Structural And Functional Insights Into α-Synuclein Fibril Polymorphism Part 2

β-Synuclein (β-syn) and γ-synuclein (γ-Syn) proteins also belong to the synuclein family [165] (Figure 1C). β-Syn is a 134 amino acid protein, earlier identified as the human homolog of bovine phosphoneuroprotein 14 (PNP14). 

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The 11-amino-acid (residues 73–83) stretch is missing in its NAC domain (Figure 1C), due to which it cannot fibrillate [11,176,177]. Earlier, it was believed that β-Syn is an inhibitor of α-Syn aggregation and prevented its neurotoxicity [11,176]. However, this notion changed after discovering missense mutations in the β-Syn gene, P123H (familial), and V70M (sporadic), known to cause DLB [178]. 

The deleterious effects of these mutations have been shown by cell- and animal-based studies [179,180]. Our group recently showed that under normal physiological conditions, fibrilization and aggregation of β-Syn and its disease-associated mutations did not occur, but an altered microenvironment, such as a decrease in pH and/or the presence of heparin, caused them to polymerize [181]. 

γ-Syn, which shares ~55% sequence homology with α-Syn, was initially identified in breast cancer malignancies encoded by a breast cancer-specific gene, BCSG1 [182]. It was reported in the peripheral central nervous system and breast cancer tissues [182,183]. It aggregates and forms fibrils in vitro [177] and in cells [184], but is comparatively slower than α-Syn [91]. 

The misfolding and fibrillation of α-Syn is a major event in several neurodegenerative disorders [185]. The misfolded α-Syn aggregates are amyloidogenic, which aberrantly accumulate in the brain, and, as a result, the patient suffers movement abnormalities that worsen over time. 

The aggregation of α-Syn is a complex phenomenon and involves the conversion of monomers to highly ordered cross-β-sheet-rich structures through the formation of several soluble on- and off-pathway oligomeric species [185,186]. The amyloid formation of α-Syn is generally monitored by thioflavin T fluorescence dye [187]. 

It follows sigmoidal growth kinetics, which consists of (i) the lag phase, involving the formation of nuclei, which eventually grow into the detectable aggregate structure in solution; (ii) the elongation phase, the conversion and subsequent growth of oligomeric species into the fibrillar structure; and (iii) the stationary phase, representing the steady-state where the monomer and fibril concentration reaches the equilibrium. 

By the end of the aggregation, α-Syn assembles into atypical long amyloid fibrils, normally characterized by electron microscopy and atomic force microscopic imaging techniques [9]. These phases of aggregation cannot be attributed to a single event or microscopic process. Instead, all the processes, viz., primary nucleation, elongation, secondary nucleation, and fragmentation, are active through all the phases of the growth curve but at different rates [188,189] (Figure 2). 

These reaction rates are governed by aggregation rate constants and the concentration of the reacting species at a given time [190]. The amyloid formation initiates with primary nucleation of the monomeric species in the solution and elongation by the addition of monomer to the growing ends of the aggregates [189]. However, primary nucleation processes are short-lived and rapidly surpassed by secondary nucleation processes [191]. 

The fragmentation of the fibrils under agitation conditions (or even under quiescent conditions depending upon the stability of amyloid fibrils) modifies the number of growing ends and significantly affects the overall growth kinetics [188,189]. 

Moreover, secondary nucleation by surface catalysis is also one of the major contributors to amyloid growth in several systems, especially under quiescent conditions [189,190]. The self-assembly and aggregation of α-Syn is a complex phenomenon and involves multiple parallel processes. Therefore, it is crucial to understand the underlying molecular events to delineate their fundamental connection with human disease.

4. Prion-like Strain Phenomena in α-Syn

Since the discovery of α-Syn as the main constituent of Lewy body pathology in 1997, the primary focus has been shifted to delineating the underlying pathogenic mechanism of PD. 

Heiko Braak [192] presented a staging system of Lewy pathology in 2003 based on the specific patterns of α-Syn spreading. According to the Braak hypothesis, the Lewy pathology initiates from the olfactory bulb and DMV and then progressively spreads to the other brain regions. 

