpha-synuclein fibril conformations (Bousset et al., 2013; Guo et al., 2013). Therefore, the development of a fast, reliable and reproducible assay to study the aggregation of alpha-synuclein and the effect of drugs in the formation of these aggregates was a pressing need (Narkiewicz et al., 2014). In the prion field, technologies have been developed to reproducibly form aggregates and transferring them to alpha-synuclein could be useful. We have recently developed Protein Misfolding Cyclic Amplification (PMCA), a technique commonly used in the prion field (Saborio et al., 2001) to efficiently generate alphasynuclein aggregates (Herva et al., 2014). The misfolded proteins produced with PMCA exhibit the biochemical characteristics of alpha-synuclein aggregates present in patient brain inclusions. PMCA-produced alpha-synuclein aggregates have a high content in ␤-sheet structures, partial resistance to Proteinase K digestion and they are filamentous (Fig. 2). Importantly, the alpha-synuclein

Fig. 2. Alpha-synuclein PMCA. (A) Comparison of alpha-synuclein fibril formation with and without PMCA samples in a 24 h reaction. (B) Immunoblotting of non-PMCA and PMCA alpha-synuclein samples following digestion with different concentrations of PK. (C) Transmission electron microscopy of PMCA alpha-synuclein fibrils in carbon coated grids ×140,000, red box: enlargement of the field showing fibrillar aggregates. Data from Herva et al. (2014).

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PMCA is very reproducible with small variations between experiments, it uses small concentrations of protein and it is a very fast method. These characteristics make it an excellent method for drug screening accordingly, we have proved that anti-amyloid compounds inhibit the formation of PMCA-fibrils. 5. Alpha-synuclein prion-like transmission It was in 2003 when Braak et al. (2003a,b) proposed the hypothesis of PD as a progressive disease that followed a predictable topographical sequence. This idea was based on post-mortem studies demonstrating that the Lewy bodies and alpha-synuclein pathology appeared initially at two sites, the olfactory bulb and the dorsal motor nucleus of the vagus nerve (Del Tredici et al., 2002; Pearce et al., 1995). From these areas the lesions ascend through the brain and progress in 6 stages being the first 3 non-symptomatic and the last 3 symptomatic (Braak et al., 2002, 2003a,b). The progressing alpha-synuclein pathology culminated in the brain with the appearance of the Lewy body inclusions (Spillantini et al., 1997) in the substantia nigra, feature considered the hallmark of PD. This distribution of pathology could have suggested that the pathology was transmitted between neurons via the spreading of the misfolded alpha-synuclein, but not supporting molecular evidence was found until 2008. Indeed, the first indication about prion-like transmission of the alpha-synuclein protein came from three studies published simultaneously in 2008 (Kordower et al., 2008; Li et al., 2008; Mendez et al., 2008). These articles discussed the examination of postmortem brains of PD patients who participated in clinical trials involving grafts of fetal nigral dopaminergic nerve cells into the brains. The aim of the transplants was to replace the loss of striatal dopamine in individuals no longer responding to levodopa treatments (Brundin et al., 2008). And the three papers reported the presence of alpha-synuclein inclusions with Lewy body appearance in the grafted tissues. The age of the grafted cells where the inclusions were found was 11–16 years, the time passed since the transplant, therefore, the possibility of the grafts developing spontaneous Lewy pathology was minimal. The most plausible explanation for the presence of Lewy bodies in grafted cells was the transmission of the disease from the host to the grafted cells during the years of close interaction. Those observations had profound implications for replacement therapies and surgical interventions. Additionally, they opened the door to a new way of understanding and studying PD where the agent inducing Lewy pathology, resembling a prion, could be transmitted from cell to cell in human brains. To strengthen these findings, controlled experiments were performed where healthy mouse cortical neuronal stem cells were injected into a transgenic mouse model of PD expressing human alpha-synuclein (Rockenstein et al., 2002). The stem cells expressed GFP that allowed their tracking and co-localization of human alphasynuclein immunoreactivity with GFP signal was found 1 week after grafting and increased after 4 weeks. Some transplanted cells even developed Lewy body inclusions. This elegant experiment demonstrated that alpha-synuclein pathology was transmitted directly from the host to the grafted cells in a prion-like fashion (Desplats et al., 2009). 6. Cellular models of alpha-synuclein pathology and/or spreading Several cellular models have been generated over the last 15 years to simulate in culture the pathological features of the dopaminergic cells dying in PD patients. The first models were established using cell lines like HEK293 where human wild type

