==== Front Neural Regen ResNeural Regen ResNRRNeural Regeneration Research1673-53741876-7958Medknow Publications & Media Pvt Ltd India 30028312NRR-13-113610.4103/1673-5374.235013ReviewSubcellular localization of alpha-synuclein aggregates and their interaction with membranes Miraglia Fabiana 12#Ricci Alessio 1#Rota Lucia 1#Colla Emanuela Ph.D.1http://orcid.org/0000-0002-4755-7450*1 Bio@SNS Laboratory, Scuola Normale Superiore, Pisa, Italy2 Department of Pharmacy, University of Pisa, Pisa, Italy* Correspondence to: Emanuela Colla, emanuela.colla@sns.it. Author contributions: FM, AR, LR took part in the literature search, initial draft and drawn the figures. EC conceived the idea, wrote, edited and reviewed the manuscript. All authors read and approved the final manuscript. # These authors contributed equally to the study. 7 2018 13 7 1136 1144 09 5 2018 Copyright: © Neural Regeneration Research2018This is an open access journal, and articles are distributed under the terms of the Creative Commons Attribution-NonCommercial-ShareAlike 4.0 License, which allows others to remix, tweak, and build upon the work non-commercially, as long as appropriate credit is given and the new creations are licensed under the identical terms.For more than a decade numerous evidence has been reported on the mechanisms of toxicity of α-synuclein (αS) oligomers and aggregates in α-synucleinopathies. These species were thought to form freely in the cytoplasm but recent reports of αS multimer conformations when bound to synaptic vesicles in physiological conditions, have raised the question about where αS aggregation initiates. In this review we focus on recent literature regarding the impact on membrane binding and subcellular localization of αS toxic species to understand how regular cellular function of αS contributes to pathology. Notably αS has been reported to mainly associate with specific membranes in neurons such as those of synaptic vesicles, ER/Golgi and the mitochondria, while toxic species of αS have been shown to inhibit, among others, neurotransmission, protein trafficking and mitochondrial function. Strategies interfering with αS membrane binding have shown to improve αS-driven toxicity in worms and in mice. Thus, a selective membrane binding that would result in a specific subcellular localization could be the key to understand how aggregation and pathology evolves, pointing out to αS functions that are primarily affected before onset of irreversible damage. alpha-synucleinoligomersaggregatessubcellular localizationmembranes bindingParkinson's diseaseneurodegenerationalpha-synucleinopathies ==== Body Introduction Parkinson’s disease (PD) is the most frequent neurodegenerative disease among α-synucleinopathies, a family of illnesses that share as a common feature the accumulation of intracellular proteinaceous inclusions made mainly of α-synuclein (αS) [for a review (Goedert et al., 2012)]. In PD, αS inclusions are predominantly present in the soma, named Lewy bodies (LB), or in neurites, named Lewy neurites (LN), of neurons of the central nervous system. Although PD has been previously considered a motor disease, the involvement of peripheral neurons, both sympathetic and parasympathetic bearing LBs/LNs has been shown in recent years (Braak et al., 2003) and has been furthermore suggested to correlate with the presence of numerous non-motor dysfunctions, which represents an important aspect of PD symptomatology and negatively impacts the quality of life of patients. Pathological accumulation of αS inclusions has been shown to correlate with the degree of neurodegeneration and dysfunction in a variety of animal models [as example flies, worms, mice (Feany and Bender, 2000; Masliah, 2000; Lakso et al., 2003)] and it is thought to be a cardinal step in the pathogenesis of the disease. Formation of αS inclusions is a complex nucleation reaction where αS, a small soluble protein, becomes trapped in an insoluble β-sheet conformation and tightly packed in long filamentous protofibrils and fibrils (Lashuel et al., 2002; Cremades et al., 2012; Tuttle et al., 2016). Chemical and structural variables such as pH, ionic salts and point mutations can influence fibril formation (Buell et al., 2014) and intermediate multiple high molecular weight (HMW) species with different structures, defined collectively as oligomers, can form during the aggregation process, raising the issue about their relative toxicity in αS driven pathology. While for long time αS toxic species have been thought to have a cytoplasmic localization, αS ability to bind membranes and associate with cellular organelles and synaptic vesicles has prompted the question about how cellular localization impacts on pathology and whether membrane interaction influences aggregation. In this review we attempt to piece together recent findings regarding the subcellular localization of αS toxic HMW species and their relationship with biological membranes. Initially we will discuss αS conformation in native and physiological conditions as well as during aggregation and then we will focus our attention on the impact of membrane binding on αS structure and cellular localization in vitro and in vivo. Finally we will evaluate the impact of subcellular localization of αS and its membrane binding preference on αS pathology in PD. αS αS, together with β-synuclein and γ-synuclein, belongs to a family of proteins called synucleins which were discovered in 1988 (Maroteaux et al., 1988). Initially observed to localize in the nucleus and in the presynaptic terminals of neurons, αS was linked to the autosomal dominant form of PD, when a missense mutation of αS, a threonine substitution to an alanine at position 53 (A53T) was found in a family pedigree with early onset PD (Polymeropoulos, 1997). At the same year, αS was found to be the main constituent of LBs/LNs, providing strong evidence that the αS gene, mutated and wild-type isoforms, is associated to familial and sporadic PD and other α-synucleinopathies (Spillantini et al., 1997). The SNCA gene, which encodes for αS protein, in humans is located in the long arm of chromosome 4 at position 22.1. Besides the A53T mutations, which is so far the most frequent and thus better characterized (Polymeropoulos, 1997), several missense mutations linked to a genetic form of PD and dementia with LBs have been mapped in SNCA gene more recently such as A30P (Krüger et al., 1998), E46K (Zarranz et al., 2004), H50Q (Appel-Cresswell et al., 2013; Proukakis et al., 2013), G51D (Lesage et al., 2013) and A53E (Pasanen et al., 2014). Furthermore, duplication or triplication of the SNCA gene have also been found and linked to familial PD, suggesting that increasing the amount of the wild-type protein is also pathogenic (Singleton et al., 2003; Ibáñez et al., 2004). All missense mutations and amplifications of the SNCA gene were associated with a dominant inheritance and an early onset of the disease compared to the sporadic forms. Since the overexpression of wild-type or mutated αS causes neurodegeneration in different animal models [as examples (Feany and Bender, 2000; Masliah, 2000; Lakso et al., 2003)] while its ablation has little or no effect in mice (Abeliovich et al., 2000), αS toxicity has been explained through a gain-of-function mechanism in which a modified version of the protein is responsible for causing neuronal demise. Because of its presynaptic localization (Maroteaux et al., 1988) and its ability to bind biological membrane, it was proposed that αS physiological function was implicated in neurotransmission. More recent findings (Burré et al., 2010; Nemani et al., 2010; Diao et al., 2013; Wang et al., 2014), have strengthened