Brain-derived and in vitro-seeded alpha-synuclein fibrils exhibit distinct biophysical profiles

The alpha-synuclein (αSyn) seeding amplification assay (SAA) that allows the generation of disease-specific in vitro seeded fibrils (SAA fibrils) is used as a research tool to study the connection between the structure of αSyn fibrils, cellular seeding/spreading, and the clinico-pathological manifestations of different synucleinopathies. However, structural differences between human brain-derived and SAA αSyn fibrils have been recently highlighted. Here, we characterize biophysical properties of the human brain-derived αSyn fibrils from the brains of patients with Parkinson’s disease with and without dementia (PD, PDD), dementia with Lewy bodies (DLB), multiple system atrophy (MSA) and compare them to the ‘model’ SAA fibrils. We report that the brain-derived αSyn fibrils show distinct biochemical profiles, which were not replicated in the corresponding SAA fibrils. Furthermore, the brain-derived αSyn fibrils from all synucleinopathies displayed a mixture of- ‘straight’ and ‘twisted’ microscopic structures. However, the PD, PDD, and DLB SAA fibrils had a ‘straight’ structure, whereas MSA SAA fibrils showed a ‘twisted’ structure. Finally, the brain-derived αSyn fibrils from all four synucleinopathies were phosphorylated (S129). Interestingly, phosphorylated αSyn were carried over to the PDD and DLB SAA fibrils. Our findings demonstrate the limitation of the SAA fibrils modelling the brain-derived αSyn fibrils and pay attention to the necessity of deepening the understanding of the SAA fibrillation methodology.


