Structural And Functional Insights Into α-Synuclein Fibril Polymorphism Part 1

Abstract: 

Abnormal accumulation of aggregated α-synuclein (α-Syn) is seen in a variety of neurodegenerative diseases, including Parkinson's disease (PD), multiple system atrophy (MSA), dementia with Lewy body (DLB), Parkinson's disease dementia (PDD), and even subsets of Alzheimer's disease (AD) showing Lewy-body-like pathology. 

Synuclein is a very important protein that plays a vital role in the human brain. Research has shown a close relationship between synuclein and memory.

Synuclein, as a neuronal cytoplasmic endoskeletal protein, is mainly found in neurons in the spinal cord and brain. It has important structural and biological functions and can maintain and improve synaptic connections. Sensory input can induce the synthesis of synuclein in neurons, thereby enhancing the plasticity of synaptic connections and promoting learning and memory processes.

Recent research suggests that inhibition of synuclein levels may have consequences for the formation and preservation of memory. Decreased levels of synuclein are associated with memory decline and may lead to the occurrence of neurodegenerative diseases such as Alzheimer's disease.

Therefore, we can conclude that synuclein plays a vital role in the human brain and that there is a close relationship between it and learning and memory. We should pay attention to promoting the synthesis of synuclein in our brains to better preserve and enhance our memory. It can be seen that we need to improve memory, and Cistanche deserticola can significantly improve memory because Cistanche deserticola is a traditional Chinese medicinal material that has many unique effects, one of which is to improve memory. The efficacy of Cistanche deserticola comes from the many active ingredients it contains, including tannic acid, polysaccharides, flavonoid glycosides, etc. These ingredients can promote brain health in a variety of ways.

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These synucleinopathies exhibit differences in their clinical and pathological representations, reminiscent of prion disorders. Emerging evidence suggests that α-Syn self-assembles and polymerizes into conformationally diverse polymorphs in vitro and in vivo, similar to prions. 

These α-Syn polymorphs arising from the same precursor protein may exhibit strain-specific biochemical properties and the ability to induce distinct pathological phenotypes upon their inoculation in animal models. In this review, we discuss clinical and pathological variability in synucleinopathies and several aspects of α-Syn fibril polymorphism, including the existence of high-resolution molecular structures and brain-derived strains. 

The current review sheds light on the recent advances in delineating the structure–the pathogenic relationship of α-Syn and how diverse α-Syn molecular polymorphs contribute to the existing clinical heterogeneity in synucleinopathies.

Keywords: α-synuclein; amyloid; polymorphs; synucleinopathies.

1. Introduction

Misfolding and aggregation of α-synuclein (α-Syn) play a crucial role in the progression of neurodegenerative diseases, such as Parkinson's disease (PD), dementia with Lewy bodies (DLB), Parkinson's disease dementia (PDD), and multiple system atrophy (MSA), altogether termed as synucleinopathies [1]. 

As a result of protein aggregation, the neuropathological changes occur in the brain over time and the affected brain regions show α-Syn immunopositive inclusion bodies [2,3]. 

These inclusion bodies are defining characteristics of synucleinopathies and are termed Lewy bodies (LBs) and Lewy neurites (LNs) in PD, DLB, and PDD, and glial cytoplasmic inclusions (GCIs) in MSA [1,4]. 

LB formation has also been shown to be the major driver of neurodegeneration [5]. It involves an interplay of fibrillation, interactions with membranous components of the cell, and posttranslational modifications (PTMs) [5–7]. 

Although inclusion bodies in synucleinopathies exhibit differences in size, shape, structure, and locations in the brain [1], the common trait is the presence of filamentous aggregates of α-Syn protein. These α-Syn filaments are highly ordered cross-β-sheet structures formed by the aggregation of the protein [8]. Numerous in vitro studies have demonstrated that α-Syn undergoes a structural transition from unordered state to cross-β-sheet-rich fibrils [9–11], which are similar to α-Syn amyloids found in the inclusion bodies in diseased patients' brains [12–16]. 

However, the aggregation of α-Syn is not a single-state conversion process; instead, it involves the interconversion of multiple conformational states and the formation of oligomeric intermediates [17]. 