this view and now it is largely accepted that αS can act as a molecular chaperon and promote synaptic transmission by facilitating clustering, recycling and docking of synaptic vesicles to the cell membrane. In addition αS has been involved in intracellular protein trafficking such as vesicles transport from the endoplasmic reticulum (ER) to Golgi (Cooper et al., 2006; Gitler et al., 2008; Thayanidhi et al., 2010; Oaks et al., 2013) and from the Golgi to endosomes/lysosomes (Chung et al., 2013; Volpicelli-Daley et al., 2014; Breda et al., 2015; Mazzulli et al., 2016). An active role in axonal transport has also been reported for αS in which the protein acts as a molecular dynamase, binding directly to microtubule and promoting their assembly and stability (Cartelli et al., 2016). Native αS Protein Structure The αS protein, an acidic protein of 140 amino acids with a predicted molecular weight of approximately 14 kDa, is expressed mainly in neurons and possibly oligodendrocytes of the CNS (Asi et al., 2014), but also, under physiological conditions, in the PNS, in circulating blood cells and in hematopoietic cells of the bone marrow (Nakai et al., 2007; Gardai et al., 2013). Biochemically and functionally the αS protein can be divided into three distinct regions (Figure 1): Figure 1 Human α-synuclein (αS) protein sequence. αS is a small protein of 140 amino acids where point mutations (in red) have been associated with familial forms of Parkinson’s disease (PD) (Polymeropoulos, 1997; Krüger et al., 1998; Zarranz et al., 2004; Appel-Cresswell et al., 2013; Lesage et al., 2013). The protein can be divided in three domains: an N-terminal domain (light blue), important for membrane binding; the non amyloid β-component (NAC) domain (yellow), important for fibril formation (El-Agnaf et al., 1998) and a C-terminal domain (blue) important for protein interaction. Seven 11-amino acids imperfect repeats (purple), a unique motif predicted to form α-helix and highly conserved, are also shown (George et al., 1995). This motif is located within the N-terminal domain and the NAC domain. Notably, missense point mutations that have been found thus far are all located within the N-terminal domain, suggesting that membrane binding may influence αS aggregation. i) the amphipathic N-terminal domain (residues 1–60), which interacts with phospholipid membranes and micelles; ii) the hydrophobic non amyloid β-component (NAC) of Alzheimer’s disease (AD) (residues 61–95), which plays a strong role in αS self-aggregation (El-Agnaf et al., 1998); iii) the acidic C-terminal domain (96–140), a major site for post translational modifications, protein truncation (Li et al., 2005) and interaction with modulators of αS aggregation such as metal cations (Binolfi et al., 2006). The overall protein contains seven imperfect 11-residues repeats with a conserved KTKEGV sequence: four included in the N-terminal region and three in the NAC core (George et al., 1995). Soluble cytosolic αS has been described as an intrinsically disorder protein due to an unfolded native conformation (Weinreb et al., 1996). In fact, although αS, purified from mouse brain by gel-filtration, elutes as a single peak with an apparent molecular mass of 63 kDa, close to a folded tetramer, mass spectrometry and circular dichroism analysis reveal a monomer conformation with a single mass of 17 kDa (larger than the expected size, probably due to an in vivo N-terminal acetylation) (Burré et al., 2013). In agreement with these latest data, NMR studies show how acetylated αS, which is the predominant form in physiological conditions, is a disordered monomer but adopts a more compact conformation in solution that shields the NAC domain from other interaction in the cytosol (Theillet et al., 2016). Thus the higher molecular mass obtained previously in native conditions after gel filtration was associated with αS’s tendency to adopt an extended conformation, thereby yielding a larger mass, rather than a tetramer structure. The unfolded and disordered monomer conformation was confirmed in rat, human brain and erythrocytes isolated under denaturing and non-denaturing conditions as well as in bacteria expressed αS, while no oligomer species were found (Fauvet et al., 2012) under physiological conditions. In contrast with previous evidence, however, Bartels and collaborators have shown how αS extracted in non-denaturating conditions and upon crosslinking in living cells (i.e., human erythrocytes, cell lines and brain tissue), is mainly a metastable homo-tetramer of 58 kDa. This αS tetramer is in a dynamic equilibrium with the unfolded monomer, which on the contrary is more susceptible to aggregation (Bartels et al., 2011). Under conventional extraction protocols, the 58 kDa tetramer disappears resulting in an increase in monomer concentration. Interestingly in the same study 80–100 kDa αS homo-oligomers (i.e., hexamers and octamers) were also detected together with the tetramer in native conditions. A homo-tetramer structure in physiological conditions was also suggested by an independent study (Wang et al., 2011), which found how subunits in the αS tetramer are held together by hydrophobic interactions and each subunit is characterized by two transient α-helices structure in the first 100 N-terminal residues, followed by a disordered C-terminal region. Thus it was postulated that αS tetramer and monomer would co-exist in native conditions and any perturbation of this dynamic equilibrium with an increased accumulation of the monomer would be associated with aggregation and pathology. In agreement with this, it was found that certain missense mutations could decrease the tetramer:monomer ratio and initiate neurotoxicity (Dettmer et al., 2015a, b). Although the tetramer model was and is still widely debated, more recent work by Burré et al. (2014) showed how αS binds synaptic vesicles in vivo not as a monomer but in a folded α-helical multimer conformation, larger than an octamer. This conformation has a defined structural orientation and occurs only upon binding with vesicles that are docked at the cell membrane. In accordance with this finding, Bartels et al. (2011) described how the αS tetramer isolated upon crosslinking from human erythrocytes had a greater lipid-binding ability than the single monomer although the NMR structure obtained by Wang and coworkers did not show any phospholipid molecule (Wang et al., 2011). Thus while tetramer and membrane-bound multimer might be in reality part of the same complex, more evidence is necessary to fully understand the physiological structure of αS. αS Aggregation Besides the controversy about αS native physiological state, it is known that transition to an aggregated β sheet conformation is the necessary step for the formation of insoluble inclusions or LBs. In its amyloid form, αS monomers form antiparallel in-register β-sandwich fold, which in turn stack into a parallel arrangement forming the fibril protofilament (Vilar et al., 2008; Tuttle et al., 2016). Protofilaments further assemble into fully mature fibrils. The aggregation process (summarized in Figure 2) is a nucleation-type reaction thought to occur in a sequential series of steps, even though “ramifications” of this path are likely to occur. The in vitro characterization of the fibrillation process revealed a precise time course, with an initial lag phase, in which the monomers convert into an oligomer-type of conformation (nucleation), a growth phase and a steady state that terminates with the accumulation of α-sheet rich amyloid fibrils (Cremades et al., 2012). Oligomers are defined in general as low-molecular weight aggregates, soluble or insoluble, that have