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or mutated alpha-synuclein was overexpressed and its effect on the cells and the formation of aggregates were studied (Engelender et al., 1999; Opazo et al., 2008; Tabrizi et al., 2000). Recently even transmission from cell to cell in a prion-like fashion was achieved using this model (Hansen et al., 2011a,b). However, the HEK293 cell line although easy to manipulate has a major drawback and it is its kidney origin and therefore the lack of neuronal attributes. Another cell line, the SH-SY5Y human neuroblastoma with neural background, has been also widely used for studying the effect of wild-type, mutated or truncated alpha-synuclein overexpression in cells (Kanda et al., 2000; Pandey et al., 2006; El-Agnaf et al., 1998). Among the toxic effects caused by the accumulation of alpha-synuclein that have been described in this cell model there is an increase in reactive oxygen species (Junn and Mouradian, 2002), autophagy enhancement (Gomez-Santos, 2003), mitochondrial damage by direct binding to cytochrome C (Elkon, 2002) calcium homeostasis deregulation (Melachroinou, 2013). Our group, using the SH-SY5Y cell model, demonstrated that impairment of the proteasome, involved in alpha-synuclein degradation (Bennett et al., 1999), increased the accumulation of alpha-synuclein in the cells (Tofaris et al., 2001). Moreover, we demonstrated that there was a fraction of non-ubiquitinated alphasynuclein that was degraded directly by the proteasome and that the accumulated alpha-synuclein formed amorphous nonfilamentous aggregates (Tofaris et al., 2001). Furthermore, studies of cellular stress induced by glucose deprivation in differentiated SH-SY5Y have shown that in addition of inducing formation of intracellular alpha-synuclein aggregates, it activates unfolded protein response (UPR) (Bellucci et al., 2011). This cellular pathway halts protein production affecting through phosphorylation a pathway that includes proteins such as PERK and eIF2␣ that has been shown to be activated also in prion diseases (Hetz and Soto, 2006). Moreover, the fact that PD patients have higher levels of phosphorylated eIF2␣ (Hoozemans et al., 2007) than healthy controls and that genetically and pharmacologically targeting the UPR pathway rescues prion mouse model from neurodegeneration (Moreno et al., 2013, 2012) might open new doors for alpha-synucleinopathies treatments. These HEK 293 and SH-SY5Y have been used also for seeding and transmission experiments having the advantage of been easily expandable and to reproduce the phenotypic features of the alphasynuclein pathology (Danzer et al., 2009; Emmanouilidou et al., 2010; Hansen et al., 2011a,b). Recently, we have demonstrated that SH-SY5Y cells overexpressing human wild type alpha-synuclein can develop alpha-synuclein aggregation by seeding with recombinant alpha-synuclein fibrils generated in vitro by PMCA (Herva et al., 2014). Importantly, these cells become chronically infected, in a prion-like manner, as they are able to transmit the pathological aggregates at least after 10 divisions. Similar results have been obtained by Aulic´ et al. (2014) in wild type SH-SY5Y. We believe that this could be an excellent model for screening of drugs able to inhibit alpha-synuclein aggregation and decrease its pathological effects. Primary neuronal cultures have also been used to study alphasynuclein toxicity. These primary cultures have been derived from rat, mouse, flies and even from human embryonic stem cells (Park and Lee, 2006; Rideout et al., 2004; Zhou et al., 2002, 2000). Besides the species, also the region of the nervous systems from which the cell derive can vary from study to study, cerebellar granular cells (Monti et al., 2007), dorsal root ganglia (Lee et al., 2006), cortical and midbrain neurons (Rideout et al., 2003) and enteric neurons (Paillusson et al., 2013) just to name a few. These primary culture cell models have been highly used to study pathological mechanisms underlying alpha-synuclein overexpression including endoplasmic reticulum (ER) stress (Jiang et al., 2010) excitatory synaptic transmission (Hüls et al., 2011)

