Structural And Functional Insights Into α-Synuclein Fibril Polymorphism Part 4

7. Phase Separation and Nucleation: Molecular Basis of Fibril Polymorphism

Soon after the discovery that α-Syn aggregation is not only linked to PD but also MSA and DLB, the key research area has been primarily focused on understanding the ability of a protein to cause clinically and pathologically diverse neurodegenerative disorders. 

As you get older, you may find that your memory is not as good as it was when you were a child. Sometimes you may even feel that your brain is "rusty" and it is difficult to remember new information. But what can be done to improve this situation? Research in recent years has shown that there is a close relationship between MSA (dopamine-saccharide complex) and memory.

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Over the past few years, the prion strain hypothesis has emerged as the leading explanation for the observed disease variability in synucleinopathies. Although a range of biophysical and biological data support the existence of α-Syn strains [29,30,32–35,53], it is still unclear how these strains originate and the factors that drive their formation in vivo. 

The probable answer could lie in delineating the aggregation pathways and discerning the molecular drivers underlying strain formation. Amyloid formation is not only governed by primary nucleation but is dominated by secondary nucleation events through most of the aggregation growth phase, as discussed earlier [189]. 

This secondary nucleation involves the elongation of new fibrils from the existing seeds by recruiting monomers from the solution. Any changes in the solution conditions of the fibrils tend to alter the rate of primary and secondary nucleation events [188,189]. For example, acidic pH enhances the binding of α-Syn monomers to the fibril surface, thereby increasing the secondary nucleation rates [189,302]. 

Since seed-induced propagation of aggregates is attributed to the fragmentation and elongation of fibril seeds, this leads us to hypothesize that the strain phenomenon is, at least in part, a consequence of secondary nucleation. Moreover, multiple species coexist at different stages of the aggregation kinetics [17,303,304], suggesting the involvement of aggregation intermediates in dictating the polymorphism. 

The mapping of the conformational space of α-Syn monomer reveals a structural subpopulation of α-Syn monomer that promotes its binding with membranes and induces the formation of various oligomers and fibrils [170]. 

Our lab recently demonstrated that the heterogeneous nucleation during the aggregation pathway forms the basis of the origin of polymorphism [252]. We generated two different polymorphs, HMFs (Helix matured fibrils) and PMFs (Pre-matured fibrils), from the aggregation intermediates formed under the same assembly conditions. 

PMFs do not have a stable amyloid core and possess random coil content along with β-sheet elements. Moreover, the structured β-sheet from residues 74–79 is absent from its NAC domain (residues 65–80), suggesting PMFs to be less ordered fibril types. On the contrary, morphologically and structurally distinct HMFs are more compact and well-ordered with a stable fibril core [252]. 

These contain highly exposed hydrophobic surfaces and are potentially more toxic than less ordered PMFs. These polymorphs display not only structural differences but also exhibit different biological activities [252]. 

A similar study involving the identification of the aggregation intermediates in the presence of a phospholipid membrane revealed that prefibrillar species contain two loop regions with residues 57 to 61 and 71 to 80 [305]. These intermediates then rearrange to fibrillar species with most of the NAC region and the N-terminus (residues 38–80) forming part of the final fibril conformation [305]. 

Studies have also been reported with aggregates of Aβ, PrP, and tau, where distinct biological properties emanate due to structural differences between the polymorphs [28,306–309]. For instance, morphologically and structurally different Aβ fibrils formed under quiescent and agitating conditions show significant differences in toxicity in primary neurons, with quiescent fibrils being more toxic compared to other ones [28]. 

A range of structurally diverse PrPSc conformations exhibits host cell tropism, with a specific set of strains preferentially targeting neurons, astrocytes, or even both [307,308]. Thus, α-Syn strains resulting from the heterogeneous nucleation in the aggregation pathway may cause clinical and pathological variations in PD by exhibiting variable cytotoxicity and different prion-like properties (Figure 5). 

Recently, the evolution of the concept of protein aggregation to a more fundamental phenomenon, namely liquid-liquid phase separation (LLPS), has significantly influenced the field and directed the research in a new direction [310–314]. Proteins are incubated in varying conditions, for example, pH, temperature, and salt conditions [315–317]. 

The phase separation events are promoted in the presence of molecular crowders, like polyethylene glycol, dextran, or ficoll, which aid in increasing the local protein concentration and facilitating droplet formation [315–318]. Thereafter, the dynamicity, maturation, and aggregation profile are investigated using a unique combination of biophysical, biochemical, and spectroscopic methods [315,317]. 