not acquired a fibrillary organization. Once the seeds are formed, αS fibrils are believed to grow through the addition of monomers rather than oligomers (Buell et al., 2014). At least two different aggregate polymorphs, fibrils and ribbons, that present different biochemical and seeding properties, have been described in vitro, depending on the aggregation protocol used (Bousset et al., 2013; Guo et al., 2013). Figure 2 α-Synuclein (αS) fibrils formation. In physiological conditions, αS has been reported to be a highly disordered monomer in a dynamic equilibrium with a multimer conformation when bound to synaptic vesicles (Weinreb et al., 1996; Burré et al., 2010, 2014; Fauvet et al., 2012; Theillet et al., 2016). Others have suggested that αS native structure is a homo-tetramer and dissolution of this latest conformation gives rise to an increase of the monomer form that drives pathology (Bartels et al., 2011; Wang et al., 2011; Dettmer et al., 2015a, b). Apart from its physiological state, it is still unclear which conformation is more susceptible to aggregation. Formation of αS fibrils is a nucleation reaction in which soluble αS monomer is converted to an insoluble β-sheet rich structure, tightly stacked in a parallel configuration, that give rise to protofibrils and fibrils (Lashuel et al., 2002; Vilar et al., 2008; Tuttle et al., 2016). During this process, a heterogeneous population of intermediate configurations, collectively called oligomers, has been described in vitro (Cremades et al., 2012; Chen et al., 2015). αS fibrils obtained with in vitro fibrillation can have a ribbon or a fibril structure (Bousset et al., 2013; Guo et al., 2013). Both oligomers and fibrils can be toxic though with different mechanisms of pathology (Danzer et al., 2007; Peelaerts et al., 2015; Fusco et al., 2017). Extensive literature has focused on the role of αS oligomers and aggregates in PD pathology. Despite the presence of fibrillar αS in LBs strongly suggests an involvement of the aggregation process in α-synucleinopathies, it has been proposed that fibrils formation could constitute an innocuous by-product or even a neuroprotective response. For instance, LBs deposition is not always associated with neurological symptoms (Braak et al., 2003), whereas in some forms of familial PD there are no signs of αS aggregation (Schneider and Alcalay, 2017). However, Peelaerts et al. (2015) showed that all the in vitro-generated αS aggregates (fibrils and ribbons) are potentially toxic and can elicit distinct histopathological phenotypes, posing a structural base for heterogeneity among α-synucleinopathies. Just as with αS fibrils, multiple forms of oligomers have been described in vitro, differing in size and morphology, including spherical, annular and tubular structures (Lashuel et al., 2002). Some of them are described as on-fibrillization pathway, while others generate amorphous, nonfibrillar assemblies. Since the fibrillation process can be influenced by numerous factors, including protein concentration, specific physicochemical conditions, the presence of certain ligands (including dopamine) and cross-linking (Buell et al., 2014), it is still unclear whether this heterogeneity in the oligomers population is due to the aggregation protocol used or has physiological relevance. More recent data obtained by directly following the oligomerization reaction using single molecule fluorescence technique showed how the oligomers population is mainly composed of two different species, named type A and type B, that form during early stages of aggregation (Cremades et al., 2012; Chen et al., 2015). These two oligomer populations seem to differ for chemical, structural and toxic properties. Type B is more resistant to protease K digestion than type A and requires a longer lag time for formation, suggesting that these species could derive from the conversion and rearrangement of type A oligomer. In addition, type B has a higher content in β-sheet structures that is instead negligible in type A (Fusco et al., 2017). In vivo, type B oligomers were shown to induce cell death in neuronal cells, via disruption of cellular ion homeostasis and production of reactive oxygen species with concomitant mitochondrial dysfunction (Danzer et al., 2007; Fusco et al., 2017), while type A were able to enter cells and induce intracellular aggregation, leading indirectly to cell death. Recently the structural basis of these different mechanisms of pathogenicity has been investigated by Fusco et al., who showed that while both oligomers bind biological membranes, only type B form a rigid β-sheet core that is able to insert into the lipid bilayer and induce directly membrane disruption and cellular toxicity (Fusco et al., 2017). Thus based on this model, both αS oligomers and aggregates are toxic. On-pathway type A oligomers are converted in compact protofibrils and fibrils and are responsible for seeding formation of new aggregates and propagate the αS pathology, while off-pathway type B oligomers are still largely detrimental, acting directly on biological membranes but do not self-propagate. In agreement with this, our group found that microsomes-associated αS toxic species behaved differently, in terms of seeding abilities of intracellular aggregates, when isolated from diseased αS transgenic (Tg) mice as opposed to aged presymptomatic littermates, suggesting the presence in vivo of at least two types of αS HMW species depending on the stage of αS pathology (Colla et al., 2018). For instance when isolated from presymptomatic mice, microsomes-associated αS oligomers induce cell death of primary neurons without seeding the formation of intracellular aggregates as opposed to microsomes-associated αS species (probably a mixture of oligomers and aggregates) isolated from diseased mice that had both properties. Thus the heterogeneity of αS toxic species, that is coming to light with recent findings shows a complicated picture of αS aggregation in which both aggregates and oligomers are toxic for cellular functions and the biochemical and functional diversity of αS toxic species is pathologically translated in at least two different and interconnected mechanisms of disease. It becomes evident that targeting one single HMW species of αS is not sufficient to stop successfully aggregation and αS-driven neurodegeneration. αS Binding to Biological Membranes and Subcellular Localization αS is known to bind lipids and biological membranes in vitro and in vivo. But does it bind them with the same efficiency? The answer is probably no. Physical properties and chemical composition of biological membranes or presence of cationic compounds able to bind lipids (Perni et al., 2017) dictate the strength of αS binding. In vitro αS binding preference is toward membranes composed of negatively charged phospholipids [such as phosphatidylethanolamine (PE), phosphatidic acid (PA) and phosphatidylinositol (PI) over phosphatidylserine (PS), or phosphatidylcholine (PC) (Middleton and Rhoades, 2010; Galvagnion et al., 2016)] or containing lipid packing defects, such as cone-head phospholipids (Ouberai et al., 2013). In addition, αS senses membrane curvature, preferring to bind to small, highly curved vesicles such as synaptic vesicles, rather than large multilamellar bodies (Middleton and Rhoades, 2010). Membrane interaction is mediated by the αS N-terminal that, upon binding, undergoes a conformational transition from random coil to α-helix, adopting a long extended one-single α-helix (Ferreon et al., 2009) in the presence of big vesicles (diameter larger than 100 nm) or two anti-parallel curved α-helices linked with a short residues chain (Chandra et al., 2003) in