Introduction
Synucleinopathies are all characterized by abnormal accumulation of the protein alpha-synuclein (Syn) in the brain.However, they are clinically and neuropathologically highly heterogeneous diseases with prominent disease-specific differences in presentation of symptoms, rate of disease progression, and the brain regions and cell types that are vulnerable to Syn deposition and neuronal death.In Parkinson's disease (PD), Parkinson's disease with dementia (PDD), and dementia with Lewy bodies (DLB), Syn aggregation is found in the neuronal soma as Lewy bodies (LBs) and in the axons and dendrites as Lewy neurites (LNs) (Figure 1) (Forno, 1988;Spillantini et al., 1997;Braak et al., 2003).Furthermore, the astroglial accumulation of Syn is a prominent but so far underdiagnosed pathology in these Lewy body disorders (Altay et al., 2022).The Syn pathology in multiple system atrophy (MSA) is primarily found in oligodendrocytes as glial cytoplasmic inclusions (GCIs) (Papp et al., 1989).
The mechanistic link between Syn and clinico-pathological diversity of the synucleinopathies is hypothesized to be the different fibrillar 'strains' of Syn in analogy to prion disease.A strain is generated when Syn monomers fold into specific fibrillar forms with distinct conformational and bio- atomic level, PD and DLB strains share a "Lewy fold" structure at the fibrillar core and comprise a single protofilament (Yang et al., 2022).MSA strains are twisted with two protofilaments intertwined, forming a different core structure to the "Lewy fold" (Schweighauser et al., 2020).At a cellular level, PD is characterized by significant neuronal loss at the brainstem, especially substantia nigra (SN), highlighted in blue.Lewy body (LB) and Lewy neurite (LN) accumulate in neurons.Syn also accumulates in astrocytes, forming astroglial pathology.In DLB, the brainstem and neocortex are the most affected regions, highlighted in green.Here, LB and LN accumulate in neurons and astroglial pathology is also observed.In MSA, the cerebellum, basal ganglia and brainstem are the most affected, highlighted in red.Here, Syn accumulates as glial cytoplasmic inclusions (GCIs) in the oligodendrocytes.The 3D structures of the Syn fibrillar cores were generated using PyMOL, and the PDB structures from Schweighauser et al. (2020); Yang et al. (2022).
logical characteristics (Melki, 2017).Under the disease condition, misfolded and aggregated Syn recruits normal, soluble endogenous Syn to aggregate, and this self-perpetuating process spreads throughout the brain-periphery axis.Several studies have generated Syn fibrils in vitro using various experimental conditions to characterize the presence of different strains (Bousset et al., 2013;Peelaerts et al., 2015;Li et al., 2018;Guerrero-Ferreira et al., 2019;Lau et al., 2020), but their relevance to human condition remains questionable.However, some studies have also examined Syn fibrils extracted from the human brain tissue of different synucleinopathies and these have demonstrated that the human-derived Syn strains exhibit distinct structures (Schweighauser et al., 2020), infectivity and bioactivity in cells (Woerman et al., 2018a(Woerman et al., , 2019;;Ayers et al., 2022) and animals (Prusiner et al., 2015;Lau et al., 2020;Holec et al., 2022;Peng et al., 2018).The intrinsic structure of Syn proteoforms may also depend on the local conditions of the brain region where they are formed and thus may vary within a single disease entity (Peng et al., 2018;Lau et al., 2020;Schweighauser et al., 2020).Future studies must comprehensively map the distinct fingerprints of human brain-derived Syn strains to the disease.
Seeding amplification assays (SAAs) have become more applicable due to their feasibility in amplifying in vitro-seeded Syn fibrils (SAA fibrils) from human biosamples.The SAA reaction incorporates a key characteristic of pathological Syn, which is its ability to induce aggregation of monomeric Syn into different complex conformers.This 'seeding' property is the critical mechanism of SAA.When pathological Syn is present in the biosample, it seeds aggregation of the recombinant Syn in the reaction, which is monitored in real-time using a fluorophore such as Thioflavin-T (ThT).SAA, together with different biosamples such as cerebrospinal fluid (CSF) (Fairfoul et al., 2016;Poggiolini et al., 2021), brain homogenates (Groveman et al., 2018;Sano et al., 2018), and skin punctures (Mammana et al., 2020;Kuzkina et al., 2021), has presented its potential as a diagnostic method but also as a research tool to amplify disease-relevant fibrils.The newly amplified SAA fibrils are assumed to encode the intrinsic properties of the original seeding fibrils, thus being representative of the disease-specific strains.Therefore, using the 'model' SAA fibrils, studies have shown disease-specific structural, biochemical and phenotypic differences, suggesting the presence of distinct conformational Syn strains in different synucleinopathies (Strohaker et al., 2019;Shahnawaz et al., 2020;Van der Perren et al., 2020;Frieg et al., 2022).Recent studies have, however, demonstrated significant differences between the human brain-derived and SAA fibrils in their structure (Lövestam et al., 2021) and seeded pathology in vivo (Van der Perren et al., 2020).Nevertheless, there is currently a limited understanding of whether the resulting SAA fibrils truly preserve the original properties of the seeding fibrils across all the major synucleinopathies.
Here, we aim to investigate the biophysical differences between 1) the brain-derived Syn fibrils in different synucleinopathies and 2) the brain-derived Syn versus the corresponding SAA fibrils.We examine the brain-derived Syn fibrils extracted from the brains of patients with PD, PDD, DLB, and MSA and the brain-derived versus SAA Syn fibrils and show striking differences in their biochemical profile, structure, and phosphorylation pattern.Our findings reveal further evidence supporting the molecular diversity among Syn fibrils from different synucleinopathies.
Also, our study highlights the limitations of the SAA fibrils to fully mirror the brain-derived Syn fibrils and the need to study further the mechanism of seeding amplification and the application of SAA fibrils.

Results
Detection of differential Syn seeding activity from brain-derived fibrils of PD, PDD,

DLB, and MSA
We extracted Syn fibrils from the human brain of patients with PD (n = 3), PDD (n = 3), DLB (n = 3), MSA (n = 3) and healthy controls (n = 3) (Table S1).The entorhinal cortex was used for PD, PDD, and DLB, and the striatum for MSA, as these are one of the main affected regions with Syn pathology in each disorder.The brain-derived fibrils were diluted 1: 1,000 and used as a seed in an SAA reaction significantly lower in PDD/DLB than in PD/MSA (Figure 2B,C).As a result, the area under the curve (AUC) was statistically higher in PDD/DLB than in PD/MSA (Figure 2D).However, the maximum rate of increase in fluorescence ( MAX ) was not statistically different among the diseases (Figure 2E).
Overall, the SAA kinetic parameters suggest PDD and DLB brain-derived Syn fibrils have a more aggressive seeding capacity than PD and MSA.