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and microglial activation using mixed cultures of neurons and glia (Zhang et al., 2005). Other studies have focused on trying to understand what changes can be triggered by alpha-synuclein aggregation in cell metabolism. Some groups have reported than promoting apoptosis, energy depriving the cells and impairing proteasome degradation, among other cell insults, increase the levels of aggregated alpha-synuclein, suggesting that maintaining cellular homeostasis is critical for alpha-synuclein (Bellucci et al., 2008; Gentile et al., 2008; Vogiatzi et al., 2008). More importantly, primary cultures have been an invaluable model to study the seeding of alpha-synuclein pathological forms and the prion-like transmission from cell to cell. One of the first reports of recombinant alpha-synuclein seeding alphasynuclein aggregation in cortical neurons is that by Danzer et al. (2009) in their study primary neuronal cultures are exposed to alpha-synuclein oligomers resulting on the accumulation of alphasynuclein inside the cells during the next 4 h. Exposure to in vitro generated alpha-synuclein fibrils has been shown to be toxic to hippocampus primary neurons (Volpicelli-Daley et al., 2011; Majd et al., 2013). Furthermore, these neurons have been used to show that pathological alpha-synuclein can be transmitted from cell to cell. Indeed, in an experiment where neuronal cells were cultured in micro-chambers and exposed to alpha-synuclein fibrils, it was possible to observe that the alpha-synuclein pathological aggregates were transmitted to neurons in a different part of the chambers connected through groves (Freundt et al., 2012). Interestingly, in addition to neurons it has been demonstrated that recombinant alpha-synuclein fibrils can also been uptaken by primary microglia and astroglia cultures causing pro-inflammatory response and cytokine release (Cao et al., 2012; Fellner et al., 2013). Alpha-synuclein seeding has also been investigated in mixed cultures of neurons and glia where different types of aggregates can be seen depending on the alpha-synuclein mutation carried on the recombinant protein to which the cells were exposed (Sacino et al., 2013). This was the first time that “strain” differences coming from wild-type and mutant alpha-synuclein forms have been reported in cell culture. Alpha-synuclein strains have been also reported by Guo et al. (2013) who have shown that two different strains are generated by serial fibrillation of wild-type alpha-synuclein in vitro. Exposure of primary hippocampal neurons to the conformationally different fibrils produced different effects. While the first samples from fibrillized alpha-synuclein promoted the formation of alphasynuclein aggregates, a later passage of the fibrillar protein had less effect on alpha-synuclein but a great effect on Tau aggregation (Guo et al., 2013). In addition to cell lines and primary cultures an exciting new model has being used, patient derived neurons. Whether they are directly reprogrammed from patient fibroblasts or derived from induced pluripotent stem cells (iPSC), they are the model closest to proper human neurons available to date. Cortical neurons derived from patients carrying mutations have been used for testing compounds against alpha-synuclein related toxicity, such as ER stress and nitrosative stress which were restored after treatment with a drug candidate selected using an alpha-synuclein toxicity yeast screen (Chung et al., 2013).iPSCderived neurons have been used from patients with Gaucher’s disease, who has been shown to often develop PD (Beavan and Schapira, 2013). In these human neurons an increase of alphasynuclein levels was found and the pathological phenotype was reverted when the mutation causing the Gaucher pathology was corrected (Schöndorf et al., 2014). Similarly, iPSCs from PD patients carrying an alpha-synuclein triplication have been shown to transmit alpha-synuclein pathology to neighbour N2a cells in a non-mixed co-culture experiment (Reyes et al., 2014). These results, not involving cell to cell contact, are in agreement with previous data from other groups showing that alpha-synuclein

aggregates are secreted by cells through exosomes into the media in a calcium-dependent manner and subsequently uptaken by naïve cells (Alvarez-Erviti et al., 2011; Emmanouilidou et al., 2010). As we have already mentioned, a similar mechanism of prion secretion through exosomes and transmission to other cells has been also described, providing yet another similarity between the two proteins. Interestingly, prion strains seem to be differently released in exosomes so it might be interesting to understand whether also different species or conformations of alpha-synuclein are released in different manners.