the case of small vesicles. In both conformations the amphipathic helices insert between the lipids polar groups at a depth corresponding to that of the glycerol group (Fusco et al., 2016). While both structures seem interchangeable and physiologically relevant (Georgieva et al., 2010), it is not clear if other conditions in vivo, besides vesicles size, would dictate a conformational change toward one structure over the other. Also, of note, is that part of the N-terminal αS region binding lipids contains the NAC domain, which is responsible for αS fibril formation (El-Agnaf et al., 1998). Interaction with membranes is known to modify not only αS conformation, but also the membranes’ physical properties, e.g., inducing changes in melting temperature (Galvagnion et al., 2016) and membrane remodelling (Jiang et al., 2013) such as lateral expansion of membrane lipids and lipid packing modifications (Ouberai et al., 2013). In addition αS binding to membrane promotes clustering of synaptic vesicles (Diao et al., 2013). How does αS preference for membranes translate in a cell context, in vivo? Physiologically, αS is believed to shift between a free, cytosolic and a membrane-bound state in a dynamic equilibrium with the membrane-bound state accounting for 10–15% of the total protein amount. In line with this, membranes of specific organelle such as the mitochondria and the ER, and synaptic vesicles have been shown to be associated at different extent with αS (Figure 3). Figure 3 Influence of subcellular localization on α-synuclein (αS) oligomers/aggregates toxicity. αS has been found to selectively bind to synaptic vesicles, endoplasmic reticulum (ER)/Golgi and the mitochondria and membrane binding seems to be part of its physiological function (Devi et al., 2008; Nakamura et al., 2008; Burré et al., 2010; Colla et al., 2012a). Although it is still unclear if association with membranes interferes with or accelerates αS aggregation, accumulation of αS toxic species selectively along these subcellular locations can primarily impact specific cellular functions such as neurotransmission, protein trafficking and mitochondrial respiration directly linked to the above-mentioned organelles. αS can bind synaptic vesicles at the synapses and this binding is believed to mediate its synaptic function in neurotransmission. For instance, αS acts as a molecular chaperon to promote SNARE-complex assembly (Burré et al., 2010), which is necessary to regulate docking of synaptic vesicles to the cell membrane. Vesicle binding is mediated not only by the interaction with acidic phospholipids such as PE, PC, PS, or cholesterol, of which the synaptic vesicles are rich, but also by specific proteins such as SNARE-protein synaptobrevin-2/vesicle-associated membrane protein 2 (VAMP2) (Diao et al., 2013). αS interaction with synaptic vesicles that occurs through a multimer conformation, promotes vesicular clustering and thus reducing synaptic vesicles trafficking and recycling (Wang et al., 2014). Moreover, αS has been found to be associated with the ER and Golgi in mice and human cells cultures (Colla et al., 2012a). Protease K sensitivity assays have shown how microsomes-associated αS is partially protected from digestion, therefore associating with the lumenal side of the microsomes (a membrane fraction including ER/Golgi and synaptic vesicles) in mice and human cell lines (Lee, 2005; Colla et al., 2012a). While no lipid binding involvement has been described yet, αS was found to bind, in αS Tg mice and human cell lines overexpressing αS, to gpr78/binding immunoglobulin protein (BIP), an ER chaperone bound to the luminal side of the ER, transiently associated with the ER translocon import pore, and directly implicated as a sensor of protein misfolding and initiator of the unfolded protein response (Bellucci et al., 2011; Colla et al., 2012a). Moreover overexpression of αS was shown to impair vesicular trafficking at the ER-Golgi level in yeast and other organisms (Cooper et al., 2006) causing accumulation of ER proteins with induction of ER stress, Golgi fragmentation and depletion of lysosomal enzymes (Oaks et al., 2013; Mazzulli et al., 2016). Interestingly, this transport defect was rescued by overexpression of proteins implicated in vesicles transit from the ER to the cell membrane such as Rab1 (ER-Golgi), Rab8 (Golgi) and Rab3A (post-Golgi) (Gitler et al., 2008) but also from endolysosomal pathway such as Rab-11A (recycling endosomal) (Breda et al., 2015), implicating a major role for αS in vesicle trafficking and recycling, outside the synapses. In addition, αS has been found to bind mitochondrial outer and inner membrane [(Devi et al., 2008; Nakamura et al., 2008) and our lab (unpublished results)]. Since most of the data were obtained from in vivo observations, it is not clear whether this binding was based on lipids, according to αS preference to cardiolipin, which is rich in the mitochondria membranes, or was also mediated by specific proteins. Interestingly a translocase of the mitochondria outer membrane has been described as responsible for the import of αS into the mitochondria and one of its subunit, TOM20, has been shown to bind αS in vivo (Di Maio et al., 2016). Moreover overexpression of αS was found to promote mitochondria dysfunction in αS Tg mice (Martin et al., 2006, 2014) and mitochondria fragmentation in vitro and in primary neurons. This last effect was dependent on the direct interaction of αS with mitochondria since disruption of αS N-terminal membrane-binding domain, rescued mitochondria morphology (Nakamura et al., 2011). Impact of Membrane Binding and Subcellular αS Localization In Vivo on αS Pathology While αS cytosolic and membrane bound-state are both physiologically relevant, it still unclear how their localization affects αS pathology and where aggregation initiates. Accumulating evidence has shown that membrane binding and lipid interaction can stimulate but also attenuate αS fibrillation (Narayanan and Scarlata, 2001; Lee et al., 2002; Jo et al., 2004; Burre et al., 2015; Galvagnion et al., 2016). In line with this controversial aspect is the observation that point mutations in αS associated with familial PD are located in the N-terminal lipid-binding domain, suggesting that lipid binding can be implicated in αS acquired pathogenicity. This is somewhat true for some point mutations such as A30P, where membrane binding is reduced while aggregation increased, but not others. In fact pathological amino acid substitutions in αS such as A53T, E46K and H50Q, lead to an increase in fibril formation without affecting lipid binding (Bussell and Eliezer, 2004; Fredenburg et al., 2007; Khalaf et al., 2014) while other mutations such as G51D, attenuate both membrane binding and aggregation (Fares et al., 2014). Thus while membrane binding and aggregation may not always be directly correlated, other factors, besides point mutations, such as intramolecular interaction between the N and C termini or protein binding to the C-terminal can influence the propensity of αS to aggregate and compensate for amino acid substitutions (Ulrih et al., 2008; Burré et al., 2010). In addition the presence of oxidative stress-induced posttranslational modifications of αS [such as nitrosylation, metal ion-catalyzed oxidation and presence of dopamine (or its oxidative metabolites) adducts] has been shown to increase oligomerization and, possibly, influence αS ability to bind vesicle membranes as a monomer or in an oligomer conformation (Binolfi et al., 2006; Xiang et al., 2013; Follmer et al., 2015; Plotegher et al., 2017). More recently, two independent papers proposed how two different small compounds, one of