Brain-derived and SAA 𝛼Syn fibrils display distinct biochemical profiles
At the end of the SAA reaction, the SAA Syn fibrils were collected by ultracentrifugation.Having established that prion strains can be characterized by distinct biochemical profiles (Lau et al., 2020;
The weakest GdnHCl (1 M) treatment completely denatured the PD and MSA brain-derived Syn fibrils, as they were no longer visible on the immunoblot (Figure 3).This result was consistent among all the three cases analyzed (Figure S2).Higher GdnHCl concentrations of 2 M and 3 M denatured PDD and DLB brain-derived Syn fibrils, respectively (Figure 3).The results illustrate that PD and MSA brain-derived Syn fibrils have weaker biochemical stability than PDD and DLB fibrils.
The disappearance of the bands on the immunoblot after GndHCl treatment limited the comparative study of the Syn fibrils between synucleinopathies.Therefore, we repeated the experiment using PK alone to improve the visualization of the biochemical profiles.The brain-derived Syn fibrils were treated with five different PK concentrations starting from 0 mg/ml, incrementing to 1 mg/ml.PD cases contained mostly monomeric Syn (15 kDa) that immediately degraded at the lowest PK concentration (1 ug/ml) (Figure 4A).In contrast, in PDD, DLB, and MSA cases, high molecular weight (MW) bands above 15 kDa were observed, indicative of Syn aggregates (Figure 4B-D).
Furthermore, lower MW bands ( 13, 10, 7 kDa) were also observed.As the PK concentration increased, the PDD and DLB fibrils gradually degraded, as the high MW bands started to disappear and low MW bands below 15 kDa began to appear (Figure 4B,C).Similarly, the MSA fibrils gradually degraded as the PK concentration increased (Figure 4D).However, low MW bands below 15 kDa were less prominent than those of PDD and DLB.
Although we experimented with an identical amount of total protein (10 ug), the amount of Syn fibril could differ in each brain-derived sample.Using immunohistochemistry, we observed a lower load of Syn pathology in PD and MSA brains than in PDD and DLB (Figure S3).In addition, the brainderived fibrils were immunogold-labelled with anti-pSyn and negatively stained for transmission electron microscopy (TEM).The samples were imaged with TEM at low magnification to observe  Syn fibril density and distribution.PD and MSA cases contained 1-3 Syn fibrils per region of interest (ROI), whereas PDD and DLB cases contained 3-5 Syn fibrils per ROI (Figure S4).Adjacent slot blots also confirmed a higher amount of Syn fibrils in PDD and DLB than in PD and MSA brainderived samples.Therefore, lower fibril concentration in the PD and MSA brains could have limited the observation of the PK-resistant peptides using immunoblotting.
The SAA fibrils revealed markedly different biochemical profiles to the brain-derived Syn fibrils.
We observed identical profiles among all SAA fibrils from PD, PDD, DLB, and MSA.PK-resistant bands of 7 and 10 kDa started to appear after treatment with 2 M GdnHCl (Figure 3).However, with increasing concentrations of GdnHCl, specific PK-resistant peptides appeared, consisting of five bands (7, 11, 12, 13, 14 kDa) (Figure 3).These findings suggest that harsher denaturing conditions are required to destabilize the SAA fibrils and expose PK digestion sites.We concluded that SAA Syn fibrils are more stable and characterized by distinct biochemical properties than the brainderived Syn fibrils.