The phenomena of phase separation of various amyloidogenic proteins have been shown to precede aggregation and fibril formation. It has been suggested that the presence of intrinsic disorder regions (IDRs), prion-like domains (PLD), and low complexity domains (LCD) promote the formation of phase-separated condensates of the amyloidogenic proteins [310,315,316,319,320]. 

Our lab recently demonstrated that the LLPS of α-Syn is a critical event in the early lag phase and precedes its aggregation under phase-separating conditions (presence of crowders, stressors, amyloid co-factors, etc.) [315]. 

The appearance of these phase-separated droplets in the lag phase of aggregation kinetics suggests that LLPs might enhance the nucleation events by increasing the local concentration of the protein molecules [315,320]. Moreover, the phase-separated α-Syn droplets undergo liquid-to-solid transition with time and result in the formation of amyloid hydrogel [315]. 

These amyloid hydrogels have been previously shown to entrap cytotoxic oligomers and fibrils [321], indicating the possibility that fibrils formed via LLPS could be toxic. However, under normal assembly conditions (without phase separation), α-Syn fibrils show very little or no cytotoxicity [322,323]. This suggests that the fibrils formed under phase-separating and non-phase-separating conditions could be different. 

Moreover, the fibril formation in dilute solutions (known to occur via primary and secondary nucleation) [189] and that within the condensates (via LLPS) are not mutually exclusive events [324], but different aggregation pathways can result in the formation of different fibrils (Figure 6).

For instance, TDP-43, a protein involved in ALS/FTD, undergoes fibrillation with or without LLPS. Still, the fibrillation kinetics in both cases are different, suggesting the involvement of complex processes in fibrillation in the presence of LPS [329]. 

Therefore, it would be highly relevant to ask a few questions like whether the fibrils formed with or without undergoing phase separation are structurally and functionally distinct from each other, or, simply put, show fibril polymorphism. Whether different LLPS conditions or mutations have any role in deciding the strain behavior of the α-Syn fibrils formed? 

For example, the pathogenic mutations in FUS have been shown to exhibit different biophysical properties compared to the wild-type protein [327]. These findings suggest that, similarly to non-phase-separating conditions where different solution conditions give rise to polymorphs [29], fibrils formed under the different phase-separating conditions could also be different (Figure 7). 

For instance, the α-Syn phase separating under different conditions, like the presence of PTMs, familial mutations, small molecules, or metal ions, can show polymorphism and form different types of fibrils (Figure 7). 

However, this phenomenon is yet to be determined as it would need extensive characterization of fibrils formed inside the droplets. We believe that the co-existence of conformationally distinct intermediate species and a multitude of aggregation pathways observed with or without LLPS could form the basis of the origin of polymorphism. 

However, more research in this field is required to delineate the contribution of heterogeneous nucleation and multiple aggregation pathways to fibril polymorphism. The development and application of various biophysical techniques could help us gain insightful information about the molecular events occurring during LLPS and subsequent droplet maturation.

8. Clinical and Therapeutic Implications of Polymorphism

The studies discussed in the review provide conclusive evidence that α-Syn can form diverse polymorphs with distinct structural and biological properties. Similar to prions, where each prion disease is encoded by a distinct conformation of the misfolded protein, each synucleinopathy is also possibly associated with a unique α-Syn structure. 

However, diverse fibril structures for the same protein pose several challenges in drug development against neurodegenerative disorders. Therapies like immunotherapy would be highly specific for a particular strain but might fail to recognize a different one. Similarly, developing small therapeutic molecules or drugs for blocking or slowing down the protein aggregation process needs to be screened for multiple conformations. Considering the complexity of fibrils and their polymorphism, targeting monomeric α-Syn could be an option. 

However, that is also challenging due to the intrinsically disordered nature of the protein. Despite these challenges, detecting and characterizing patients' derived α-Syn strains will open a window of opportunities for deeper understanding and characterization of synucleinopathies. It will facilitate the development of new therapies and more robust classification systems of synucleinopathies. 

In this context, highly sensitive techniques like PMCA [330], real-time quaking-induced conversion assay (RT-QuIC) [331] and HANdai amyloid burst inducer (HANABI) [332] have significantly contributed to amplifying α-Syn aggregates from CSF of patients' brains. PMCA and RT-QuIC have been used to discriminate between PD/MSA and PD/DLB patient-derived α-Syn strains, respectively [52,333]. 

