α-synuclein is a small, highly abundant, and highly conserved presynaptic protein with intimate links to many neurodegenerative diseases and is one of the more intensively studied human proteins. In fact, since its discovery in 1991 ¹ , followed by the demonstration of its natively unfolded nature ² , and especially after finding in 1997 a potential relationship between α-synuclein aggregation and the pathology of Parkinson’s disease (PD) ^{3, 4} , this protein has attracted the close attention of many researchers specializing in various scientific areas. According to the Web of Science database, as of 17 February 2017, there were 15,367 publications about this protein, reflecting collective efforts of 37,066 researchers affiliated with 6,326 organizations from 93 countries/territories, and more than 3,140 papers were published on α-synuclein during the past 2 years alone. Figure 1 represents a more detailed view of these Web of Science data by showing the cumulative number of publications for the past 26 years and the annual number of publications dedicated to this protein.

Despite immense efforts, this 25-year-old enigmatic protein continues to keep multiple secrets related to its structure, function, dysfunction, multipathogenicity, and pathology transmission. The aforementioned considerations constitute a perfect stage for an important question related to α-synuclein: what is so special about this protein that makes it a multipathogenicity carrier? The goal of this article is to shed some light on α-synuclein-related mysteries by presenting the most recent advances in our understanding of this protein and its aggregation.

To better understand why researchers continue to study this protein, let’s consider what multipathogenicity means for α-synuclein. Although originally considered a potential cause of PD (where aggregated α-synuclein is present in the form of intracellular inclusions, Lewy bodies [LBs] and Lewy neurites [LNs] ^{5, 6} ), this protein is known to be involved in the pathogenesis of a diverse group of neurodegenerative diseases collectively known as synucleinopathies. Some of these maladies include Alzheimer’s disease (AD), neurodegeneration with brain iron accumulation type 1, pure autonomic failure, Down’s syndrome, amyotrophic lateral sclerosis-parkinsonism-dementia complex of Guam, multiple system atrophy (MSA), and several LB disorders (that, in fact, might represent a clinical continuum ⁷ ), such as sporadic and familial PD, dementia with LBs (DLB), diffuse LB disease, the LB variant of AD, and PD dementia ^{4, 8– 15} . These (and potentially many other) neurodegenerative diseases can be considered α-synuclein-related brain amyloidoses, since all of them are characterized by the presence of common pathological intracellular inclusions containing α-synuclein in selectively vulnerable neurons and glia and since the onset and progression of their clinical symptoms, as well as the degeneration of affected brain regions, are linked to the formation of abnormal filamentous aggregates containing α-synuclein ^{9, 15– 21} . Importantly, accumulated evidence indicates that α-synuclein-based aggregation and deposition of LBs can affect multiple areas of the peripheral and central nervous systems, such as the dorsal raphe nucleus, dorsal nucleus of the vagus nerve, hypothalamic nuclei, intermediolateral nucleus, locus coeruleus, nucleus basalis of Meynert, and substantia nigra ²² , whereas LNs can be found in the basal ganglia, cerebral cortex, dorsal nucleus of the vagus nerve, and sympathetic ganglia as well as in the intramural autonomic ganglia of the gastrointestinal tract ^{23, 24} . In line with these earlier observations, a recent study of the inclusion pathologies in PD and DLB confirmed the abundant presence of LBs and LNs in the dorsal motor vagal and solitary nuclei, locus coeruleus, parabrachial nuclei, pedunculopontine and raphe nuclei, periaqueductal gray, prepositus hypoglossal, substantia nigra, reticular formation, and ventral tegmental area and demonstrated the presence of LN in brainstem fiber tracts and the existence of LBs and LNs in cranial nerve, premotor oculomotor, precerebellar, and vestibular brainstem nuclei ²⁵ . This study also supported an important notion that α-synuclein deposition-related pathological processes can spread and do so transneuronally along anatomical pathways ²⁵ . Recently, application of the α-synuclein proximity ligation assay revealed the presence in the post-mortem brain tissue from PD patients of previously unrecognized pathology in the form of extensive diffuse deposition of α-synuclein oligomers that were often localized, in the absence of LBs, to neuroanatomical regions mildly affected in PD ²⁶ .