Brain-derived and SAA 𝛼yn fibrils show structural differences
Next, we used TEM to investigate the structure of the brain-derived and SAA Syn fibrils.The brain-derived Syn fibrils from PD, PDD, and DLB exhibited two structures in TEM: straight and twisted (Figure 5A, Figure S5).The fibrils from MSA were predominantly straight, with the rare presence of twisted type.Therefore, we only considered straight fibrils when comparing the dimensions of the fibrils among synucleinopathies.The length of the fibrils from different synucleinopathies ranged between 68 to 885 nm.Only the PDD fibrils were significantly longer than DLB and MSA (Figure 5B).The width ranged from 7 to 21 nm, and there were significant differences between the diseases.PD had the widest fibrils, followed by MSA/DLB and PDD (Figure 5C).
On the other hand, the SAA fibrils demonstrated a single dominant structure.PD, PDD, and DLB SAA fibrils were all straight, and MSA SAA fibrils were all twisted (Figure 5A).Unlike the brainderived fibrils, the SAA fibrils were highly clustered.The length of the SAA fibrils ranged from 104 to 1008 nm, and PDD/DLB SAA fibrils were significantly longer than those of PD/MSA (Figure 5D).
The width ranged from 8 to 25 nm, and the widths of the MSA SAA fibrils were distributed into two groups due to the alternating widths arising from the twisted structure.Unlike the brain-derived fibrils, the average widths of the SAA fibrils were similar between different synucleinopathies (Fig- ure 5E).
Overall, in DLB, the SAA fibrils were significantly longer and wider than the brain-derived fibrils (Figure S6).SAA fibrils from PDD were wider than the brain-derived fibrils but not different in length.The dimensions of the brain-derived and SAA fibrils from PD and MSA were not significantly different.However, it was difficult to compare the dimensions of the MSA fibrils as the brain-derived and SAA fibrils had different morphologies.Our observations demonstrate that brain-derived and SAA Syn fibrils exhibit distinct TEM structures.

Brain-derived and SAA 𝛼Syn fibrils have distinct phosphorylation patterns
To confirm the identity of the fibrils imaged by TEM, we used immunogold with Syn fibril conformation specific (MJFR-14) and pS129-specific (anti-pSyn) antibodies.Both brain-derived and SAA fibrils from all four synucleinopathies were probed with MJFR-14, confirming that the fibrils were Syn fibrils (Figure 6A).The brain-derived fibrils were also strongly labeled with anti-pSyn.
MJFR-14 and anti-pSyn, however, did not label the twisted brain-derived fibrils from PD, PDD, and DLB (Figure 6B).The SAA fibrils from PD and MSA were not labeled with anti-pSyn, but PD and MSA SAA fibrils were weakly labeled (Figure 6A).
We performed a slot blot using identical antibodies to confirm the immunogold-labelling results.
On the slot blot, DLB brain-derived Syn fibrils showed the strongest signal with MJFR-14 and anti-pSyn, followed by PDD, MSA, and PD (Figure 6C).Interestingly, the anti-pSyn signal from the SAA fibrils was markedly different from the brain-derived fibrils.In parallel to the anti-pSyn immunogold results (Figure 6A), PDD SAA fibrils showed the highest anti-pSyn signal (Figure 6D).The DLB SAA fibrils were also weakly phosphorylated, while those of PD and MSA were negative.Our results demonstrate that the brain-derived and SAA Syn fibrils display different patterns of S129 phosphorylation.