Employing these techniques would aid in monitoring the disease progression over time and help in the early and specific diagnosis of synucleinopathies. Furthermore, the clinical and pathological differences and patient-to-patient heterogeneity observed in PD and related disorders may impact how synucleinopathy patients should be treated. As of now, all PD patients receive the same type of treatment, and no distinction is made between the patients depending on how they have acquired the disease and what symptoms they present. 

Therefore, we anticipate that drugs and therapeutic agents designed to target pathogenic α-Syn species may involve using either a single conformational-based drug or a cocktail of drugs against diverse polymorphs or strains that are populated in the diseased human brain. Furthermore, several key parameters should be considered on a case-by-case basis, like disease profile, pathological and clinical symptoms, types and nature of the protein strains involved, and disease progression rate while treating patients with synucleinopathies. 

Therefore, identifying potential therapeutic targets and designing conformational-based drugs is the next big step toward developing drugs against PD and related disorders. Establishing the link between the propagation of a strain and the disease phenotype will provide valuable insights into developing effective strategies for combating neurodegenerative disorders. 

Another challenge is delivering drugs to the brain to treat neurodegenerative disorders, mainly facing setbacks due to restrictive blood-brain barriers [32]. Numerous attempts have been made to deliver drugs through nanocarriers, direct drug delivery methods, transient disruption of the blood-brain barrier, and stem cell therapies [33–37]. However, none of the treatments have been able to overcome the current challenges fully [32].

9. Concluding Remarks and Open Questions

The prion-like strain behavior of α-Syn is still an enigmatic phenomenon. It is surprising how a single protein without any proper structure can fold in many different ways and adopt conformations that result in various pathologies. 

Many unanswered questions need further investigation, even after an impressive amount of work on α-Syn polymorphs and strains. For instance, what drives the formation of α-Syn strain under given cellular and environmental conditions? How do these strains target different cell types and brain regions? Do α-Syn strains evolve, change, or adapt with time depending upon the host factors? 

How does the presence of other proteins, ligands, membranes, or co-factors influence strain formation? Do α-Syn strains have the ability to cross-see with each other and result in mixed pathologies? Do α-Syn strains interfere and block the propagation of each other, similarly to prions? 

Is it possible, or do we have techniques sensitive enough to discriminate between PD and MSA strains at an early stage of diagnosis? Considering the expansion of the prion strain phenomenon to several other amyloidogenic proteins, it may also be necessary to understand the key molecular events in strain biology and design effective strategies.

Author Contributions: Conceptualization, S.M., and S.K.M. investigation (literature review); S.M.; L.G.; R.B. and A.S.S.; writing-original draft preparation, S.M.; L.G.; R.B. and A.S.S.; writing-review and editing, S.M.; L.G.; R.B.; A.S.S. and S.K.M.; visualization, S.M.; project administration, S.M., and S.K.M.; supervision, S.M., and S.K.M.; funding acquisition, S.K.M., S.M., and S.K.M. are equally contributing corresponding authors. All authors have read and agreed to the published version of the manuscript.

Funding: This research was funded by the Department of Biotechnology (DBT) [BT/PR22749/BRB/10/ 1576/2016], Government of India.

Institutional Review Board Statement: Not applicable.

Informed Consent Statement: Not applicable.

Data Availability Statement: Not applicable.

Acknowledgments: The authors acknowledge the Department of Biotechnology (DBT), Government of India for the financial support. We thank Pradeep Kadu for his help in making figures. S.M. is thankful to the University Grants Commission of India for her fellowship.

Conflicts of Interest: The authors declare no conflict of interest.

Abbreviations

α-synuclein (α-Syn), Parkinson's disease (PD), multiple system atrophy (MSA), dementia with Lewy body (DLB), Parkinson's Disease Dementia (PDD), Lewy bodies (LBs), Lewy neurites (LNs), Alzheimer's disease (AD), Glial cytoplasmic inclusions (GCIs), post-translational modifications (PTMs), wild-type (WT), cryo-electron microscopy (cryo-EM), solid-state NMR (ssNMR) spectroscopy; transmission electron microscopy (TEM), atomic force microscopy (AFM), circular dichroism (CD), dorsal motor nucleus of the vagus nerve (DMV), enteric nervous system (ENS); β-Synuclein (βsyn); γ-synuclein (γ-Syn); Protein misfolding cyclic amplification (PMCA); Liquid-Liquid phase separation (LLPS).

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