Although there is experimental and clinical evidence supporting Braak's hypothesis, it is uncertain whether it is applicable and/or accurately describes the progression of PD in all patients. For instance, there are cases in which patients do not show Lewy pathology in DMV or ENS, while other brain regions are severely affected [193–198]. 

Even in some cases, no link has been observed between the severity of Lewy pathology and clinical symptoms in PD [195]. Therefore, it is suggested to only apply Braak's hypothesis to a subset of the population [198] as not all PD patients adhere to the staging system proposed by Braak [199]. Intriguingly, the reports of Lewy pathology in fetal neuronal grafts after fourteen years of transplantation into the striatum of the PD patient provided direct proof of cell-to-cell transmission and the spreading of α-Syn pathology proposed by Braak [200,201]. 

Studies using in vitro and cell model systems later suggested that α-Syn aggregates are infectious, can move from one cell to another, and seed the aggregation of their soluble endogenous counterpart in the recipient cells, explaining the phenomenon observed in grafted neurons [18,21,23]. This prion-like transmission of α-Syn aggregate from one region to another is also implicated in DLB and PDD patients, suggesting that the spread of Lewy pathology is the shared property of α-Syn aggregates in synucleinopathies [202–205]. 

However, the clinical and pathological features of these synucleinopathies are highly variable and heterogeneous [206,207], [146,208]. One might ask, why, despite being linked to the aggregation of the same protein, the distribution of α-Syn pathology and the manifestation of disease symptoms are different amongst synucleinopathies. 

This could be explained by the prion-like strain phenomenon of α-Syn, in which the same precursor protein forms different fibrils that result in distinct pathology.

4.1. Concept of Prion Strains

The last few decades of research have suggested that proteins/peptides with various structures and sequences can form a common fold of cross-β-sheet-rich structure of amyloid [209–213]. These proteins/peptides form amyloids with a common aggregation framework, i.e., through a nucleation-dependent polymerization mechanism [214,215]. 

However, each protein/peptide may also undergo a distinct aggregation pathway to form a unique amyloid structure. Recent high-resolution structural studies with ssNMR and cryo-EM have indeed suggested that each protein packs uniquely and forms different structures for the cross-β-sheet fold [56–58,61,216]. Not only that, but, surprisingly, one protein can form multiple different structural folds [40,59]. 

Thus, these proteins can adopt various conformations from the same amino acid sequence, giving rise to several proteinopathies and, therefore, not confirming the one protein–one structure hypothesis [217]. For instance, tau folds differently in Alzheimer's and Pick's disease [40,218,219]. Different TAR DNA-binding protein (TDP-43) aggregates exist in the brains of Frontotemporal lobar degeneration (FTLD-TDP) subtypes, showing morphological differences across the subtypes [220]. 

This protein's ability to misfold and display conformational diversity can lead to severe consequences, such as neurodegeneration [221]. This phenomenon of a protein forming different amyloids associated with various phenotypic properties is well-known for prions [46,222]. Prions are infectious protein particles that show conformational heterogeneity and can be transmitted from one individual to another [223]. 

A myriad of evidence shows epidemiological and clinicopathological diversity in human prion diseases, such as Kuru disease, Gerstmann–Straussler–Scheinker syndrome, and Creutzfeldt–Jakob disease [224], as well as non-human prion diseases, such as bovine spongiform encephalopathy (BSE) in cattle, scrapie in sheep and goats, etc. [225]. 

The normal cellular prion protein (PrPC) undergoes conversion from α-helical to β-sheet-rich conformation (PrPSc), which is an insoluble, PK-resistant, and infectious form. PrPSc propagates and aggregates following two widely accepted mechanisms/models, i.e., the template-assisted and nucleation polymerization model. 

A pathogenic prion acts as a template in the template-assisted model and provides a surface for converting an endogenous normal prion protein to its misfolded pathogenic form [226]. In the nucleation–polymerization model, monomeric PrPSc combine and form a stable nucleus, also called a seed. These seeds keep on recruiting PrPC and convert them to their pathogenic counterparts [226]. 