Discussion
Strain-like properties of the brain-derived Syn fibrils from different synucleinopathies.
Findings in this study provide evidence of distinct 'strain-like' properties of the brain-derived Syn fibrils from different synucleinopathies.First, faster seeding kinetics was observed in the brainderived Syn fibrils from PDD and DLB than those from PD and MSA, suggesting a distinct 'strain' characterized by an aggressive seeding capacity.In contrast, those from PD and MSA are discrete that pSyn enhances Syn aggregation in vitro (SH-SY5Y cells) and in vivo (rodent models) (Smith et al., 2005;Karampetsou et al., 2017).However, contradicting findings have also been reported, suggesting a neuroprotective role of pSyn (Ma et al., 2016;Ghanem et al., 2022).Although the biological role of pSyn is unclear, we report that a higher amount of pSyn may contribute to the more aggressive seeding kinetics observed in PDD and DLB brain-derived Syn fibrils than those of PD and MSA.
Further evidence of 'strain-like' property was exhibited by the distinct biochemical profiles of the brain-derived Syn fibrils from different synucleinopathies.The brain-derived Syn fibrils from PD and MSA were more susceptible to GndHCl denaturation than PDD and DLB, indicating conformational instability of PD and MSA brain-derived Syn fibrils.Nevertheless, it must be noted that the rapid denaturation of PD fibrils might be due to a low concentration of Syn fibrils, as demonstrated by a lower Lewy pathology load in the entorhinal cortex.A previous study also showed that human brain-derived Syn fibrils from DLB were more stable than MSA by using GdnHCl treatment (Lau et al., 2020).Interestingly, the less stable MSA fibrils propagated faster in TgM83 mice than the more stable DLB fibrils.Such inverse correlation between conformational stability and disease propagation has been characterized in prion disease (Legname et al., 2006).Therefore, our findings on the differential conformational stability could reflect the strain-specific characteristics of Syn fibrils.Subsequently, it could be related to the fast disease progression of MSA compared to PDD and DLB.
Subsequently, by assessing the PK-resistance of the Syn fibrils, we illustrate further the molecular diversity of Syn fibrils.The sizes of the low MW PK-resistant fragments differed between diseases and cases within each disease.These distinct PK-resistant peptides suggest the presence of conformational variants indicative of Syn fibrillar strains within a single disease entity.Similarly, Guo and colleagues reported evidence of distinct Syn fibrillar conformers in different PDD patients' brains (Guo et al., 2013).Interestingly, MSA fibrils also showed distinct structural features compared to those from PD, PDD, and DLB.The fibrils from MSA were predominantly 'straight', and those from PD, PDD, and DLB were 'straight' and 'twisted'.While the 'straight' brain-derived fibrils were densely decorated with MJFR-14 and anti-pSyn antibodies, confirming their identity as a phosphorylated Syn fibril, the 'twisted' fibrils were not recognised by these proteins.Similarly, Spillantini and colleagues also reported a finding of 'twisted' fibril from the cingulate cortex of a DLB brain with no labeling by the Syn-specific antibody (Spillantini et al., 1998).Further ultrastructure analysis is essential to reveal the identity of the unlabeled twisted fibrils.
The methodological limitations of the structural characterisation in this study should be considered.Cryo-electron microscope (EM) ultrastructure revealed the twisted structures of the MSA brain-derived Syn fibrils and single twisted protofilament structures in PD, PDD and DLB brains (Yang et al., 2022).Our TEM structures may differ due to the selection of different brain regions compared to those used in the literature and the limitation of the resolving power of TEM.Future cryo-EM structural studies of Syn fibrils from not only different synucleinopathies but also different brain regions and subtypes of PD are required to establish a more profound understanding of the molecular diversity of the Syn conformers in synucleinopathies.
Distinct biophysical properties between brain-derived and SAA fibrils.