Obviously, although the diversity of synucleinopathies and related symptoms potentially can be attributed to the complexity of the organization of brain and nervous system (different motor and non-motor symptoms are the manifestation of the malfunction of different brain and nervous system regions), this complexity, in general, cannot be used to explain the cause of the multifaceted, α-synuclein-related pathology at the molecular level. In fact, since α-synuclein is highly expressed throughout the brain, accounting for as much as 1% of the total protein in soluble cytosolic brain fractions ²⁷ , and since any given synucleinopathy possesses unique spatiotemporal characteristics (happens at a specific time due to the malfunction of a specific brain region), some other factors, likely related to α-synuclein and its interaction with the environment (such as mutations in the SYNC gene, presence of alternatively spliced isoforms, post-translational modifications (PTMs), toxic insult, oxidative stress, presence of dopamine, metal ions, specific binding partners, etc.), should be taken into account.

Recent studies provided further support for an interesting molecular link between the synucleinopathies and other neurodegenerative diseases by demonstrating the ability of α-synuclein to interact with and regulate proteins specific to several degenerative maladies. For example, it was shown that α-synuclein can interact and co-aggregate with mutant huntingtin, a protein related to Huntington's disease ²⁸ , and with tau protein, the aggregation of which is associated with various tauopathies, including AD ²⁹ . A cooperation of α-synuclein with β-amyloid (Aβ) was shown to block SNARE-dependent vesicle fusion ³⁰ , whereas interaction with Aβ led to the inhibition of Aβ deposition and reduced plaque formation ³¹ . Finally, α-synuclein was also shown to engage in interaction with autophagy/beclin1 regulator 1, a protein related to MSA pathogenesis, and this binding was dramatically enhanced by the α-synuclein phosphorylation at serine 129 ³² . Note that the aforementioned interactions of α-synuclein with the proteins associated with other neurodegenerative diseases can be grouped into two classes – interactions leading to the co-aggregation of α-synuclein with said proteins and interactions leading to the modulation of functionality of proteins targeted by α-synuclein.

Many papers in the field start with an introductory sentence stating that α-synuclein is a small, highly conserved presynaptic protein with unknown function. This statement is a bit odd, taking into account all the efforts of numerous researchers working on α-synuclein. In fact, according to the PubMed database (as of 16 December 2016), there were more than 7,150 papers mentioning synuclein function, many of those papers were dedicated to the detailed investigation of what this protein can do, and, as a result, many potential functions were ascribed to α-synuclein. The explanation of this contradiction (many functions are described, but function is unknown) is in the logistics of the classical structural biology relying on the influential “one gene – one enzyme – one function” hypothesis, according to which each gene encodes a single protein that has a unique biological function and is responsible for a single step in a metabolic pathway ³³ . In line with this hypothesis, distortion of normal protein function (in the form of loss of a normal function or gain of a pathological function) might represent the molecular basis of a proteinopathy.

However, since α-synuclein was shown to have not one but many functions (see below), this immediately brought significant uneasiness to functional data interpretation, leading to the logical conclusion that the observed multifunctionality indicates the lack of a unique function and, therefore, could be unreal. Although, from the viewpoint of the “one protein – one function” model, these observations provide grounds for the “protein with unknown function” epithet, an alternative (and much more intriguing and lucrative) hypothesis stating that α-synuclein is indeed a multifunctional protein, which can, in fact, do everything ascribed to it (and, probably, much more than that). In this case, problems with different α-synuclein-based functions might be directly or indirectly related to the pathogenesis of different synucleinopathies. In other words, a spectrum of functions can cause a spectrum of dysfunctions that might lead to a spectrum of diseases.

