Structural And Functional Insights Into α-Synuclein Fibril Polymorphism Part 3

4.3. α-Syn Strains in Human Synucleinopathy Samples

The structural and functional differences observed in recombinant strains can be validated by identifying and characterizing fibrils directly from synucleinopathy patient samples. 

Recombinant strains refer to microbial species that have been recombined at the genetic level. Huge progress has been made in this area, providing us with many opportunities. At the same time, scientists are constantly studying the impact of recombinant strains on humans and the environment. Research results in recent years have shown that recombinant strains have a positive impact on human memory.

Studies have found that recombinant strains can affect human cognitive ability and memory by improving the metabolic activity of microorganisms in the intestines and increasing the beneficial flora in the intestines. This communication mechanism of the gut-brain axis is called "gut-brain interaction."

Specifically, the recombinant strain can promote the repair of the blood-brain barrier by increasing the beneficial flora in the intestine, thereby promoting the normal functioning of the brain. Additionally, the recombinant strain was able to increase the release of dopamine and neuropeptides, which are critical for long-term memory and learning.

Therefore, scientists suggest that people should eat more foods rich in probiotics and prebiotics, such as yogurt, coffee, and whole-grain foods. In addition, people can also supplement the probiotics in the intestines by consuming some specific recombinant strains, thereby further promoting the effect of gut-brain interaction.

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The first evidence of a brain-derived strain came from a seminal study by Prusiner et al. [270], which demonstrated that brain extracts from MSA are transmissible to transgenic mice and cells, resulting in abundant α-Syn pathology [270]. In contrast, this was not observed using brain extracts from PD, suggesting that the PD-derived strain may differ from MSA [270]. 

Then comes the question, what might lead α-Syn to adopt a different conformation in MSA or PD? In vitro, various solution conditions (like the presence or absence of salt) give rise to fibrils with different structural and functional properties [29]. Similarly, α-Syn is also exposed to several microenvironments in vivo, affecting its aggregation [271]. 

The dopaminergic neurons in PD and the oligodendrocytes affected in MSA belong to different cell lineages and have distinct cellular environments. Lee and co-workers demonstrated that distinct intracellular environments of two cell lines impart strain formation in MSA and PD [34]. α-Syn fibrils derived from GCIs in oligodendrocytes (GCI-α-Syn) and LBs in neurons (LB-α-Syn) of diseased brains differ significantly and exhibit distinct seeding abilities [34]. 

GCI-α-Syn strain is highly effective in seeding α-Syn aggregation compared to LB-α-Syn, thereby contributing to the aggressiveness of MSA [34]. α-Syn aggregates have also been detected in biological fluids like cerebrospinal fluid (CSF) and plasma of PD patients [272,273]. α-Syn aggregation begins years before the onset of actual disease symptoms and, thus, the detection of these aggregates at early stages may enable the identification and characterization of a particular strain in these fluids. 

In this context, the amplification of α-Syn aggregates from brain extracts of PD and MSA patients using the protein misfolding cyclic amplification (PMCA) technique has been recently developed. This technique involves the amplification of misfolded proteins in vitro, like DNA amplification by PCR [274]. It consists of alternate cycles of incubation and sonication, resulting in amyloid replication. 

First, the trace amount of amyloid is incubated with an excess of native protein to induce polymer growth. Then, the sample mixture is subjected to sonication, which will break down the fibrils, resulting in several nuclei. 

Each newly formed nucleus will then act as a seed in the next cycle and further induce the growth of fibrils. This way, after each cycle, the number of seeds will increase exponentially and will allow the detection of the minute amount of misfolded aggregates present at the beginning [274]. Soto and co-workers used PMCA to amplify the α-Syn aggregates from the CSF of the patients diagnosed with PD and MSA [52]. 

They found that PD- and MSA-derived fibrils exhibit different biophysical and biochemical properties and correspond to distinct conformational strains of α-Syn [52]. Even α-Syn aggregates amplified from PD and MSA brain homogenates have been shown to exert variable toxicity and neurodegeneration in human dopaminergic neurons, reflecting different disease severity observed in PD and MSA patients due to different strains of α-Syn [275]. 

These findings conclusively suggest that synucleinopathies can be distinguished based on the type of α-Syn strain present in the brain. However, the complexity in detecting aggregates arises when patient-to-patient heterogeneity is observed in the same disease. This heterogeneity in α-Syn aggregates amplified from PD patients' brain extracts is greater than in MSA brain extracts [276]. 