In order to assess whether the SAA fibrils truly replicate the brain-derived fibrils (the seed), we compared the biophysical characteristics of the brain-derived Syn fibrils and their respective SAA fibrils.First, by examining the resistance to GdnHCl and PK digestion, we illustrated a prominent biochemical difference between the brain-derived and SAA fibrils.High concentrations (4-5 M) of GdnHCl completely denatured the brain-derived fibrils, whilst SAA fibrils were still resistant.This indicates that the SAA fibrils are more stable and PK-resistant than their respective brain-derived fibrils.Furthermore, the SAA fibrils from all four synucleinopathies had identical patterns of PK resistance.The SAA amplifies fibrils with uniform biochemical features, losing the intrinsic properties of the original seed fibril and failing to replicate the biochemical properties of the original brain-derived fibrils.
The differences in biochemical stability might arise from the distinct distribution of the fibrils.
SAA fibrils were clustered in negative-stain TEM, whereas the brain-derived fibrils were distributed in single filaments (Figure 5).Also, this study used a PIPES-based buffer to amplify the SAA fibrils (Poggiolini et al., 2021).As the microenvironmental context of Syn amplification substantially affects the newly amplified fibrils (Peng et al., 2018;Gustavsson et al., 2021), the artificial in vitro environment can favor the formation of specific three-dimensional structures that are more conformationally stable.
Next, we showed that brain-derived fibrils display a mixture of straight and twisted structures, while SAA fibrils have a unified structure: straight (PD, PDD, DLB) or twisted (MSA).The striking structural difference may be due to a dominant seeding fibril within the mixture of brain-derived fibrils.Interestingly, straight fibrils were dominant in MSA brains, but the corresponding SAA fibrils were twisted.The commonality of a particular structure in a fibrillar mixture may not be a determinant of a dominant seeding fibril.Other factors might contribute, such as metal ions (Uversky et al., 2001) and post-translational modifications (PTMs), which also affect seeding efficiency (Balana et al., 2023).Also, our PIPES-based SAA reaction buffer with 150 mM NaCl is likely to have influenced the SAA fibril structure.However, the discrete structure of the MSA SAA fibrils compared to those of PD, PDD, and DLB implies that a complex matrix of intrinsic factors within the brain-derived fraction impacts seeding amplification and the resulting fibrillar structure.
A recent cryo-EM analysis of the MSA brain-derived and SAA fibrils revealed striking structural differences (Schweighauser et al., 2020;Lövestam et al., 2021).A few significant differences included the protofilament fold, the inter-protofilament interface and the geometry of specific residues that shape the hydrophobic core.As the TEM has restricted resolution, future cryo-EM analyses are imperative to connect the structure to the disease heterogeneity and progression.
Lastly, we investigated the differences in the phosphorylation level between the brain-derived and SAA Syn fibrils to identify differences in the PTM patterns.The brain-derived fibrils generally showed a higher phosphorylation level than the SAA fibrils.PDD and DLB SAA fibrils were weakly phosphorylated and decorated with pSyn immunogold labels at the rare end of the fibrils (Fig- ure 6).Combining these findings, we can argue the possibility that the SAA fibrils extended from pSyn species dissociated from the brain-derived fibrils.Therefore, the amount of initial pSyn fibrils in the seed might be a critical determinant of the phosphorylation state of the resulting SAA fibrils.
In summary, the distinct biochemical profiles and structures of the brain-derived Syn fibrils from different synucleinopathies provide supporting evidence of molecular diversity of brain-derived Syn fibrils among different synucleinopathies and individual patients.Moreover, the SAA fibrils failed to adopt the biochemical, structural and PTM properties of the seed brain-derived Syn fibrils, which addresses a significant limitation of SAA in replicating the intrinsic biophysical properties of the seed fibrils.Therefore, our finding highlights the necessity of re-evaluating the SAA seeding mechanism and its capacity to generate disease-relevant Syn fibrils used in different in vitro and in vivo models.Furthermore, it remains essential to investigate the human-derived Syn fibrils in the different brain regions and in other peripheral sites to explore Syn strains and how they may affect the progression of pathology.They also remain the most disease-specific fibrils used in cellular and animal models.