Early work in this direction showed that functions of α-synuclein can range from fatty acid binding ³⁴ to interaction with plasma membranes and formation of membrane channels or modification of their activity ³⁵ , association with mitochondria causing mitochondrial dysfunction ³⁵ , metal binding ^{36– 39} , interaction with pesticides and herbicides ^{40– 42} , synaptic vesicle release and trafficking ³⁴ , positive and negative regulation of neurotransmitter release ⁴³ , regulation of certain enzymes and transporters ³⁴ , control of neuronal survival ³⁴ , regulation of the neuronal apoptotic response ⁴⁴ and protection of neurons from various apoptotic stimuli ⁴⁴ , and promiscuous interaction with hundreds of unrelated proteins and other binding partners ^{34, 45– 47} . In the past 2 years, the phenomenon of α-synuclein multifunctionality was further confirmed and elaborated by adding a long list of new functions. Note that the inventory below is far from being exhaustive, since over 1,100 articles dedicated to synuclein function were published during the last 2 years, and, therefore, it is physically impossible to cover all new developments in this commentary owing to the space restrictions.

Also, a set of recent studies was dedicated to the analysis of various means of regulation of α-synuclein, its normal and pathological functions, and aggregation. It was shown that this protein can be regulated, at the expression level, by the microRNAs miR-34b and miR-34c ¹¹⁰ as well as miR-19b, miR-29a, and miR-29c ¹¹¹ , by promoter methylation in the α-synuclein gene SNCA ¹¹² , and by other means of epigenetic-mediated regulation ¹¹³ . Aggregation of α-synuclein can be controlled by antibodies at substoichiometric concentrations (as low as 1:1000 antibody to protein ratio) ¹¹⁴ . Different monoclonal anti-C-terminal antibodies were shown to differently interact with different forms of α-synuclein (monomeric or aggregated), with the antibodies with the strongest binding to aggregated protein also being the strongest inhibitors of α-synuclein fibrillation and membrane permeabilization ¹¹⁵ . Aggregation of α-synuclein can be modulated via interaction with the moonlighting 14-3-3 proteins that act as ATP-independent anti-aggregation “holdases” ¹¹⁶ , by caspase-1-mediated truncation ¹¹⁷ , by the small secretory chaperone proSAAS ¹¹⁸ , or by Geum urbanum extract ¹¹⁹ . The aggregation, toxicity, levels, and secretion of α-synuclein were shown to be controlled by endocytic recycling pathway components, such as Rab8b, Rab11a, Rab13, and Slp5 ¹²⁰ . Also, interaction with an anti-amyloidogenic agent, ginsenoside Rg1, was shown to inhibit the fibrillation and toxicity of α-synuclein and disaggregate preformed fibrils ¹²¹ . The extracellular α-synuclein can be sorted in extracellular vesicles in a SUMOylation-dependent manner ¹²² , whereas the extracellular release of this protein is controlled by the DnaJ/Hsc70 chaperone complexes ¹²³ . The nuclear accumulation and toxicity of α-synuclein can be regulated by a novel protein, TRIM28 ¹²⁴ , that controls the fate specification of the neural cells ¹²⁵ , whereas loss of lysosomal β-glucocerebrosidase activity promotes global intracellular accumulation and toxicity of α-synuclein ¹²⁶ .

Not only is monomeric α-synuclein characterized by structural polymorphism but also its oligomeric and insoluble aggregated forms are polymorphic at both structural and functional levels. For example, application of the cryo-electron microscopy image reconstruction technique revealed that oligomers that are kinetically trapped during α-synuclein fibril formation are characterized by different sizes, β-sheet contents, and levels of exposed hydrophobicity ¹⁶⁸ . Also, two morphologically different oligomeric α-synuclein forms were found in human post-mortem PD brain tissue ¹⁶⁹ . The presence of two different types of oligomeric species formed by the wild-type α-synuclein and its familial PD-related mutants A30P, E46K, and A53T was described using hydrogen/deuterium exchange monitored by mass spectrometry ¹⁷⁰ . Although generated at specific conditions, oligomers of the wild-type α-synuclein and its familial PD-related mutants A30P, A53T, E46K, H50Q, and G51D have similar global structures and are composed of a similar number of monomers, but they interact differently with biological membranes ¹⁷¹ .