One of the remarkable properties of prions is that they can misfold into diverse conformations, each giving rise to distinct clinical, histological, and pathological profiles. These aggregates with different conformations and pathological behavior are referred to as 'strains' [45,227]. The pioneer observations on the presence of prion strains came from the study by Pattison and Milson, 1961 [228], wherein they experimentally produced scrapie in goats and observed distinct clinical manifestations of the disease owing to different strains. 

In another study, Fraser and Dickinson were able to distinguish different strains of scrapie in infected mice models depending on the extent of damage in different regions of the brain [229]. Later on, a plethora of reports showed the existence of PrPSc strains [227,230–233] and methods to distinguish them, such as Proteinase K (PK) digestion [234,235], electron paramagnetic resonance (EPR), and NMR spectroscopy [43]. 

These conformations of PrPSc vary in terms of different types of secondary structure elements, such as α-helix, β-strand, β-turn, or different structural folds or different packing [227,230,232,235,236]. For instance, prion protein from the Syrian hamster refolds into both α-helical and β-sheet structures, as well as various intermediates in aqueous solution [236]. 

These are not only structurally but also functionally distinct from each other [43] and have been identified in different human and animal prion disorders [233,237]. Despite resulting from the aggregation of the same prion protein, the prion diseases differ from each other concerning the disease onset/incubation period, progression, and histopathological lesions in the infected brain [224,225]. This has been collectively termed as 'prion strain phenomena' [45,46,238]. 

One of the major contributors to this strain diversity/variation is the ability of the infectious agents/prions to infect certain species [44,239,240] and cross the 'species barrier', thereby generating a variety of strains with distinguishable biochemical and pathological characteristics. One such case is vCJD (variant Creutzfeldt–Jakob disease), resulting from the interspecies transmission of BSE prion from cattle to humans [44,240], which is the only known case of non-human prions being transmitted to humans. 

Also, different prion strains have been known to coexist with each other, as seen in the case of sCJD (sporadic Creutzfeldt–Jakob disease) [241,242], showing distinct biochemical properties in different regions of the brain. Prion strains are also known to extend their incubation period upon co-infection with other strains, thus showing a phenomenon of 'competition', as observed in various studies [243–245]. 

All the above-mentioned features of prion strain phenomena and the potential for the generation of new prion strains have emerged not only as a serious scientific challenge but also as a threat to general public health. 

Recent studies, however, have suggested that this strain property of amyloids is not only limited to prions but also other amyloids associated with various neurodegenerative disorders such as Alzheimer's and Parkinson's [19,246,247], [18,248]. Various evidence has been provided from in vitro and in vivo studies to demonstrate the prion-like strain behavior of α-Syn, as discussed below.

4.2. α-Syn Strains Generated In Vitro

Growing evidence of prion-like strains of α-Syn associated with clinical and pathological variations observed in synucleinopathies has been reported in the past few years [29–35,249]. 

These strains have been defined as distinct and stable conformational assemblies of a single protein that can self-replicate and propagate in vivo and result in different disease phenotypes. Guo et al. discovered two 'strains' of α-Syn pre-formed fibrils (puffs), through de novo fibrilization, termed 'strain A', and repetitive seeding fibrilization in vitro, termed 'strain B'. 

These two strains exhibited distinct conformations and striking differences in cross-seeding tau protein in primary neuronal cultures and in vivo [30]. Besides documenting the evidence of α-Syn fibril strains for the first time, these findings also identified the cross-seeding behavior of α-Syn. Later, several groups took advantage of the chameleon property of α-Syn [9] and screened numerous growth conditions to generate α-Syn strains in vitro. Under different growth conditions, several conformationally stable and unstable states of protein were observed [250]. 

The conformations, which are not thermodynamically stable or cannot establish stable intermolecular interactions, cannot grow into amyloid fibrils. These are referred to as growth-incompetent states (Figure 3). On the other hand, growth-competent states of α-Syn can grow and form different fibrils depending on the growth conditions (Figure 3). 