synthesis, NPT100-18A and the other naturally produced, squalamine, could reduce αS oligomers/aggregates content and subsequently rescue behavioural deficits in mice and worms, by interfering with αS binding to membranes (Wrasidlo et al., 2016; Perni et al., 2017). Notably, while NPT100-18A and squalamine have a different origin, they both bind only to membrane-associated αS, inducing a rearrangement of the protein structure that would lead to a displacement from the lipids, therefore reducing the amount of aggregation-prone αS. Thus while these results greatly suggest that aggregation might initiate from a pathological conversion of the membrane-bound αS fraction, because αS membrane-binding is required for αS function, development of therapeutic strategy that would block aggregation by interfering with αS membrane binding has to be taken with caution. In vivo subcellular localization of αS and association with specific membranes can determine pathobiology processes connected to aggregation and neuronal degeneration. Our group and others (Colla et al., 2012a, b; Fagerqvist et al., 2013) have shown that αS aggregates can be selectively associated with the secretory pathway including the ER and Golgi, in pathogenic conditions in αS Tg mice but not with other organelles such as the mitochondria. Notably in absence of pathology, aged Tg mice already accumulated specifically oligomer species associated with the ER, Golgi and synaptic vesicles before neuronal degeneration appearance. When compared to other αS species that co-precipitate at lower g, microsomes-associated αS oligomers (i.e., HMW species associated with membranes from the secretory pathway) obtained from healthy aged Tg mice with no overt accumulation of αS insoluble aggregates, were increasingly harmful and able to induce apoptosis, after exogenous administration to murine neuronal cultures (Colla et al., 2018). Additionally, αS oligomers have been found to decrease axonal transport and influence microtubule stability, a condition that could exacerbate synaptic vesicles traffic dysfunction (Prots et al., 2013). At the synapse, exogenous administration of large oligomers of recombinant αS was shown to bind synaptic vesicles through synaptobrevin-2 causing vesicle docking inhibition to the membrane (Choi et al., 2013) and to lower synapsin-I/II abundance (Larson et al., 2017). Although direct measurement of ER-Golgi traffic was not assessed in these conditions, it is plausible that accumulation of toxic species of αS might affect the whole protein transport system from the ER to the membrane. Moreover, electrophysiology studies showed that αS oligomers impair long-term potentiation (Diogenes et al., 2012; Martin et al., 2012) and reduces neuronal excitability (Kaufmann et al., 2016). αS oligomers have been shown to be particularly toxic to mitochondria. Mitochondrial damage such as inhibition of complex I activity with subsequently increase of reactive oxygen radical production and oxidative stress (Devi et al., 2008; Cremades et al., 2012), alteration of membrane potential and Ca2+ homeostasis, induction of mitochondrial fragmentation (Nakamura et al., 2011), mitochondrial protein import impairment (Di Maio et al., 2016), and, more recently externalization of cardiolipin to the outer mitochondrial membrane, a process that stimulates mitophagy in response to cellular stress (Ryan et al., 2018), has been associated with accumulation of toxic αS. High-ordered αS structure such as a small oligomer but not the monomer was found to be able to associate and cluster artificial mitochondria and induced fragmentation, a process similar to that described in the case of synaptic vesicles (Diao et al., 2013). In addition cardiolipin has been shown to associate and promote refolding of αS fibrils in vitro, a process negatively affected and reduced by the presence of PD-related αS point mutations (Ryan et al., 2018). Because of most of those evidence were obtained in vitro, it is not clear whether mitochondria could be a primary site of aggregation or if mitochondrial dysfunction, due to toxic αS, might be the result of a secondary generalized spreading of αS aggregates in the neuron. Thus while more evidence will be necessary to fully understand the influence of membrane binding on αS pathology, it is plausible to hypothesize that the initial pathologic transition of αS toward a toxic conformation might occur in proximity of the membranes in the above mentioned specific locations and then spread to other sites in neurons. Conclusions αS has been found to shift between a free native conformation and a membrane-bound state in a dynamic equilibrium. What dictates this transition is not clear but αS has been found to associate with specific membranes in neurons such as that of synaptic vesicles and some cellular organelles, like the ER/Golgi and the mitochondria. Although it is still not clear whether membrane-bound αS is more susceptible to aggregation or binding to membrane prevents the pathologic conversion to toxic species, initial phases of αS aggregation might begin selectively along those specific membranes and compromise, as has been reported, processes linked to these sites such as synaptic transmission, protein folding and trafficking, energy production. Initial damage from these sites would spread to other cellular functions, exacerbating αS aggregation and lead ultimately to neuronal demise. In line with this, compounds that would modify αS interaction with membranes might inhibit the initiation of αS aggregation and clarify whether membranes are necessary for the initiation of αS aggregation. At the same time though, a widespread inhibition of αS binding to membranes might result in a decrease in cellular functions mediated by αS, therefore the use of such strategies in mammals might be difficult and not directly result in a pathology improvement. A way to bypass this problem could be to implement strategies that would act on the initial phases of cellular dysfunction described above. New data will be necessary in the near future to clarify the impact that membrane binding and subcellular localization have on αS toxicity and to understand how to intervene in the early phases of the αS aggregation process before a generalized damage occurs. Conflicts of interest: The authors declare that they have no competing interests. Financial support: This work has been supported by the Italian Ministry of University and Research (MIUR) through the Career Reintegration grant scheme (RLM Program for Young Researcher) and from Scuola Normale Superiore. Copyright transfer agreement: The Copyright License Agreement has been signed by all authors before publication. Plagiarism check: Checked twice by iThenticate. Peer review: Externally peer reviewed. Open peer review reports: Reviewer 1: Hailong Song, University of Missouri Columbia, USA. Comments to authors: The authors provided a comprehensive review with nice illustrations and detailed explanation of some current research. The scientific significance, quality, and novelty of this review are high. In this manuscript, the authors reviewed αS’s association with membrane binding and its subcellular localization to further understand the regulation of αS contributing to PD pathophysiology. Specifically, this review included αS conformation, the impact of membrane binding on alpha-synuclein structure and cellular localization, and the subsequent alpha-synuclein pathology in PD. Overall, the authors provided a comprehensive review with nice illustrations and detailed explanation of some current research. The scientific significance, quality, and novelty of this review are high. Reviewer 2: Darrin Jason