Strohaker et al. reported that the fibrils derived from PD and MSA do not exhibit markedly distinct structural properties [276], in contrast to findings reported by Soto and co-workers [52]. 

The possible reason for the contrasting observations could be the differences in the PMCA protocols used by the two groups [277]. Additionally, Strohaker et al. used a much smaller sample size than Soto's group [52,276]. Other factors, like the genetic background of the patients, the age of the selected patients, a load of α-Syn aggregates in different patients, the presence of other components in the extracts, and the region of the brain from where extraction was done, could also be responsible for these differences [276]. 

Similar contradictions also exist in the field of AD pathology. Recent findings on brain-derived tau samples have suggested that patient-to-patient heterogeneity in the tau fibril conformations exists within the same disease, AD [278]. However, Goedert and colleagues observed the same type of tau conformation in all AD cases analyzed so far, suggesting that tau fibrils from a single disease (like AD or Pick's disease) adopt a common structural fold [218,219]. 

Although the reasons and factors that drive this structural specificity in tauopathies are unclear, it could be due to multiple isoforms of tau, PTMs, interactions with other protein molecules, co-factors, etc. 

Recently, Scheres and Godert presented a hierarchal classification of tau fibrils from different tauopathies based on the folds of their filaments [279]. Whether a similar classification exists for α-Syn fibrils isolated from synucleinopathy samples remains to be determined. Recently, a great effort has been made to solve the structure of α-Syn derived from the human brain by Schweighauser et al., using Cryo-EM [280]. 

The group found that α-Syn filaments from the brain of DLB patients do not twist and are thinner than those derived from the brain of MSA patients [280], consistent with the previous findings [3]. The lack of twists in fibrils derived from DLB precluded the determination of 3D structure by cryo-EM and the differences in α-Syn fibrils derived from MSA and DLB patients were drawn based on two-dimensional class averaging [280]. 

Although we need more high-resolution structures derived from synucleinopathy patients to reach a definite conclusion, the present reports certainly strengthen the claims on the existence of distinct fibril types of α-Syn. 

Moreover, the structures of α-Syn filaments from PD cases are not yet available, but solving them in the future can significantly help to understand the disease mechanism and generate therapeutic approaches against synucleinopathies.

5. High-Resolution Structural Models of Existing α-Syn Fibril Strains

Various biophysical techniques have been used so far, like ssNMR, micro-electron diffraction, EPR, circular dichroism (CD), hydrogen/deuterium exchange NMR (HDXNMR), and cryo-EM, to determine the structure of α-Syn fibrils at different resolutions. 

These techniques have laid the foundation of molecular-level polymorphism in fibrils. The β-sheet structure of the fibril core using ssNMR revealed two fibrils, form A and form B, by sequential assignment of 48 residues of the core [56]. 

The study elucidated the presence of two fibril polymorphs that may have formed due to different mechanisms for fibrillation [56]. Likewise, the two contrasting fibril structures, 'ribbons' and 'fibrils', generated in vitro showed differences in the length, distribution, and number of β-sheet elements in their fibril structure analyzed by ssNMR [29]. 

However, despite several attempts, how α-Syn fibril polymorphs differ in atomic structure has remained largely unknown. The revolution in structural polymorphism came after the structure of α-Syn fibril was solved using cryo-EM at the atomic level resolution. Stahlberg and the group revealed that the α-Syn fibrils (residues 1–121) consist of two identical protofilaments [61]. 

The β-sheets from each protofilament interact and stabilize the structure via hydrophobic zipper geometry [61]. Notably, the residues 50–57 located at the protofilament interface are also the site of familial PD mutants (A53T/V/E), H50Q, and G51D [61]. In this regard, it was predicted that these mutations might change the fibrillar structure, resulting in different fibril types.

Subsequent cryo-EM studies of the structure of full-length α-Syn have shown a difference in chirality and a helical twist [281] compared to the C-terminal truncated α-Syn fibril structure (residues 1–121) [61]. It was believed that these differences in the structures of full-length (1–140) and C-terminally truncated fibrils could be due to fibril polymorphism. 

The direct proof of these theories was obtained by recent seminal studies that used cryo-EM to delineate the models of α-Syn fibril polymorphs. Li et al. identified two fibril polymorphs, 'rod' and 'twister', with a common protofilament kernel structure but different inter-protofilament interfaces [57]. Twister polymorphs display an ordered bent-β-arch motif whereas rod polymorphs recruit some additional residues to form a 'Greek-key' motif, as reported by other groups as well [55,61,281]. 