The resulting fibrils under different assembly conditions not only possess different biochemical and biophysical properties, like resistance to proteases, cytotoxicity, seeding ability, etc. [29–32,34–36,51,53] but also imprint their architecture on the daughter fibrils depending on the growth condition and the nature of seeds (Figure 3). 

Bousset et al. indeed generated two structurally and functionally different α-Syn strains, named 'fibrils' and 'ribbons', using different physiological salt concentrations [29]. Fibrils caused more cytotoxicity, whereas the ribbons were found to be more effective in inducing α-Syn inclusions in vivo [33]. Similar differences were obtained with strains generated by Suzuki et al. [51], where one strain caused the accumulation of abundant phosphorylated and ubiquitinated α-Syn aggregates in cultured neurons and mice due to its ability to interact with proteasome complexes, whereas the other strain failed to do so [51]. 

Nowadays, varying experimental conditions have rather become a common strategy to generate strains in vitro. In cases where the criteria to be called a 'strain', i.e., should be a structural variant of the protein aggregates, exhibit the ability to self-propagate and serially transmit the disease over the next generations and cause clinical and phenotypical disease variations, are not completely fulfilled, it would be more appropriate to call the fibrils as 'polymorphs.' 

These polymorphs may show striking differences in their morphology and structure [28,56,251], nucleation rates [252], seeding and membrane binding ability in cells [36], etc. However, as a functional consequence of these structural variations in polymorphs, they may or may not result in distinct clinical subtypes of diseases. There are also cases of intrasample polymorphism, which can arise irrespective of whether fibrils are generated in vitro [39,57,253–256] or derived from brain extracts [40,257,258]. 

While these may possess certain commonalities, like a similar monomeric fold or a common structure, they also show marked differences in morphology, β-strand arrangement, or biochemical properties [57,256]. Thus, different assembly conditions can generate structurally and functionally distinct fibrillar assemblies, which may either propagate as a unique α-Syn strain or may simply form polymorphs.

Several reports have claimed the presence of α-Syn aggregates in the gastrointestinal tract [259–263] and a key role of the vagal nerve in spreading these aggregates from the gut to the brain [74,76,192,264,265]. Recently, curli-expressing E. coli has been shown to promote α-Syn pathology in the gut and the brain of mice overexpressing human α-Syn [78]. Exposure to microbial amyloids may induce polymorphism. 

Instead, it would be interesting to ask if different forms of α-Syn (polymorphs) originate from the gut, spread to the brain regions via retrograde vagal transport, and cause pathology in a strain-specific manner. In this context, structural and functional differences have been observed between α-Syn fibrils formed in the presence and absence of bacterial endotoxin lipopolysaccharide (LPS) [31]. 

LPS is known to modulate α-Syn aggregation by stabilizing the α-helical intermediates formed during its aggregation pathway, resulting in fibrils with variable cytotoxicity and altered internalization behavior [266]. Furthermore, α-Syn has been shown to undergo numerous PTMs, like phosphorylation, methionine oxidation, acetylation, nitration, etc., which are directly associated with its aggregation and cytotoxicity [94,115,267,268]. 

As pS129 is the most common PTM and the main form of α-Syn in the inclusion bodies, it may cause strain formation in α-Syn in vivo. Ma et al. showed that phosphorylation at Ser129 enables the protein to form a distinct strain that differs structurally with higher cytotoxicity and different propagation properties in vitro and in cells compared to the wild-type counterpart (without phosphorylation) [269]. 

Not only phosphorylation but also N-terminal acetylation may impart fibril polymorphism [267], suggesting that even a slight modification in the α-Syn amino acid sequence can significantly impact its fibril structure. The alteration in the fibril structure may further influence the propagation of fibrils in vivo by selectively targeting distinct cell types and cellular populations within the brain [32]. 

These strain-specific differences are then faithfully preserved over the generations [28,29,52] and lead to clinical differences in disease onset, neurological illness, lesion profile, etc. [32] 

Overall, these studies suggest that in a complex and crowded milieu such as that of a cell, subtle environmental and cellular changes, the presence of co-factors or other proteins, and alterations in the protein's primary sequence may lead to the formation of different polymorphs or strains, resulting in different disease outcomes.

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