Several recent observations are in line with the idea that oligomeric/fibrillar forms of α-synuclein might be functionally different, indicating the presence of a functional polymorphism among the aggregated polymorphs. For example, it was found that fibrillar α-synuclein can trigger inflammatory responses, whereas oligomeric forms of this protein were unable to initiate these cascades ¹⁷² . Although both oligomeric and fibrillar forms of α-synuclein are able to generate free radicals, only the oligomeric protein can reduce endogenous glutathione and cause subsequent neuronal toxicity ¹⁷³ . Also, microtubule assembly was shown to be efficiently inhibited by fibrillar α-synuclein, and this fibril-mediated inhibitory effect was greater than that of the oligomeric species (protofibrils) ¹⁷⁴ .

Slight changes in the environmental conditions during the fibrillation process were shown to cause the formation of morphologically different amyloid fibrils with distinctive molecular characteristics that can be inherited by the next generation of fibrils through self-propagation ¹⁷⁵ . Under specific conditions, pure forms of structurally different fibrillar polymorphs of α-synuclein were obtained and characterized structurally and functionally ¹⁷⁶ . In addition to different morphologies, these polymorphs were characterized by different structures, different levels of toxicity, different in vivo and in vitro seeding and propagation properties ¹⁷⁶ , different molecular arrangement in the unit cell, and distinct dynamic features ¹⁷⁷ . Subsequent structural and physical characterization of several fibrillar polymorphs assembled from α-synuclein supported the notion that the same protein can assemble into a variety of large particles with fibrillar shapes, that fibrillar polymorphs are morphologically and nanomechanically different, and that exceptional structural and physical polymorphism is present within the fibrillar form of this protein ¹⁷⁸ . These findings provide mechanical foundation for better understanding of the nature of different α-synuclein strains ¹⁷⁹ that define the ability of the aggregated α-synuclein to be engaged in different neurodegenerative diseases ¹⁷⁶ .

It is important to note that the phenomenon of polymorphism of amyloid fibrils is not an exclusive property of α-synuclein but was described for other amyloidogenic proteins ^{180, 181} and was linked to the phenomenon of strains originally found in mammalian prion protein ^{182– 185} and now described for α-synuclein ¹⁷⁹ , yeast and fungal prions ^{186– 188} , transthyretin ¹⁸⁹ , and insulin ¹⁹⁰ as well as Aβ ¹⁹¹ and tau protein ¹⁹² . Strain phenomenon is related to the peculiarities of amyloid propagation and transmission, where different aggregated forms of a protein can cause the development of different pathologies. In relation to the subject of this article, the ability of α-synuclein to aggregate into distinct high-molecular-weight assemblies was proposed to be associated with the ability of this protein to cause different synucleinopathies ¹⁷⁹ . This idea is supported by the direct observation of the induction of different synucleinopathies after the administration of the different α-synuclein strains (oligomers, ribbons, and fibrils) by injection into rat brain ¹⁹³ .