The existence of polymorphs in rod and twister forms suggests that differences in the packing of the same kernel structure can lead to polymorphism. Similar observations have also been made for other amyloid proteins, like β-amyloid and tau, where the protofilaments with the same kernel structure but different packing arrangements lead to polymorphic structures [40,282]. 

Further, the two new polymorphic forms of α-Syn fibrils generated in vitro, named polymorphs 2a and 2b, respectively, are different from previously reported polymorphs 1a and 1b [57,61,281]. In polymorph 1a [61], the interactions between the residues at the protofilament interface are mediated by the formation of hydrophobic steric-zipper geometry, whereas in polymorphs 2a and 2b, they are mediated by salt bridges [41,61]. 

The closer inspection of the structural differences between polymorphs 1a/1b and new polymorphs 2a/2b revealed further differences in the arrangement of the β-arch motifs, which change the interface of the protofilaments between polymorphs 1 and 2 [41]. 

These studies strengthen the hypothesis that the same precursor protein α-Syn can assemble into multiple fibril polymorphs in vitro, which radically differ from each other in terms of atomic resolution. Goedert and his colleagues recently studied cryo-EM structures of tau fibrils derived from Alzheimer's and Pick's disease patients' brains [40,218,219]. 

They found distinct folds of tau filaments in both diseases, indicating different conformers of tau exist in different tauopathies. The same group reported two types of α-Syn filaments, type I and type II, from the brains of individuals suffering from MSA [280]. They found that each filament is made up of two non-identical protofilaments. 

The cavity formed by the close packing of the protofilaments encloses additional molecules that are yet to be determined [280]. The 2D class averaging also revealed different fibrils from MSA and DLB patients, suggesting the existence of distinct conformers associated with synucleinopathy. 

Further, it would be interesting to ask whether the patient-derived fibrils exhibit any level of similarity with the fibrils generated in vitro. Molecular-level characterization and comparison of brain-derived fibril samples with fibrils generated in vitro revealed that these two are structurally different [276,280]. The major difference between the MSA-derived and synthetic filaments is the size and packing of the protofilaments in MSA fibrils [280]. 

Moreover, researchers use harsh conditions to generate and isolate synthetic fibrils like agitation, salt concentration, etc., which affect the packing and β-sheet arrangement of filaments [10,283–285]. As a result, it becomes difficult to correlate the in vitro results with in vivo scenarios. 

On the other hand, one may only get pre-formed fibrils from disease-extracted samples but may not understand how they have originated and what factors have governed the formation of different fibrils in different brain samples. It is challenging to isolate the transient toxic species or intermediates from the brain samples to understand the disease pathogenesis. 

Consequently, we need to rely on in vitro samples to delineate the mechanisms of fibril formation, pathways, and kinetics analysis. Similarly, we have to test the same using brain-derived fibrils to develop a more extensive knowledge base. Overall, the high-resolution structures of α-Syn polymorphs could aid researchers in their quest for potential therapeutic targets. 

However, a thorough investigation is required to understand the impact of cellular conditions, mutations, PTMs, presence of co-factors, etc., on α-Syn fibril structure and the link of different fibril polymorphs with the clinical variability observed in PD.

6. Familial Mutations of α-Syn Form Distinct Fibril Conformations

α-Syn oligomerization and aggregation are associated with PD pathogenesis. Seven familial missense mutations have been discovered so far in the SNCA gene, associated with early- and late-onset PD [83–90]. 

Among these PD-associated mutations, E46K, H50Q, A53T, and newly discovered A53V mutants accelerate the rate of α-Syn aggregation, whereas A30P, G51D, and A53E mutations slow down the aggregation kinetics in vitro [91,93,96]. However, the link between the rate of aggregation (in vitro) and the age of the disease onset (in vivo) is not straightforward [103]. 

Although oligomers formed during the early stages of aggregation kinetics are potentially toxic [286], only A30P shows faster oligomerization and delayed conversion of oligomers into fibrils [102]. G51D, on the other hand, exhibits slow oligomerization and slow fibril formation [287,288], yet is associated with the early onset of the disease. 

Due to this complexity in the behavior of familial mutants, it is challenging to set up a unifying mechanism by which they cause the disease. Previous reports have suggested that α-Syn adopts a helical structure upon binding with membranes in vivo [289,290]. 