Lee, University of Toronto, Canada. Funding: This work has been supported by the Italian Ministry of University and Research (MIUR) through the Career Reintegration grant scheme (RLM Program for Young Researcher) and from Scuola Normale Superiore. ==== Refs 1 Abeliovich A Schmitz Y Fariñas I Choi-Lundberg D Ho WH Castillo PE Shinsky N Verdugo JM Armanini M Ryan A Hynes M Phillips H Sulzer D Rosenthal A Mice lacking α-synuclein display functional deficits in the nigrostriatal dopamine system Neuron 2000 25 239 252 10707987 2 Appel-Cresswell S Vilarino-Guell C Encarnacion M Sherman H Yu I Shah B Weir D Thompson C Szu-Tu C Trinh J Aasly JO Rajput A Rajput AH Jon Stoessl A Farrer MJ Alpha-synuclein p.H50Q, a novel pathogenic mutation for Parkinson’s disease Mov Disord 2013 28 811 813 23457019 3 Asi YT Simpson JE Heath PR Wharton SB Lees AJ Revesz T Houlden H Holton JL α-synuclein mRNA expression in oligodendrocytes in MSA: αSyn mRNA Expression in MSA Oligodendrocytes Glia 2014 62 964 970 24590631 4 Bartels T Choi JG Selkoe DJ α-Synuclein occurs physiologically as a helically folded tetramer that resists aggregation Nature 2011 477 107 110 21841800 5 Bellucci A Navarria L Zaltieri M Falarti E Bodei S Sigala S Battistin L Spillantini M Missale C Spano P Induction of the unfolded protein response by α-synuclein in experimental models of Parkinson’s disease: α-Synuclein accumulation induces the UPR J Neurochem 2011 116 588 605 21166675 6 Binolfi A Rasia RM Bertoncini CW Ceolin M Zweckstetter M Griesinger C Jovin TM Fernández CO Interaction of α-synuclein with divalent metal ions reveals key differences: a link between structure, binding specificity and fibrillation enhancement J Am Chem Soc 2006 128 9893 9901 16866548 7 Bousset L Pieri L Ruiz-Arlandis G Gath J Jensen PH Habenstein B Madiona K Olieric V Böckmann A Meier BH Melki R Structural and functional characterization of two α-synuclein strains Nat Commun 2013 4 2575 24108358 8 Braak H Del Tredici K Rüb U de Vos RAI Jansen Steur ENH Braak E Staging of brain pathology related to sporadic Parkinson’s disease Neurobiol Aging 2003 24 197 211 12498954 9 Breda C Nugent ML Estranero JG Kyriacou CP Outeiro TF Steinert JR Giorgini F Rab11 modulates α-synuclein-mediated defects in synaptic transmission and behaviour Hum Mol Genet 2015 24 1077 1091 25305083 10 Buell AK Galvagnion C Gaspar R Sparr E Vendruscolo M Knowles TPJ Linse S Dobson CM Solution conditions determine the relative importance of nucleation and growth processes in α-synuclein aggregation Proc Natl Acad Sci 2014 111 7671 7676 24817693 11 Burré J Sharma M Südhof TC α-Synuclein assembles into higher-order multimers upon membrane binding to promote SNARE complex formation Proc Natl Acad Sci 2014 111 E4274 E4283 25246573 12 Burre J Sharma M Sudhof TC Definition of a molecular pathway mediating α-synuclein neurotoxicity J Neurosci 2015 35 5221 5232 25834048 13 Burré J Sharma M Tsetsenis T Buchman V Etherton MR Sudhof TC Alpha-synuclein promotes SNARE-complex assembly in vivo and in vitro Science 2010 329 1663 1667 20798282 14 Burré J Vivona S Diao J Sharma M Brunger AT Südhof TC Properties of native brain α-synuclein Nature 2013 498 E4 E6 23765500 15 Bussell R Jr Eliezer D Effects of Parkinson’s disease-linked mutations on the structure of lipid-associated α-synuclein Biochemistry 2004 43 4810 4818 15096050 16 Cartelli D Aliverti A Barbiroli A Santambrogio C Ragg EM Casagrande FV Cantele F Beltramone S Marangon J De Gregorio C Pandini V Emanuele M Chieregatti E Pieraccini S Holmqvist S Bubacco L Roybon L Pezzoli G Grandori R Arnal I α-Synuclein is a novel microtubule dynamase Sci Rep 2016 6 33289 27628239 17 Chandra S Chen X Rizo J Jahn R Südhof TC A broken α-helix in folded α-Synuclein J Biol Chem 2003 278 15313 15318 12586824 18 Chen SW Drakulic S Deas E Ouberai M Aprile FA Arranz R Ness S Roodveldt C Guilliams T De-Genst EJ Klenerman D Wood NW Knowles TPJ Alfonso C Rivas G Abramov AY Valpuesta JM Dobson CM Cremades N Structural characterization of toxic oligomers that are kinetically trapped during α-synuclein fibril formation Proc Natl Acad Sci 2015 112 E1994 E2003 25855634 19 Choi BK Choi MG Kim JY Yang Y Lai Y Kweon DH Lee NK Shin YK Large α-synuclein oligomers inhibit neuronal SNARE-mediated vesicle docking Proc Natl Acad Sci 2013 110 4087 4092 23431141 20 Chung CY Khurana V Auluck PK Tardiff DF Mazzulli JR Soldner F Baru V Lou Y Freyzon Y Cho S Mungenast AE Muffat J Mitalipova M Pluth MD Jui NT Schüle B Lippard SJ Tsai LH Krainc D Buchwald SL Identification and rescue of α-synuclein toxicity in Parkinson patient-derived neurons Science 2013 342 983 987 24158904 21 Colla E Coune P Liu Y Pletnikova O Troncoso JC Iwatsubo T Schneider BL Lee MK Endoplasmic reticulum stress is important for the manifestations of α-synucleinopathy in vivo J Neurosci 2012a 32 3306 3320 22399753 22 Colla E Jensen PH Pletnikova O Troncoso JC Glabe C Lee MK Accumulation of toxic α-synuclein oligomer within endoplasmic reticulum occurs in α-synucleinopathy in vivo J Neurosci 2012b 32 3301 3305 22399752 23 Colla E Panattoni G Ricci A Rizzi C Rota L Carucci N Valvano V Gobbo F Capsoni S Lee MK Cattaneo A Toxic properties of microsome-associated α-synuclein species in mouse primary neurons Neurobiol Dis 2018 111 36 47 29246724 24 Cooper AA Gitler AD Cashikar A Haynes CM Hill KJ Bhullar B Liu K Xu K Strathearn KE Liu F Cao S Caldwell KA Caldwell GA Marsischky G Kolodner RD Labaer J Rochet JC Bonini NM Lindquist S α-synuclein blocks ER-Golgi traffic and Rab1 rescues neuron loss in Parkinson’s models Science 2006 313 324 328 16794039 25 Cremades N Cohen SIA Deas E Abramov AY Chen AY Orte A Sandal M Clarke RW Dunne P Aprile FA Bertoncini CW Wood NW Knowles TPJ Dobson CM Klenerman D Direct observation of the interconversion of normal and toxic forms of α-synuclein Cell 2012 149 1048 1059 22632969 26 Danzer KM Haasen D Karow AR Moussaud S Habeck M Giese A Kretzschmar H Hengerer B Kostka M Different species of α-synuclein oligomers induce calcium influx and seeding J Neurosci 2007 27 9220 9232 17715357 27 Dettmer U Newman AJ Soldner F Luth ES Kim NC von Saucken VE Sanderson JB Jaenisch R Bartels T Selkoe D Parkinson-causing α-synuclein missense mutations shift native tetramers to monomers as a mechanism for disease initiation Nat Commun 2015a 6 7314 26076669 28 Dettmer U Newman AJ von Saucken VE Bartels T Selkoe D KTKEGV repeat motifs are key mediators of normal α-synuclein tetramerization: Their mutation causes excess monomers and neurotoxicity Proc Natl Acad Sci 2015b 112 9596 9601 26153422 29 Devi L Raghavendran V Prabhu BM Avadhani NG Anandatheerthavarada HK Mitochondrial import and accumulation of α-synuclein impair complex i in human dopaminergic neuronal cultures and Parkinson disease brain J Biol Chem 2008 283 9089 9100 18245082 30 Di Maio R Barrett PJ Hoffman EK Barrett CW Zharikov A Borah A Hu X McCoy J Chu CT Burton EA Hastings TG Greenamyre JT α-Synuclein binds to TOM20 and inhibits mitochondrial protein import in Parkinson’s disease Sci Transl Med 2016 8 342ra78 31 Diao J Burré J Vivona S Cipriano DJ Sharma M Kyoung M Südhof TC Brunger AT Native α-synuclein induces clustering of synaptic-vesicle mimics via binding to phospholipids and synaptobrevin-2/VAMP2 eLife 2013 2 e00592 23638301 32 Diogenes MJ Dias RB Rombo DM Vicente Miranda H Maiolino F Guerreiro P Nasstrom T Franquelim HG Oliveira LMA Castanho MARB Lannfelt L Bergstrom J Ingelsson M Quintas A Sebastiao AM Lopes LV Outeiro TF Extracellular α-synuclein oligomers modulate synaptic transmission and impair LTP via NMDA-receptor activation J Neurosci 2012 32 11750 11762 22915117 33 El-Agnaf OM Bodles AM Guthrie DJ Harriott P Irvine GB The N-terminal region of non-A