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.

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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 fl ies, 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 fi lamentous proto fi brils and fi brils (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 in fl uences aggregation.

In this review we attempt to piece together recent fi ndings 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).

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 fi bril 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 in fl uence αS aggregation.

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 ampli fi cations 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 modi fi ed version of the protein is responsible for causing neuronal demise.

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).

Native αS Protein Structure

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 fi ndings (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).

Biochemically and functionally the αS protein can be divided into three distinct regions (Figure 1):

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 modi fi cations, protein truncation (Li et al., 2005) and interaction with modulators of αS aggregation such as metal cations (Binol fi et al., 2006).

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 fi bril proto fi lament(Vilar et al., 2008; Tuttle et al., 2016). Proto fi laments further assemble into fully mature fi brils.

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- fi ltration, 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 con-formation 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 fi rst 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 de fi ned structural orientation and occurs only upon binding with vesicles that are docked at the cell membrane. In accordance with this fi nding, 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

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).

The aggregation process (summarized in Figure 2) is a nucleation-type reaction thought to occur in a sequential series of steps, even though “rami fi cations” of this path are likely to occur. The in vitro characterization of the fi brillation 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 defi ned in general as low-molecular weight aggregates, soluble or insoluble, that have not acquired a fi brillary 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, fi brils 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).

Extensive literature has focused on the role of αS oligomers and aggregates in PD pathology. Despite the presence of fi brillar 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 ( fi brils 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- fi brillization pathway, while others generate amorphous, non fi brillar assemblies. Since the fi brillation process can be in fl uenced by numerous factors, including protein concentration, speci fi c 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 fl uorescence 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 fi ndings 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.

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稻米的品质，除受品种的遗传特性支配外，外界环境如气候、土壤、肥料及栽培措施等也有一定的影响[9]。因此，良好的栽培技术，不仅可使优质稻米的品质不致变劣，而且在某些情况下也有可能使品质有所提高。在施用公主岭霉素防治水稻稻瘟病的同时，稻米的品质也发生了改变。施用公主岭霉素的处理，稻米的外观品质、食味品质和营养品质均有一定程度的提升。该研究中，施用公主岭霉素后稻米出糙率降低，不完善粒降低，食味值提升，外观性状提升，硬度降低，黏度升高，平衡度升高，脂肪含量降低，直链淀粉含量提高(表5)。

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 effi-ciency? 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 fi bril 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).

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 speci fi c 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 speci fi c membranes and compromise, as has been reported, processes linked to these sites such as synaptic transmission, protein folding and traffi cking, 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.

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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 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).

四是增强应对强大网络安全事故的防控能力。在网络攻击中，谁是真正的入侵者，谁是真正的受害者，受害程度究竟有多大，以目前技术而言很难确定。但是，美国将网络攻击与战争画上等号，并与其盟国不时把一些“黑客入侵”、“网络间谍”事件与中国挂钩，我们未来面临“互联网战争”的风险进一步加大。首先我们要建立国家级网络安全危机管理机制。其次，要继续推动中美网络安全合作与对话机制的发展。

Figure 2 α-Synuclein (αS) fi brils 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 fi brils is a nucleation reaction in which soluble αS monomer is converted to an insoluble β-sheet rich structure, tightly stacked in a parallel con fi guration, that give rise to proto fi brils and fi brils(Lashuel et al., 2002; Vilar et al., 2008; Tuttle et al., 2016). During this process, a heterogeneous population of intermediate con fi gurations, collectively called oligomers, has been described in vitro (Cremades et al., 2012; Chen et al., 2015). αS fi brils obtained with in vitro fi brillation can have a ribbon or a fi bril structure (Bousset et al., 2013; Guo et al., 2013). Both oligomers and fi brils can be toxic though with different mechanisms of pathology (Danzer et al., 2007; Peelaerts et al., 2015; Fusco et al., 2017).

Figure 3 In fl uence 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.

地震具有强大的破坏性,对电力设施的损坏十分严重,造成极大的生命财产损失[5-7].在2008年四川汶川发生的大地震中,输电线路的主要破坏表现为滑坡、滚石等造成的输电线路保坎受损、塔身砸坏、变形、倾斜和倾倒,多条线路由于地震导致故障跳闸、停运.其中,110 kV线路倒塔20多基,局部受损16基;500 kV茂谭线8基、220 kV茂永线2基铁塔因滑坡损毁;同时,茂县山区的220 kV线路所经地形发生巨大变化,16基铁塔全部损毁[8-11].