Another analogy of aggregated α-synuclein to prion protein is its “infectivity”, i.e. the ability to be efficiently transmitted between neurons, thereby supporting the pathological spread within the affected brain during disease progression (e.g. as described by Braak's staging criteria of PD ^{194, 195} ). Although original models describing interneuronal propagation suggested some passive release of aggregated α-synuclein from injured, degenerated, or dead neurons, a recent study showed that intact, relatively healthy neurons are engaged in efficient cell-to-cell passage of α-synuclein ¹⁹⁶ . It was also indicated that endosomal processing might serve as a point of convergence between the transcellular propagation of aggregated α-synuclein and the intracellular trafficking of this protein ¹⁹⁷ . A crucial role of high-affinity binding of preformed α-synuclein fibrils to the lymphocyte-activation gene 3 protein is for the initiation of endocytosis and transmission of aggregated α-synuclein, thereby propagating its toxicity and leading to the loss of dopamine neurons and the development of biochemical and behavioral deficits in vivo ¹⁹⁸ . Importantly, it is not only exogenous fibrillar forms of α-synuclein that are able to promote cellular pathologies, since soluble amyloid oligomers that precede LB formation were also linked to the cell-to-cell transmission of α-synuclein pathology ¹⁹⁹ .

The knowledge of the peculiar behavior of α-synuclein accumulated over the past two decades provides an illustration of an important notion that the complexity of a biological system is mostly determined by its proteome size and not by the genome size ²⁰⁰ . In fact, the number of functionally different proteins found in eukaryotic organisms is substantially higher than the number of genes. The functional diversification of proteinaceous products of a gene is achieved by several means, including the allelic variations (i.e. single or multiple point mutations, indels, SNPs) at the DNA level, alternative splicing, and other pre-translational mechanisms affecting mRNA, complemented by a wide spectrum of various PTMs of a polypeptide chain. As a result of all of these events, a set of distinct protein molecules can be created from a single gene, giving rise to the proteoform concept ²⁰¹ . Recently, it was proposed that in addition to the aforementioned means that increase the chemical variability of a polypeptide chain, the protein’s structural diversity can be further increased by some other mechanisms, such as intrinsic disorder and function ²⁰² . In this view, a correlation between protein structure and function is considered a “protein structure–function continuum”, where a given protein exists as a dynamic conformational ensemble containing multiple proteoforms (conformational/basic, inducible/modified, and functioning) characterized by diverse structural features and miscellaneous functions ²⁰² . In application to α-synuclein, the protein structure–function continuum idea is further enhanced by the presence of remarkable structural and functional variability of its self-aggregated forms. Therefore, due to its ability to oligomerize and aggregate, an intrinsically disordered α-synuclein is characterized by the enormously expanded set of proteoforms where each of the various monomeric, oligomeric, and insoluble species is further split into numerous conformational/basic, inducible/modified, and functioning proteoforms. In other words, as illustrated by Figure 2, consideration of α-synuclein through the proteoform prism provides a logical explanation for its remarkable structural, functional, and dysfunctional multifaceted nature.

Outlined in this article, the proteoform-centric view of α-synuclein represents an important shift in understanding the biology and pathology of this protein. In fact, despite the enormous amount of effort expended by the scientific community to find a single most important physiological function of α-synuclein, there is no clear understanding of the unique biological role of this protein as of yet. However, as it follows from a great deal of the published work on α-synuclein, the applicability of the “one sequence – one structure – one function” paradigm to this protein is questionable. In other words, it seems that the search for a single physiological function of α-synuclein may be entirely misplaced, since the intrinsically disordered nature of the protein, which defines the structural polymorphism of the conformational ensemble of its monomeric form, bestows multifunctionality upon α-synuclein. Logical consequences of this structural heterogeneity and multifunctionality of a monomeric protein are the structural polymorphism of aggregated states and a “spectrum of dysfunctions” that define a range of diseases. Therefore, the enormous structural and functional complexity of α-synuclein has serious implications for future studies to understand the biology of this protein and for the development of future therapeutic strategies targeting α-synuclein.

Aβ, β-amyloid; AD, Alzheimer’s disease; DLB, dementia with Lewy bodies; H3K9, histone H3 lysine-9; IDP, intrinsically disordered protein; LB, Lewy body; LN, Lewy neurite; MSA, multiple system atrophy; NAC, non-amyloid-β component; PD, Parkinson’s disease; PP2A, protein phosphatase-2A; PTM, post-translational modification.