7. Animal models of alpha-synuclein pathology and/or spreading There is a great variety of animal models where attempts have been made to mimic PD pathology, however no model described so far presents all PD pathological features and they can be used according to the specific aspect of pathology investigated. There are models where cell death is induced using toxic substances such as MPTP (Jeon et al., 1995; Tatton and Kish, 1997), 6-OHDA (Przedborski et al., 1995; Sarre et al., 2004), and Rotenone (Betarbet et al., 2000; Inden et al., 2007; Pan-Montojo et al., 2010). These models reproduce some of the PD-like features but the speed of cell death and dysfunction leave open the question about the relevance of the toxic model in a disease that progresses in man over decades. A second type of model is the genetic one, where mutations in proteins associated with the development of PD have been expressed in transgenic mice or delivered using viral vectors. In particular several models have been produced expressing wild-type and mutant alpha-synuclein. In some mouse models high level of alpha-synuclein expression is obtained using viral vectors and achieving both alpha-synuclein accumulation and neuronal death (Decressac et al., 2012; Lauwers et al., 2003; Lo Bianco et al., 2002). Interestingly, PD features in the brain were also recapitulated by injecting a viral vector in the vagus nerve (Ulusoy et al., 2013). More stable models have been generated in transgenic mice carrying wild-type or mutated forms of human alpha-synuclein like the A53T or A30P or overexpressing human alpha-synuclein under the control of a variety of promoters (Bezard et al., 2013). In our group, based on the fact that the C-terminally truncated form of alpha-synuclein shows a faster aggregation rate in vitro (Crowther et al., 1998) a model of human 1–120 alpha-synuclein was generated. In this model, where human 1–120 alpha-synuclein is expressed under the Tyrosine hydroxylase promoter, we have reported alpha-synuclein expression and pathology in the olfactory bulb and the substantia nigra (Tofaris et al., 2006). In the striatum of these mice alpha-synuclein accumulation is associated with SNARE complex protein redistribution and deficit in dopamine release (Garcia-Reitböck et al., 2010). Using this model we have also identified a role for alpha-synuclein in dopaminergic cell development (Garcia-Reitboeck et al., 2013). Strikingly, although the transmission and spreading of alphasynuclein pathology has been a working hypothesis for many years, the definite experimental demonstration of transmission, by intracranial inoculation, date back to just 2–3 years. Baron’s group performed inoculations of pathological brain homogenate from an A53T overexpressing mouse model in their late stages of the disease into a A53T animal, where pathology had not yet developed and they observed a faster protein aggregation and degeneration in the recipient animals (Mougenot et al., 2012). This experiment although strongly suggesting that it was the misfolded alpha-synuclein the one causing the faster disease in the mice, could not prove that it was not due to any other factor in the brain homogenates.

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However, shortly after this publication and using the same A53T mouse, Luk et al. (2012b) demonstrated that misfolded alpha-synuclein was sufficient to speed up the disease. The demonstration was achieved by injecting intracranial recombinant human alpha-synuclein misfolded in vitro instead of being extracted from pathological mouse brain. Furthermore, the same group inoculated mouse recombinant alpha-synuclein in wild-type mice obtaining similar results (Luk et al., 2012a). Another group corroborated these findings by injecting misfolded human recombinant alphasynuclein into wild-type mice (Masuda-Suzukake et al., 2013) and despite the existence of some scepticism on the validity of the immunohistochemical results (Sacino et al., 2014), this study confirmed pathological features in the inoculated animals also based on behavioural changes. In addition, samples from patients that died with a diagnosis of an alpha-synucleinopathies have been proved to induce pathology when injected into the brain of transgenic and wild type animals (Masuda-Suzukake et al., 2013; Watts et al., 2013). Likewise, transmission of the alpha-synuclein pathology has been recently evidenced not only to mice but also to a species closer to humans such as rhesus monkeys. This was achieved by injecting Lewy bodies enriched preparations from PD patients and no pathology was observed when using the soluble fractions of the same preparations (Recasens et al., 2014). 8. Conclusions Up to date there is no evidence of alpha-synuclein transmission interspecies but there are definitely enough evidences to demonstrate cell to cell propagation within an organism, therefore, including alpha-synuclein under the prion denomination seems appropriate, because injection of aggregated protein leads to spreading of pathology also in wild-type models. When doing Boolean searching combining alpha-synuclein and prions there are approximately 125 articles retrieved in PubMed, and half of them are reviews. This points out to the fact that studying alpha-synuclein spreading and PD as a prion disease is a hot topic still in need of more experimental work. We believe that there are good models to work in vitro, in cell culture and in vivo, each of them with advantages and disadvantages, among them the use of patient derived neuronal cultures seems to be the one that could give results closer to the human condition and help to understand the toxic factors leading to alpha-synuclein pathology. The models should allow also answering fundamental questions regarding the progression of PD. In particular, why in a diseased person only some cells are susceptible while others are not to alpha-synuclein toxicity and maybe spreading? Or what makes a cell resistant? Identification of models where to answer to these questions will provide a system for testing compounds that would enhance the resistance or decrease the cell susceptibility to alpha-synuclein toxicity. Acknowledgements The recent work by Herva et al., mentioned in this review was supported by a grant from the Parkinson’s UK, and funding by the UK Medical Research Council. MEH is recipient of a NC3Rs David Sainsbury fellowship. References Alvarez-Erviti, L., Seow, Y., Schapira, A.H., Gardiner, C., Sargent, I.L., Wood, M.J.A., Cooper, J.M., 2011. Lysosomal dysfunction increases exosome-mediated alphasynuclein release and transmission. Neurobiol. Dis. 42, 360–367.

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