Patient Collection
Brain tissues from patients with PD and PDD were obtained from the Parkinson's UK Brain Bank  S1.

Extraction of 𝛼Syn fibrils from the human brain
Sarkosyl insoluble Syn fibril was extracted from the brains of PD (n=3), PDD (n=3), DLB (n=3), MSA (n=3) patients and healthy controls (n=3).The entorhinal cortex was selected for PD, PDD, and DLB, and the striatum was selected for MSA.The extraction protocol was adapted from the method presented by Schweighauser and colleagues (Schweighauser et al., 2020).Brain tissues (0.5 g) were homogenized in an extraction buffer of 10 mM Tris-HCl, 0.8 M NaCl, 10% sucrose and 1 mM EGTA (pH 7.5).Sarkosyl was added to a final concentration of 2% and incubated for 30 min at 37 °C.
Homogenates were centrifuged at 10,000 g for 10 min at 4 °C.The supernatants were centrifuged at 100,000 g for 60 min at 4 °C.The pellet was washed, resuspended in 500 µl/g of extraction buffer, and centrifuged at 500 g for 1 min at 4 °C.The resulting supernatants were diluted 1/3 in buffer consisting of 50 mM Tris-HCl, 0.15 M NaCl, 10 % sucrose, and 0.2% sarkosyl (pH 7.5).The diluted supernatant was centrifuged at 100,000 g for 60 min at 4 °C.The resulting pellet was washed and resuspended in 250 µl/g of 30 mM Tris-HCl (pH 7.5), being the sarkosyl-insoluble fraction.The protein concentration was determined using the bicinchoninic acid assay (BCA) (Thermo Scientific).
The sarkosyl insoluble fraction was aliquoted and stored at -80 °C.
Syn seeding amplification assay (SAA) The brain-derived Syn fibrils were diluted 1:1,000 in 30 mM Tris-HCl (pH 7.5), and 2 µl was added to 98 µl of the reaction mixture consisting of 100 mM PIPES (pH 7), 150 mM NaCl, 0.1 mg/ml of recombinant Syn (rPeptide) and 10 µM thioflavin-T (ThT) to a final reaction volume of 100 µl.Before use, the recombinant Syn was filtered through a 100 kDa molecular weight cut-off (MWCO) filter.The reaction mixture was loaded onto a black 96-well plate with a clear bottom (Nalgene Nunc).The plate was sealed with a plate sealing film (Thermo Scientific) and incubated at 42 °C in a BMG FLUOstar Omega plate reader for 100 h with cycles of 1 min shaking (400 rpm double orbital) and 1 min rest.ThT fluorescence measurement (450 nm excitation and 480 nm emission) was taken every 30 min.Each sample was run in six replicates.The reaction end-products were ultracentrifuged at 100,000 g for 1 h at 4 °C and collected as SAA fibril.

Figure 1 .
Figure 1.Distinct Syn strains are associated with different neuropathological and clinical hallmarks of PD, DLB, and MSA.Syn misfolds and aggregates into fibrils with characteristic conformations.At the

Figure 2 .
Figure 2. Syn SAA seeded with Syn fibrils from PD, PDD, DLB, and MSA brains.(A) SAA was performed with sarkosyl insoluble Syn fibrils extracted from PD (n = 3), PDD (n = 3), DLB (n = 3), and MSA (n = 3) brains.The curves represent an average of six replicates.(B) Time to reach 50% of the maximum fluorescence ( 50 ).(C) The lag phase was taken at the time point where each positive reaction exceeded the threshold (RFU ≥ 5 SD).(D) Area under the curve (AUC).(E) The largest increase of fluorescence per unit of time (V MAX ).(B-E) Plotted values represent the six replicates of the three cases for each disease (N = 18).RFU, relative fluorescence unit; SD, standard deviation.*P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.005

Figure 3 .
Figure 3. Brain-derived and SAA Syn fibrils exhibited distinct biochemical profiles.Brain-derived and SAA Syn fibrils were subjected to denaturation with increasing concentration of GdnHCl (0-5 M) and to PK digestion (1 µg/ml).The antibody clone 42 (BD Biosciences) was used to reveal the PK-resistant peptides.Immunoblots of one representative case from PD, PDD, DLB, and MSA are presented.The brain-derived and SAA fibrils are on the top and bottom rows, respectively.The molecular weights of the protein standards are shown in kilodaltons (kDa).Black arrows mark the five PK-resistant peptides revealed in the SAA fibrils.SAA, seeding amplification assay; GndHCl, guanidine-hydrochloride; PK, proteinase-K.

Figure 4 .
Figure 4. Proteinase-K degradation patterns of the brain-derived Syn fibrils.The brain-derived Syn fibrils from (A) PD, (B) PDD, (C) DLB, and (D) MSA were subjected to increasing concentrations of PK at 0, 0.001, 0.01, 0.1, and 1 mg/ml, represented by the escalating triangular bar.Western blot was performed using the antibody clone 42 (BD Biosciences).The molecular weights of the protein standards are shown in kilodaltons (kDa).