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 speci fi c 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 fi brillation (Narayanan and Scarlata, 2001; Lee et al., 2002; Jo et al.,2004; Burre et al., 2015; Galvagnion et al., 2016).

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 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 fi bril 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, in fl uence αS ability to bind vesicle membranes as a monomer or in an oligomer conformation (Binol fi et al., 2006; Xiang et al.,2013; Follmer et al., 2015; Plotegher et al., 2017).

2011年11月1日起开始施行的《太湖流域管理条例》（以下简称《条例》）是我国第一部关于重要江河湖泊流域综合管理和保护的行政法规，标志着太湖流域综合治理工作纳入了法制轨道，为保障太湖流域防洪安全、供水安全、生态安全，推动流域经济社会持续发展提供有力的法制支撑。日前，本刊记者就《条例》的贯彻实施情况采访了水利部太湖流域管理局叶建春局长。

2.4 Logistic回归分析 对死亡与存活病例间有统计学差异的变量进行Logistic回归分析。结果显示：血小板计数是脓毒症患者死亡的独立预测因素(OR=1.010，P=0.000)。

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 de fi cits 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 speci fi cally 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).

How does αS preference for membranes translate in a cell context, in vivo? Physiologically, αS is believed to shiftbetween 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 speci fi c organelle such as the mitochondria and the ER, and synaptic vesicles have been shown to be associated at different extent with αS (Figure 3).

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 arti fi cial 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 fi brils 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 in fl uence 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 speci fi c locations and then spread to other sites in neurons.

制定激励青年教师进行信息技术学习的政策 各省市、地区、学校可以针对青年教师培训提供一些奖励方法，调动青年教师参与信息技术教师网络研修社区，提高参与技术支持的专业发展活动的积极性[7]。难免有一些青年教师抱着无所谓的态度参加培训，认为培训的结果不会对他们的教学产生影响，从而让培训流于形式。对此，相关管理部门可以制定一系列激励制度，激起青年教师学习信息技术的热情，引起他们的兴趣，更好地提高信息技术应用能力。■

Conclusions

清末在新式教育起步时，浙江钱塘县私塾俞氏就敏感地意识到时势的变化，劝导自己的学生骆憬甫：“现在的时代，光光做策论是不够的。英文、算学、物理、化学、地理、历史、体操、图画等，家塾哪里学得到？而且也请不到这样多才多艺的名师。……我刚从杭州回来，知道安定学堂、杭州府中学堂都在招考，这是一个绝好的机会。……奉劝你俩赶快去报名投考，勿再过家塾生活以埋没一世！”[16]经历了两次乡试失败的骆憬甫在老师劝导下，在1905年秋考入了杭州府中学堂，开始了他的人生转折。

Author contributions: FM, AR, LR took part in the literature search,initial draft and drawn the fi gures. EC conceived the idea, wrote, edited and reviewed the manuscript. All authors read and approved the fi nal manuscript.

Con fl icts 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.

在光华闪耀的粉蓝色或棕色珍珠母贝星空中，白色珍珠母贝月亮展现从新月到满月的各种形态。两轮月亮在6点钟位置的镂空拱形钻石格栅下方旋转，如同捉迷藏一般，玩趣十足。格栅由九道金质镶钻轮幅组成，开放区域显示当前月相。钻石格栅的整个表面细密镶嵌93颗圆形明亮式切工钻石，中央点缀一颗醒目钻石。鉴于空间极小，爪镶难以实现，精细微镶再次彰显海瑞温斯顿宝石镶嵌师的高超技艺。

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Peer review: Externally peer reviewed.

Open access statement: This is an open access journal, and articles are distributed under the terms of the Creative Commons Attribution-Non-Commercial-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.

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式中，A为车体的振动加速度；f为车体的振动频率；F(f)为与振动频率有关的修正系数，详见文献[9]。经过计算，将不同车速交会下的车辆运行横向与垂向平稳性指标列于表3。由计算结果可知，在各种工况下的车辆横向平稳性指标均大于垂向平稳性指标，说明车体的横向振动受到会车流场的影响较垂向振动更大。

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 scienti fi c signi fi cance, quality, and novelty of this review are high.

Reviewer 2: Darrin Jason Lee, University of Toronto, Canada.

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