These oligomeric intermediates are highly heterogeneous, transient, and metastable and are suggested to be the most toxic species during the fibrillation pathway [17]. However, the transient and highly heterogeneous nature of these oligomeric species makes it challenging to explain their structure–toxicity relationship [17]. 

On the contrary, amyloid fibrils formed as the end-product of the aggregation process are primarily involved in the prion-like propagation of aggregates and contribute significantly to the spread of the disease pathology [18–23]. 

Nonetheless, there have been studies that have found it difficult to induce the α-Syn pathology in mice following intracerebral administration of fibrils [24], conceivably due to the size and dimension of full-length mature fibrils. Instead, several groups have suggested that non-fibrillar or oligomeric species are involved in the spread of α-Syn pathology [25–27]. 

However, considering the toxic nature of oligomers, it is difficult to establish the link between the toxicity and spread by the oligomers. Therefore, the molecular mechanism governing the interplay of oligomers and fibrils needs to be fully understood. 

Further, the polymorphic nature of amyloid fibrils has added another complexity to the field. Detailed structural models of amyloid fibrils and aggregation intermediates have revealed that amyloid fibrils exhibit polymorphism at the molecular level, i.e., a single peptide or protein can form a range of distinct, self-propagating fibrillar assemblies [28]. 

Recent findings from biochemical and structural studies involving cell lines, animal models, and human brain extracts have provided initial evidence that structural variations in amyloid fibrils can be responsible for the observed disease variations [29–35]. 

The fibril growth conditions, such as buffer composition, salts, temperature, etc., profoundly affect the morphology and biological activity of α-Syn fibrils formed in vitro [29,31,33,36–38]. 

This suggests that changes in the solution conditions alter the molecular interactions between the polypeptide chains, leading to different fibril types. Polymorphism can also be observed due to differences in the pattern of inter or intra-residue interactions, the number of amino acid residues constituting protofilaments, their packing, and orientation [39–41]. 

Several other factors are also responsible for the polymorphic nature of amyloids; however, our understanding of how a particular solution condition leads to the formation of different fibril structures is limited. The existence of polymorphs has been linked to the strain phenomenon in prions, where different strains of PrP protein are associated with a range of clinical phenotypes observed in prion diseases [42–44]. 

In vitro, prion strains are characterized by differences in protease resistance, glycosylation profile, electrophoretic mobility, seeding ability, etc. In vivo, they are distinguished based on the clinical signs and symptoms, lesion profile, disease onset, and incubation period [45–47]. 

An increasing body of evidence suggests that α-Syn also exhibits prion-like strain phenomena, which explains its association with various neurodegenerative diseases with distinct clinical and pathological phenotypes [35,48–50]. Lee and co-workers showed the generation of synthetic strains of α-Syn capable of differentially cross-seeding tau for the first time [30]. 

This study formed the basis of later investigations involving α-Syn strains [29,32,33,36,49,51–54]. Recent advances in solid-state NMR (ssNMR) spectroscopy and cryo-electron microscopy (cryo-EM) have further contributed to understanding the molecular-level polymorphism in α-Syn fibrils [55–59]. These fibril polymorphs can be distinguished based on the fibril diameter, presence of twists, number, and packing of protofilaments, side-chain interactions, the secondary and tertiary structure arrangement, etc. [41,57,60,61]. 

Cryo-EM structures of fibrils of wild-type (WT) α-Syn and its mutational variants have also provided novel insights into how disease-associated point mutants of α-Syn alter the fibril structure of the WT protein, suggesting polymorphism within the mutants. Further, characterizing and solving the structure of patient-derived strains can provide direct proof of the existence of α-Syn strains responsible for disease heterogeneity in synucleinopathies.

Overall, various previous studies have suggested that different strains of α-Syn are responsible for the clinical variations observed in synucleinopathies and possibly explain the association of α-Syn aggregates with disease heterogeneity [32–35,52,53]. 

Yet, there are questions about the origin of polymorphism in vivo, propagation of strain-specific properties of fibrils, and factors governing the formation of strains that need attention. 