Any single amino acid change in the N-terminus domain of α-Syn may alter the membrane-binding ability and increase the cytosolic concentration of the protein by promoting faster aggregation [291,292]. The membrane-binding data of familial α-Syn mutants from our laboratory [92] and others [110,288,292,293] have shown that H50Q, A53T, and E46K mutants exhibit increased membrane binding, while A53E, G51D, and A30P mutants exhibit decreased membrane binding. 

This suggests that, similarly to aggregation, the membrane-binding capability does not correlate with increased disease propensities by familial α-Syn mutations. Therefore, there is a lack of correlation between the aggregation and membrane-binding ability with the actual disease pathogenesis caused by the familial mutants of α-Syn. 

This raises the question of how a point mutation in a natively unstructured protein shows drastic differences in the disease onset and progression. We believe it could be possible that different α-Syn mutants produce different types and amounts of oligomers and also may uniquely alter the seeding capacity of wild-type protein [103]. 

That is why mutants affect not only the overall aggregation rate of the protein but also the microscopic steps involved in the amyloid formation, i.e., initiation and amplification of α-Syn through the secondary nucleation process [294]. Intriguingly, Lazaro et al. found that, despite having identical oligomerization propensity in cultured cells, A30P, E46K, H50Q, G51D, and A53T exhibit distinct abilities to form inclusions [295]. A30P showed a decreased propensity to form inclusions in cells, whereas the E46K and G51D mutants displayed an opposite effect [295]. 

Again, the inclusion formation in cells [295] did not correlate with the aggregation propensity of mutants in vitro [12,91–93,102]. Thus, addressing these questions about how wild-type α-Syn and its mutants contribute to the early and late onset of PD becomes important to understanding the differential pathogenesis of synucleinopathies. 

Fibril formation is highly sensitive to changes in the local and/or global microenvironment of the protein. This suggests that a single amino acid change can result in polymorphism due to different site-specific conformational dynamics, as shown for the wild type and fibrils of E46K, A30P, and A53T [296]. In this context, Knowles and coworkers recently studied the systematic comparison of α-Syn and its disease-associated mutants using biophysical techniques [297]. PD mutants generate fibril polymorphs with distinct morphology and secondary structures compared to the wild-type protein [297]. 

Indeed, several reports have independently confirmed that different α-Syn mutants form fibrils with characteristic morphology revealed by transmission electron microscopy (TEM) and atomic force microscopy (AFM) studies, unique X-ray diffraction (XRD) patterns, and differences in secondary structure elements (Figure 4A, B).

Furthermore, the interface of the two protofilaments in the α-Syn fibril structure is formed by residues 50–57, which also harbor three familial mutations [61]. This suggests that even a single-point mutation can alter the dynamics and packing of the protofilaments. 

A closer inspection of the fibrils formed by mutants by cryo-EM [298–300] unveiled the plasticity of such fibrils in terms of twists, the number of interacting protofilaments, packing arrangement, secondary structure elements, and quaternary shape, etc. (Figure 4C, D). Boyer and his group studied H50Q mutants and found narrow (1c) and wide fibrils (1d) with one or two protofilaments, respectively [300]. 

Despite sharing the same conserved kernel structure as reported previously for wild-type α-Syn, the mutant fibrils displayed a new protofilament arrangement and hydrogen-bond networks [300]. Further, A53 lies in the center of the interface of the interacting protofilaments in wild-type α-Syn [61] and is also a hot spot for many point mutations [16,89,90]. 

Cryo-EM studies with N-terminally acetylated A53T mutant revealed no change in the fold of wild-type α-Syn [299]. However, the mutation disrupts the residue interactions and re-arranges the orientation of the protofilament interface, thereby resulting in a different type of fibrils [299]. This could also be the case with the other two A53 mutations, i.e., A53E and A53V, but this possibility is yet to be discovered. 

Cryo-EM modelling of fibrils formed by E46K mutation has also supported the prevailing hypothesis. It revealed the formation of fibril polymorphs with distinct protofilament packing and interfaces compared to wild-type α-Syn [298,301]. Besides, it formed a more stable and pathogenic variant of wild-type α-Syn [298]. Overall, these studies suggest that protofilament packing and the interface are critical in determining fibril structure. 

Each familial mutation may behave as a strain of α-Syn, uniquely altering the structure and dynamics of the resulting fibrils. These differences in the fibril structure may lead to different clinical and pathological outcomes, thereby contributing to disease heterogeneity in synucleinopathies.

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