beta component of Alzheimer’s disease amyloid is responsible for its tendency to assume beta-sheet and aggregate to form fibrils Eur J Biochem 1998 258 157 163 9851705 34 Fagerqvist T Lindström V Nordström E Lord A Tucker SM Su X Sahlin C Kasrayan A Andersson J Welander H Näsström T Holmquist M Schell H Kahle PJ Kalimo H Möller C Gellerfors P Lannfelt L Bergström J Ingelsson M Monoclonal antibodies selective for α-synuclein oligomers/protofibrils recognize brain pathology in Lewy body disorders and α-synuclein transgenic mice with the disease-causing A30P mutation J Neurochem 2013 126 131 144 23363402 35 Fares MB Ait-Bouziad N Dikiy I Mbefo MK Jovi i A Kiely A Holton JL Lee SJ Gitler AD Eliezer D Lashuel HA The novel Parkinson’s disease linked mutation G51D attenuates in vitro aggregation and membrane binding of α-synuclein, and enhances its secretion and nuclear localization in cells Hum Mol Genet 2014 23 4491 4509 24728187 36 Fauvet B Mbefo MK Fares MB Desobry C Michael S Ardah MT Tsika E Coune P Prudent M Lion N Eliezer D Moore DJ Schneider B Aebischer P El-Agnaf OM Masliah E Lashuel HA α-Synuclein in central nervous system and from erythrocytes, mammalian cells, and Escherichia coli exists predominantly as disordered monomer J Biol Chem 2012 287 15345 15364 22315227 37 Feany MB Bender WW A Drosophila model of Parkinson’s disease Nature 2000 404 394 398 10746727 38 Ferreon ACM Gambin Y Lemke EA Deniz AA Interplay of α-synuclein binding and conformational switching probed by single-molecule fluorescence Proc Natl Acad Sci 2009 106 5645 5650 19293380 39 Follmer C Coelho-Cerqueira E Yatabe-Franco DY Araujo GDT Pinheiro AS Domont GB Eliezer D Oligomerization and membrane-binding properties of covalent adducts formed by the interaction of α-synuclein with the toxic dopamine metabolite 3, 4-dihydroxyphenylacetaldehyde (DOPAL) J Biol Chem 2015 290 27660 27679 26381411 40 Fredenburg RA Rospigliosi C Meray RK Kessler JC Lashuel HA Eliezer D Lansbury PT Jr The impact of the e46k mutation on the properties of α-synuclein in its monomeric and oligomeric states Biochemistry 2007 46 7107 7118 17530780 41 Fusco G Chen SW Williamson PTF Cascella R Perni M Jarvis JA Cecchi C Vendruscolo M Chiti F Cremades N Ying L Dobson CM De Simone A Structural basis of membrane disruption and cellular toxicity by α-synuclein oligomers Science 2017 358 1440 1443 29242346 42 Fusco G De Simone A Arosio P Vendruscolo M Veglia G Dobson CM Structural ensembles of membrane-bound α-synuclein reveal the molecular determinants of synaptic vesicle affinity Sci Rep 2016 6 27125 27273030 43 Galvagnion C Brown JWP Ouberai MM Flagmeier P Vendruscolo M Buell AK Sparr E Dobson CM Chemical properties of lipids strongly affect the kinetics of the membrane-induced aggregation of α-synuclein Proc Natl Acad Sci 2016 113 7065 7070 27298346 44 Gardai SJ Mao W Schüle B Babcock M Schoebel S Lorenzana C Alexander J Kim S Glick H Hilton K Fitzgerald JK Buttini M Chiou SS McConlogue L Anderson JP Schenk DB Bard F Langston JW Yednock T Johnston JA Elevated alpha-synuclein impairs innate immune cell function and provides a potential peripheral biomarker for Parkinson’s disease PLoS One 2013 8 1 21 45 George JM Jin H Woods WS Clayton DF Characterization of a novel protein regulated during the critical period for song learning in the zebra finch Neuron 1995 15 361 372 7646890 46 Georgieva ER Ramlall TF Borbat PP Freed JH Eliezer D The lipid-binding domain of wild type and mutant a-synuclein: compactness and interconversion between the broken and extended helix forms J Biol Chem 2010 285 28261 28274 20592036 47 Gitler AD Bevis BJ Shorter J Strathearn KE Hamamichi S Su LJ Caldwell KA Caldwell GA Rochet JC McCaffery JM Barlowe C Lindquist S The Parkinson’s disease protein alpha-synuclein disrupts cellular Rab homeostasis Proc Natl Acad Sci U S A 2008 105 145 150 18162536 48 Goedert M Spillantini MG Del Tredici K Braak H 100 years of Lewy pathology Nat Rev Neurol 2012 9 13 24 23183883 49 Guo JL Covell DJ Daniels JP Iba M Stieber A Zhang B Riddle DM Kwong LK Xu Y Trojanowski JQ Lee VMY Distinct α-synuclein strains differentially promote tau inclusions in neurons Cell 2013 154 103 117 23827677 50 Ibáñez P Bonnet AM Débarges B Lohmann E Tison F Agid Y Dürr A Brice A Pollak P Causal relation between α-synuclein locus duplication as a cause of familial Parkinson’s disease Lancet 2004 364 1169 1171 15451225 51 Jiang Z de Messieres M Lee JC Membrane remodeling by α-synuclein and effects on amyloid formation J Am Chem Soc 2013 135 15970 15973 24099487 52 Jo E Darabie AA Han K Tandon A Fraser PE McLaurin J α-Synuclein-synaptosomal membrane interactions: Implications for fibrillogenesis Eur J Biochem 2004 271 3180 3189 15265037 53 Kaufmann TJ Harrison PM Richardson MJE Pinheiro TJT Wall MJ Intracellular soluble α-synuclein oligomers reduce pyramidal cell excitability: Impact of α-synuclein on neocortical neurons J Physiol 2016 594 2751 2772 26915902 54 Khalaf O Fauvet B Oueslati A Dikiy I Mahul-Mellier AL Ruggeri FS Mbefo MK Vercruysse F Dietler G Lee SJ Eliezer D Lashuel HA The H50Q mutation enhances α-synuclein aggregation, secretion, and toxicity J Biol Chem 2014 289 21856 21876 24936070 55 Krüger R Kuhn W Müller T Woitalla D Graeber M Kösel S Przuntek H Epplen JT Schöls L Riess O Ala30Pro mutation in the gene encoding α-synuclein in Parkinson’s disease Nat Genet 1998 18 106 108 9462735 56 Lakso M Vartiainen S Moilanen A-M Sirviö J Thomas JH Nass R Blakely RD Wong G Dopaminergic neuronal loss and motor deficits in Caenorhabditis elegans overexpressing human α-synuclein J Neurochem 2003 86 165 172 12807436 57 Larson ME Greimel SJ Amar F LaCroix M Boyle G Sherman MA Schley H Miel C Schneider JA Kayed R Benfenati F Lee MK Bennett DA Lesné SE Selective lowering of synapsins induced by oligomeric α-synuclein exacerbates memory deficits Proc Natl Acad Sci 2017 114 E4648 E4657 28533388 58 Lashuel HA Petre BM Wall J Simon M Nowak RJ Walz T Lansbury PT Alpha-synuclein, especially the Parkinson’s disease-associated mutants, forms pore-like annular and tubular protofibrils J Mol Biol 2002 322 1089 1102 12367530 59 Lee HJ Intravesicular localization and exocytosis of α-synuclein and its aggregates J Neurosci 2005 25 6016 6024 15976091 60 Lee HJ Choi C Lee SJ Membrane-bound -synuclein has a high aggregation propensity and the ability to seed the aggregation of the cytosolic form J Biol Chem 2002 277 671 678 11679584 61 Lesage S Anheim M Letournel F Bousset L Honoré A Rozas N Pieri L Madiona K Dürr A Melki R Verny C Brice A for the French Parkinson’s Disease Genetics Study Group G51D α-synuclein mutation causes a novel Parkinsonian-pyramidal syndrome: SNCA G51D in Parkinsonism Ann Neurol 2013 73 459 471 23526723 62 Li W West N Colla E Pletnikova O Troncoso JC Marsh L Dawson TM Jäkälä P Hartmann T Price DL Lee MK Aggregation promoting C-terminal truncation of alpha-synuclein is a normal cellular process and is enhanced by the familial Parkinson’s disease-linked mutations Proc Natl Acad Sci U S A 2005 102 2162 2167 15684072 63 Maroteaux L Campanelli JT Scheller RH Synuclein: a neuron-specific protein localized to the nucleus and presynaptic nerve terminal J Neurosci 1988 8 2804 2815 3411354 64 Martin LJ Pan Y Price AC Sterling W Copeland NG Jenkins NA Price DL Lee MK Parkinson’s disease α-synuclein transgenic mice develop neuronal mitochondrial degeneration and cell death J Neurosci Off J Soc Neurosci 2006 26 41 50 65 Martin LJ Semenkow S Hanaford A Wong M The mitochondrial permeability transition pore regulates Parkinson’s disease development in mutant α-synuclein transgenic mice Neurobiol Aging 2014 35 1132 1152 24325796 66 Martin ZS Neugebauer V Dineley KT Kayed R Zhang