The present review focuses on the polymorphic nature of α-Syn and describes its role in the disease pathogenesis of PD and related disorders. It discusses the evidence demonstrating that α-Syn can assemble into distinct fibril strains and could be the primary drivers for the disease heterogeneity in synucleinopathies.

2. Clinical and Pathological Features of Synucleinopathies

The aggregation of α-Syn protein is associated with PD and other neurodegenerative disorders, collectively termed synucleinopathies. These include MSA, DLB, PDD, and less characterized neuroaxonal dystrophies [4,48]. 

PD is the most common among all synucleinopathies and has been a prime focus of α-Syn research conducted over the decades. α-Syn misfolds and accumulates in the form of fibrillar inclusion bodies in synucleinopathies. However, the appearance and location of these inclusion bodies vary in different synucleinopathies [62]. 

For instance, neuronal inclusions are present in PD, PDD, and DLB, whereas glial inclusions are formed in MSA [4] and less characterized axonal spheroids in neuro-axonal dystrophies [4]. The existence of diverse clinical and pathological profiles in synucleinopathies raises a fundamental question of how the aggregation of a single protein leads to different diseases.

2.1. Parkinson's Disease (PD)

PD is the second most widespread and complex neurological disorder after Alzheimer's disease (AD) [63]. It is a prevalent, chronic, and progressive neurodegenerative disorder, affecting approximately 1% to 4% of the general population over 60 and 80 years of age [64–66]. 

It involves the accumulation of eosinophilic, round, cytoplasmic LBs and LNs, accompanied by the degeneration of dopaminergic neurons in the substantia nigra pars compacta (SNpc) region of the midbrain [3,67]. 

The loss of dopaminergic neurons results in a decrease in the level of the neurotransmitter dopamine [68], which results in abnormal brain functioning and impairment in motor functioning that leads to PD symptoms. Four cardinal symptoms, such as bradykinesia, resting tremor, postural instability, and rigidity are considered for clinical diagnosis of PD [67]. 

In addition to these motor symptoms, non-motor symptoms like insomnia, constipation, cognitive dysfunction, autonomic failure, and depression are also observed in PD patients [67]. Several studies claim that these nonmotor symptoms and many gastrointestinal (GI) tract problems [69–73] in patients involve the enteric nervous system (ENS) affected in the early stages of PD. 

Experimental data suggest that misfolded α-Syn spreads in a prion-like fashion from ENS to CNS through innervations of the dorsal motor nucleus of the vagus nerve (DMV) [74–76]. However, the factors that cause α-Syn to misfold and aggregate in ENS are not fully known. One of the reported factors is the high prevalence of Enterobacteriaceae within the GI tract that produce extracellular amyloids termed curli fibers [77]. 

These curli amyloids are used for host attachment, tissue invasion, and biofilm production by bacteria. Despite the functional roles of curli fibers, curli-producing Escherichia coli induces GI dysfunction and motor impairment in mice overexpressing α-Syn [78]. 

The amyloidogenic subunit of curli fibrils (CsgA) interacts and accelerates the aggregation of α-Syn and curli expression is indeed required to induce α-Syn associated behavioral deficits [78]. However, further research is needed to decipher the role of bacterial amyloids in promoting α-Syn aggregation and tracing the origin of PD along the gut-to brain axis. In the past few years, the genetics of PD have been studied markedly. 

The SNCA gene has been identified as one of the major genes linked to sporadic and familial PD [63,79]. Mutations in parkin and LRRK2 are the other common genes associated with recessively and dominantly inherited PD, respectively [63,79]. The duplication [80] and triplication [81] of the SNCA gene, the gene encoding for α-Syn, causes the early onset of parkinsonism. 

Along with the multiplication of the SNCA gene [82], single-point mutations are also associated with familial autosomal parkinsonism. To date, seven missense mutations are known to be associated with familial PD: A30P [83], E46K [84], H50Q [85,86], G51D [87], A53T [88], A53E [89], and the newly discovered A53V [90]. Aggregation and amyloid formation of these familial mutants have been extensively studied in vitro [12,16,91–101]. 

A30P, A53E, and G51D slow down the aggregation of WT α-Syn, whereas E46K, A53T, H50Q, and A53V accelerate the same [10,91–95,99,101,102]. However, their aggregation rate in vitro does not correlate with the disease onset, suggesting an interplay of oligomerization and fibrillation in vivo, which dictates the disease progression and onset in familial forms of PD [103]. 