Figure 5 .
Figure 5. Transmission electron microscopy revealed different structures of brain-derived and SAA Syn fibrils.(A) Electron microscope image of negatively stained brain-derived (BD) and SAA fibrils from PD, PDD, DLB, and MSA brains.(B,C) The lengths and widths of brain-derived fibrils.(D,E) The lengths and width of SAA fibrils.MSA SAA fibrils were twisted with alternating widths, resulting in two clusters of measurements.A total of 30 fibrils from each case were measured and plotted (N = 90).Scale bar = 50 nm.*P ≤ 0.05, **P ≤ 0.01, **** P ≤ 0.001.

'Figure 6 .
Figure 6.Brain-derived and SAA Syn fibrils showed distinct phosphorylation patterns.(A) Electron microscope image of brain-derived and SAA Syn fibrils from PD, PDD, DLB, and MSA brains labeled with fibril conformation-specific (MJFR-14) and anti-pSyn (pS129) antibodies.(B) The twisted Syn fibrils from PD, PDD, and DLB brains were not labeled for MJFR-14 and pS129.Scale bar = 50 nm.(C,D) Semi-quantification of Syn fibrils and pSyn confirm the different patterns of Syn phosphorylation between brain-derived and SAA Syn fibrils.The amount of Syn fibrils and pSyn in (C) brain-derived and (D) SAA fibrils was determined using a slot blot.Two µg of proteins were filtered on a nitrocellulose membrane and probed with MJFR-14 and pS129 antibodies.The semi-quantitative measurement was done by averaging the measurements of three cases from each disease.Error bars indicate ± one standard deviation (SD).* P ≤ 0.05.
group has also shown different SAA kinetics in the CSF samples of PD patients divided into four different subtypes based on their baseline and progressive motor and non-motor symptoms (Lawton et al., 2018), suggesting Syn strain heterogeneity within the same disease (Poggiolini et al., 2021).Finally, unlike PDD and DLB, MSA Syn fibrils gradually disappeared upon PK treatment without being digested into small fragments.The lack of PKresistant peptides in MSA indicates a readily digestible property of MSA Syn fibrils.Together with its conformational instability, these biochemical properties might indicate a specific Syn strain in MSA.

(
Imperial College London, UK) in accordance with approved protocols by the London Multicentre Research Ethics Committee.Brain tissues from patients with DLB, MSA and healthy controls were collected from the Oxford Brain Bank (OBB, University of Oxford, UK) in accordance with approved protocols by the South Central -Oxford C Research Ethics Committee (ref 23/SC/0241).All participants had given prior written informed consent for the brain donation.Both brain banks comply with the requirements of the Human Tissue Act 2004 and the Codes of Practice set by the Human Tissue Authority (HTA licence numbers 12275 for the Imperial and 12217 for OBB).The clinicopathological demographics of the patients and healthy controls are summarized in Table

Figure S1 .Figure S2 .
Figure S1.Raw data of the Syn SAA reaction seeded with Syn fibrils from PD, PDD, DLB, MSA, and healthy control brains.The curves represent the average of 6 replicates, and error bars indicate ± one standard deviation (SD).RFU, relative fluorescence unit.

Figure S6 .
Figure S4.pS129 immunogold TEM images of the brain-derived Syn fibrils.The brain-derived Syn fibrils were labeled with anti-pSyn (pS129) and imaged at low magnification to compare the relative quantities of the pSyn fibrils between the diseases.All three cases were imaged for each disease (A) PD, (B) PDD, (C) DLB, and (D) MSA.The amount of pSyn fibrils was examined using slot blots.Red arrows indicate labeled fibrils.Scale bar = 200 µm.

Table S1 . Clinicopathological summary of the patients included in this study
. The table summarizes the clinical and pathological reports of the patients included in this study.PD, Parkinson's disease; PDD, Parkinson's disease with dementia; DLB, dementia with Lewy body; MSA, multiple system atrophy; CTR, control; ECtx, entorhinal cortex; BG, basal ganglia; M, male; F, female; AD, Alzheimer's disease; CVD, cardiovascular disease; Syn, -synuclein.