W Reese LC Taglialatela G α-Synuclein oligomers oppose long-term potentiation and impair memory through a calcineurin-dependent mechanism: relevance to human synucleopathic diseases: Cognitive effects of α-synuclein oligomers J Neurochem 2012 120 440 452 22060133 67 Masliah E Dopaminergic loss and inclusion body formation in α-synuclein mice: implications for neurodegenerative disorders Science 2000 287 1265 1269 10678833 68 Mazzulli JR Zunke F Isacson O Studer L Krainc D α-Synuclein-induced lysosomal dysfunction occurs through disruptions in protein trafficking in human midbrain synucleinopathy models Proc Natl Acad Sci 2016 113 1931 1936 26839413 69 Middleton ER Rhoades E Effects of curvature and composition on α-synuclein binding to lipid vesicles Biophys J 2010 99 2279 2288 20923663 70 Nakai M Fujita M Waragai M Sugama S Wei J Akatsu H Ohtaka-Maruyama C Okado H Hashimoto M Expression of α-synuclein, a presynaptic protein implicated in Parkinson’s disease, in erythropoietic lineage Biochem Biophys Res Commun 2007 358 104 110 17475220 71 Nakamura K Nemani VM Azarbal F Skibinski G Levy JM Egami K Munishkina L Zhang J Gardner B Wakabayashi J Sesaki H Cheng Y Finkbeiner S Nussbaum RL Masliah E Edwards RH Direct membrane association drives mitochondrial fission by the Parkinson disease-associated protein α-synuclein J Biol Chem 2011 286 20710 20726 21489994 72 Nakamura K Nemani VM Wallender EK Kaehlcke K Ott M Edwards RH Optical reporters for the conformation of α-synuclein reveal a specific interaction with mitochondria J Neurosci 2008 28 12305 12317 19020024 73 Narayanan V Scarlata S Membrane binding and self-association of α-synucleins Biochemistry 2001 40 9927 9934 11502187 74 Nemani VM Lu W Berge V Nakamura K Onoa B Lee MK Chaudhry FA Nicoll RA Edwards RH Increased expression of α-synuclein reduces neurotransmitter release by inhibiting synaptic vesicle reclustering after endocytosis Neuron 2010 65 66 79 20152114 75 Oaks AW Marsh-Armstrong N Jones JM Credle JJ Sidhu A Synucleins antagonize endoplasmic reticulum function to modulate dopamine transporter trafficking PLoS One 2013 8 e70872 23967127 76 Ouberai MM Wang J Swann MJ Galvagnion C Guilliams T Dobson CM Welland ME α-Synuclein senses lipid packing defects and induces lateral expansion of lipids leading to membrane remodeling J Biol Chem 2013 288 20883 20895 23740253 77 Pasanen P Myllykangas L Siitonen M Raunio A Kaakkola S Lyytinen J Tienari PJ Pöyhönen M Paetau A A novel α-synuclein mutation A53E associated with atypical multiple system atrophy and Parkinson’s disease-type pathology Neurobiol Aging 2014 35 1 5 23954174 78 Peelaerts W Bousset L Van der Perren A Moskalyuk A Pulizzi R Giugliano M Van den Haute C Melki R Baekelandt V α-Synuclein strains cause distinct synucleinopathies after local and systemic administration Nature 2015 522 340 344 26061766 79 Perni M Galvagnion C Maltsev A Meisl G Müller MB Challa PK Kirkegaard JB Flagmeier P Cohen SI Cascella R Chen SW Limbocker R Sormanni P Heller GT Aprile FA Cremades N Cecchi C Chiti F Nollen EA Knowles TP A natural product inhibits the initiation of α-synuclein aggregation and suppresses its toxicity Proc Natl Acad Sci 2017 114 E1009 E1017 28096355 80 Plotegher N Berti G Ferrari E Tessari I Zanetti M Lunelli L Greggio E Bisaglia M Veronesi M Girotto S Dalla Serra M Perego C Casella L Bubacco L DOPAL derived α-synuclein oligomers impair synaptic vesicles physiological function Sci Rep 2017 7 40699 28084443 81 Polymeropoulos MH Mutation in the α-synuclein gene identified in families with Parkinson’s disease Science 1997 276 2045 2047 9197268 82 Prots I Veber V Brey S Campioni S Buder K Riek R Böhm KJ Winner B α-Synuclein oligomers impair neuronal microtubule-kinesin interplay J Biol Chem 2013 288 21742 21754 23744071 83 Proukakis C Dudzik CG Brier T MacKay DS Cooper JM Millhauser GL Houlden H Schapira AH A novel α-synuclein missense mutation in Parkinson disease Neurology 2013 80 1062 1064 23427326 84 Ryan T Bamm VV Stykel MG Coackley CL Humphries KM Jamieson-Williams R Ambasudhan R Mosser DD Lipton SA Harauz G Ryan SD Cardiolipin exposure on the outer mitochondrial membrane modulates α-synuclein Nat Commun 2018 9 817 29483518 85 Schneider SA Alcalay RN Neuropathology of genetic synucleinopathies with parkinsonism: review of the literature: neuropathology of genetic Parkinson‘s disease Mov Disord 2017 32 1504 1523 29124790 86 Singleton AB Farrer M Johnson J Singleton A Hague S Kachergus J Hulihan M Peuralinna T Dutra A Nussbaum R Lincoln S Crawley A Hanson M Maraganore D Adler C Cookson MR Muenter M Baptista M Miller D Blancato J α-Synuclein locus triplication causes parkinson’s disease Science 2003 302 841 14593171 87 Spillantini MG Schmidt ML Lee VM Trojanowski JQ Jakes R Goedert M α-synuclein in Lewy bodies Nature 1997 388 839 840 9278044 88 Thayanidhi N Helm JR Nycz DC Bentley M Liang Y Hay JC Alpha-synuclein delays endoplasmic reticulum (ER)-to-Golgi transport in mammalian cells by antagonizing ER/Golgi SNAREs Mol Biol Cell 2010 21 1850 1863 20392839 89 Theillet F-X Binolfi A Bekei B Martorana A Rose HM Stuiver M Verzini S Lorenz D van Rossum M Goldfarb D Selenko P Structural disorder of monomeric α-synuclein persists in mammalian cells Nature 2016 530 45 50 26808899 90 Tuttle MD Comellas G Nieuwkoop AJ Covell DJ Berthold DA Kloepper KD Courtney JM Kim JK Barclay AM Kendall A Wan W Stubbs G Schwieters CD Lee VMY George JM Rienstra CM Solid-state NMR structure of a pathogenic fibril of full-length human α-synuclein Nat Struct Mol Biol 2016 23 409 415 27018801 91 Ulrih NP Barry CH Fink AL Impact of Tyr to Ala mutations on α-synuclein fibrillation and structural properties Biochim Biophys Acta BBA-Mol Basis Dis 2008 1782 581 585 92 Vilar M Chou H-T Luhrs T Maji SK Riek-Loher D Verel R Manning G Stahlberg H Riek R The fold of α-synuclein fibrils Proc Natl Acad Sci 2008 105 8637 8642 18550842 93 Volpicelli-Daley LA Gamble KL Schultheiss CE Riddle DM West AB Lee VM Formation of α-synuclein Lewy neurite-like aggregates in axons impedes the transport of distinct endosomes Mol Biol Cell 2014 25 4010 4023 25298402 94 Wang L Das U Scott DA Tang Y McLean PJ Roy S α-Synuclein multimers cluster synaptic vesicles and attenuate recycling Curr Biol 2014 24 2319 2326 25264250 95 Wang W Perovic I Chittuluru J Kaganovich A Nguyen LT Liao J Auclair JR Johnson D Landeru A Simorellis AK Ju S Cookson MR Asturias FJ Agar JN Webb BN Kang C Ringe D Petsko GA Pochapsky TC Hoang QQ A soluble α-synuclein construct forms a dynamic tetramer Proc Natl Acad Sci U S A 2011 108 17797 17802 22006323 96 Weinreb PH Zhen W Poon AW Conway KA Lansbury PT NACP, a protein implicated in Alzheimer’s disease and learning, is natively unfolded Biochemistry 1996 35 13709 13715 8901511 97 Wrasidlo W Tsigelny IF Price DL Dutta G Rockenstein E Schwarz TC Ledolter K Bonhaus D Paulino A Eleuteri S Skjevik ÅA Kouznetsova VL Spencer B Desplats P Gonzalez-Ruelas T Trejo-Morales M Overk CR Winter S Zhu C Chesselet MF A de novo compound targeting α-synuclein improves deficits in models of Parkinson’s disease Brain 2016 139 3217 3236 27679481 98 Xiang W Schlachetzki JCM Helling S Bussmann JC Berlinghof M Schäffer TE Marcus K Winkler J Klucken J Becker CM Oxidative stress-induced posttranslational modifications of a-synuclein: Specific modification of alpha-synuclein by 4-hydroxy-2-nonenal increases dopaminergic toxicity Mol Cell Neurosci 2013 54 71 83 23369945 99 Zarranz JJ Alegre J Gómez-Esteban JC Lezcano E Ros R Ampuero I Vidal L Hoenicka J Rodriguez O Atarés B Llorens V Gomez Tortosa E Del Ser T Muñoz DG De Yebenes JG The new mutation, E46K, of α-synuclein causes Parkinson and lewy body dementia Ann Neurol 2004 55 164 173 14755719