Apart from genetic factors, ~95% of cases of PD are sporadic [63,104] and are associated with cellular and environmental risk factors. These include the presence of polyamines, chaperons, glycosaminoglycans, membranes, metal ions, exposure to pesticides, and heavy metals, etc. [6,105–114]. These risk factors uniquely modulate the misfolding and aggregation of α-Syn associated with PD pathogenesis [94,115].

2.2. Multiple System Atrophy (MSA)

MSA is a rare sporadic neurodegenerative disease and becomes progressively chronic with an autonomic failure along with symptoms of parkinsonism or cerebellar ataxia [116,117]. The prevalence of the disease is 2.4–4.9 cases per 100,000 population [117]. 

MSA affects both genders equally, and the incidence is more prevalent in people above 60 years of age [118,119]. MSA is a more devastating and aggressive neurological disorder than other synucleinopathies because of more rapid clinical progression with a much shorter disease duration (6–9 years) than PD (~12 years) [117,119]. Many patients diagnosed with PD are found to have MSA after autopsy [120]. 

This misdiagnosis of MSA happens due to overlapping symptoms of the two disorders, suggesting that the prevalence of MSA is more than the estimation [117]. MSA was previously described by three clinical syndromes, striatonigral degeneration, olivopontocerebellar atrophy, and Shy-Drager syndrome, formerly thought of as separate disorders [121,122]. 

Later, it was found that these syndromes often coexist clinically and pathologically and give the impression of a common underlying disease, which was termed MSA. Clinically, MSA patients display numerous combinations of symptoms like parkinsonism, cerebellar ataxia, progressive autonomic failure, and pyramidal signs. 

Based on that, they are categorized into two main clinical subtypes: (i) the parkinsonian subtype (MSA-P), with parkinsonism as a predominant feature, and (ii) the cerebellar subtype (MSA-C), with cerebellar ataxia as a major trait [123–125]. The occurrence of MSA-P and MSA-C ranges from 2:1 to 4:1, respectively [126–128]. 

However, the MSA-C subtype is majorly found and predominates in the Japanese population [118]. The histopathological hallmark of MSA is the presence of GCIs formed in the oligodendrocytes in the brain, which show strong immunoreactivity with α-Syn [129]. This makes it pathologically distinct from other synucleinopathies as it shows an abnormal accumulation of α-Syn protein in oligodendrocytes, unlike PD and DLB, where α-Syn inclusions are found in neurons [130]. 

Although some MSA patients have shown the presence of α-Syn aggregates in the nucleus and cytoplasm of neurons [131], these neuronal inclusions are less prevalent than GCIs in MSA. Furthermore, the mechanism of aberrant accumulation of α-Syn in glial cells is unclear, as there is no or minimal expression of α-Syn in mature oligodendrocytes [132–135]. 

A few reports have suggested the possibility of transmission/translocation of α-Syn from neurons to oligodendrocytes [136,137]. However, this mechanism is not completely known, and the exact origin of α-Syn inclusions in oligodendrocytes remains obscure.

2.3. Dementia with Lewy Bodies (DLB)

DLB is the second most common α-synucleinopathy after PD [138–140]. The incidence and prevalence rates of DLB are not accurate because of overlapping symptoms with AD, PDD, vascular dementia, and other synucleinopathies. 

It is estimated that its prevalence is approximately 0.4%, i.e., 400 people per 100,000 population in the elderly [141], accounting for 5% of all dementia cases and between 1–4 people per 1000 population [142]. Initially, DLB was identified as dementia syndrome [143]. Later, the inclusion bodies from DLB patients were found to be highly immunoreactive to α-Syn [3]. After that, it was categorized as one of the main types of synucleinopathies. 

While PD is characterized by a decline in motor abilities, DLB is mainly characterized by dementia. Instead, a DLB patient may or may not suffer from Parkinsonism [144]. Unlike PD, the LBs in DLB patients are mainly localized and distributed in the cytoplasm of cortical neurons of the diseased brain [145]. Cortical LBs are eosinophilic, rounded, and generally lack the halo structure seen in classical LBs. 

Clinically, it is characterized by dementia, memory impairment, parkinsonism, and changes in behavior, sleep, and autonomic bodily and cognitive functions [143]. Many DLB patients also show significant Aβ deposition in the cortical area, along with the formation of LBs [146,147]. Several compelling pieces of evidence support the synergistic relationship between Aβ and α-Syn [148–154]. 

In vitro studies have demonstrated that α-Syn and Aβ can cross-seed, form hetero-oligomers, and promote the aggregation of each other [148,151,152,154]. Consequently, shorter disease duration and more rapid decline have been observed in patients with AD pathology and dementia [155,156]. 

A recent study provided direct experimental evidence of the effect of co-pathology where Aβ plaques promoted the seeding and spreading of α-Syn in mice with abundant Aβ pathology [157]. Still, clinical and pathological studies suggest that DLB overlaps more with PDD than AD [158]. Overall, the synucleinopathies are associated with abnormal deposition of α-Syn but still vary in terms of clinical and pathological phenotypes. 

Despite several studies, the reason for this clinicopathological divergence remains a puzzle. 3. Misfolding and Aggregation of α-Syn Monomeric α-Syn is an intrinsically disordered protein and tends to adopt multiple conformational states affected by solution conditions like pH, temperature, ionic strength, viscosity, etc. [9]. 

For instance, the presence of alcohols (ethanol) or fluoroalcohols (TFE or HFiP) induces the formation of β-sheet or α-helical partially folded structures of α-Syn, depending on the concentration and the type of alcohol used [9]. α-Syn was first isolated from the antisera raised against the cholinergic vesicle from Torpedo californica, an electric ray [159]. 

Due to its location at the nuclear envelope and presynaptic terminal, it was named synuclein [159,160]. α-Syn protein is encoded by the SNCA gene mapped to the human chromosome 4q21.3-q22 [160]. α-Syn was also discovered by Ueda et al. [161] during the study of amyloid plaques from the brains of patients with Alzheimer's, in which they identified a non-amyloid-β component (NAC) in the plaques, which was derived from a precursor protein, NACP [161]. It was detected in all the tissues except the liver, and the highest concentration was found in the brain [161]. 

Later, it was found that NACP is a natively unstructured and human homolog of α-Syn [162–164]. Extensive biophysical and structural characterization revealed that α-Syn is a 140 amino acid protein with a molecular weight of ~14.4 kDa and pKa of 4.7 [165]. 

It is known to be involved in neurotransmitter release, vesicle trafficking, and SNARE complex assembly in the brain, though its exact physiological role is still obscure [160,165]. α-Syn consists of three domains, N-terminal, NAC, and C-terminal domains (Figure 1A). 

The N-terminal of α-Syn (residues 1–60) is an amphipathic, lysine-rich, and lipid-binding domain, which interacts with the membranes [109]. It contains 11 aa repeats, including conserved KTKEGV hexameric motifs [109]. These repeats are conserved across species as well as among three synuclein members. Although α-Syn remains unordered in an aqueous solution, it adopts a helical structure involving N-terminus upon association with negatively charged small unilamellar vesicles or detergent micelles [109,166,167]. 

Interestingly, all the familial mutations of αSyn also occur in the N-terminus region [83–90] (Figure 1B). The NAC domain of α-Syn (residues 61–95) forms the protein's hydrophobic core and is prone to aggregation. This domain is responsible for the conversion of α-Syn from an unordered state to β-sheet-rich fibrils [168,169]. NAC is also part of the membrane-binding domain of the protein [167]. 

The conformational ensemble of α-Syn monomer indeed consists of structures that are similar to the membrane-bound state of α-Syn [170]. These contain partially folded helices involving N-terminuses and NAC domains similar to the 1XQ8 model [170], suggesting that such a type of folding might also be present in the early stages of aggregation. 

The C-terminal domain (residues 95–140) is flexible and predominantly consists of negatively charged amino acids [165]. The disordered carboxy-terminal part is also involved in the nuclear localization of α-Syn protein and its interaction with metal, small molecules, and proteins [171–175].

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