The effect of truncation on prion-like properties of α-synuclein

Increasing evidence suggests that α-synuclein (αS) aggregates in brains of individuals with Parkinson's disease and dementia with Lewy bodies can spread in a prion-like manner. Although the initial αS nuclei are pivotal in determining αS fibril polymorphs and resulting phenotypes, it is not clear how the initial fibril seeds are generated. Previous studies have shown that αS truncation might have an important role in αS aggregation. However, little is known about how this truncation influences αS's propagation properties. In the present study, we generated αS fibrils from a series of truncated human αS constructs, characterized their structures and conformational stabilities, and investigated their ability to convert the conformation of full-length αS in vitro, in cultured cells, and in WT mice. We show that both C- and N-terminal truncations of human αS induce fibril polymorphs and exhibit different cross-seeding activities. N-terminally 10- or 30-residue–truncated human αS fibrils induced more abundant αS pathologies than WT fibrils in mice, whereas other truncated fibrils induced less abundant pathologies. Biochemical analyses of these truncated fibrils revealed that N-terminal 10- or 30-residue truncations of human αS change the fibril conformation in a manner that increases their structural compatibility with WT mouse αS fibrils and reduces their stability. C-terminally 20-residue–truncated fibrils displayed enhanced seeding activity in vitro. Our findings imply that truncation of αS can influence its prion-like pathogenicity, resulting in phenotypic diversity of α-synucleinopathies.

(PD) 3 , dementia with Lewy bodies (DLB), and multiple system atrophy (MSA) are associated with formation of abnormal aggregates of ␣-synuclein (␣S), a natively unfolded 14-kDa protein consisting of 140 amino acids. These diseases are neuropathologically characterized by the deposition of filamentous ␣S aggregates with cross-␤ structure (2), which are abnormally phosphorylated (3) and partially ubiquitinated (4). ␣S and various other neurodegenerative disease-related proteins, including ␤-amyloid (A␤), tau, and TAR-DNA-binding protein-43 (TDP-43), can propagate through neural networks in a similar manner to abnormal prion proteins in prion diseases, in which abnormal insoluble protein acts as a seed or template for converting soluble protein to abnormal form (5,6). Although in vitro (7,8) and in vivo (9 -11) experiments using detergentinsoluble fraction of diseased brains and aggregated recombinant proteins as seeds have provided support for the "prion-like propagation hypothesis," it is not yet clear how ␣S aggregates propagate through the diseased brain.
In MSA, ␣S is mainly deposited in glial cells, whereas ␣S aggregates are found in neuronal cells in PD and dementia with Lewy bodies (DLB) (12). Such phenotypic differences suggest the existence of different "strains" of pathogenic ␣S, whose aggregate structures determine clinical phenotypes, as in prion diseases (13,14). Although the differences in cross-seeding activity between MSA and PD brain extracts in heterozygous mice transgenic for A53T human ␣S suggest that structural differences exist (11,15), the mechanisms that cause such structural variations among sporadic ␣-synucleinopathies remain unclear.
In Lewy bodies, not only full-length (FL), but also truncated ␣S is accumulated (16). Truncation is a common posttranslational modification that is not specific to the diseased brain, and about 15% of ␣S is truncated even in the brain of healthy individuals (17). In the diseased brain, both N-terminally and C-terminally truncated ␣S species are present (18). Overexpression of truncated ␣S or coexistence of truncated and FL ␣S can enhance aggregation and neurodegeneration both in vitro (19,20) and in vivo (21,22). Although it has been suggested that 3 The abbreviations used are: PD, Parkinson's disease; ␣S, ␣-synuclein; MSA, multiple system atrophy; Hu, human; Mo, mouse; FL, full-length; FLA, seeded full-length ␣S aggregates; TEM, transmission electron microscopy; ThT, thioflavin T; PK, proteinase K; CBB, Coomassie Brilliant Blue; Str, striatum; SN, substantia nigra; Amy, amygdala; ACC, anterior cingulate cortex; buffer A, 50 mM Tris-HCl, pH 7.5, 1 mM EGTA, 1 mM DTT; NAC, non-amyloid-␤ component.
cro ARTICLE truncation has an important influence on ␣S aggregation (19,23,24), very few studies have focused on the effect of truncation on prion-like propagation.
In the present study, we systematically investigated the structural polymorphism of truncated ␣S fibrils and examined whether or not these fibrils can act as templates for FL ␣S aggregation, using in vitro and in vivo experimental models. We found that differently truncated human ␣S fibrils exhibit a variety of cross-seeding activities in vitro and in vivo. Our results imply that truncation of ␣S in the initial nucleation phase might not only influence aggregation, but also result in phenotypic diversity.

Fibril formation from truncated ␣S proteins
To investigate the effects of C-and N-terminal truncations on ␣S aggregation, we prepared a series of wildtype (WT) and truncated human ␣S (Fig. 1A). The constructs were expressed in Escherichia coli, and the purified soluble ␣S proteins were analyzed by SDS-PAGE (Fig. 1B). After agitation in the presence of 150 mM KCl, the formation of fibrillar aggregates was confirmed by negative-staining transmission electron microscopy (TEM) (Fig. 1C). The fibrils were 6 -10 nm in Truncation induces ␣-synuclein fibril polymorphs diameter and did not branch. In agreement with a previous report (25), human C-terminally truncated ␣S fibrils (⌬C20 and ⌬C30) tended to be shorter than WT fibrils and were bundled laterally, whereas human N-terminally truncated ␣S formed long fibrils similar to WT fibrils (26). These fibrils bound to thioflavin T (ThT), which is commonly used to detect amyloid formation, and showed enhanced fluorescence (Fig. S1).
To examine whether these truncated ␣S fibrils could act as seeds for FL ␣S aggregation, seeding assay against human WT ␣S monomer was performed by incubation under quiescent conditions in the presence or absence of 1 mol % of preformed human ␣S fibril seeds in vitro (Fig. 1D). WT and all truncated ␣S fibrils induced the aggregation of FL ␣S protein, but the ThT fluorescence was not increased in the absence of ␣S fibril seeds. We found that ⌬C20 seeds drastically increased the rate of fibril assembly (t1 ⁄2 ϭ 2 Ϯ 0 h), compared with WT seeds (t1 ⁄2 ϭ 26 Ϯ 6 h). ⌬N10 seeds and ⌬N30 also induced fibril assembly, but more slowly (t1 ⁄2 ϭ 76 Ϯ 3 and 58 Ϯ 1 h, respectively).

Effect of truncation on induction of phospho-␣S pathology in WT mice
To examine the cross-seeding activity of human truncated ␣S fibril seeds in WT mouse brain, we injected 750 pmol human WT and truncated ␣S fibril seeds into the right striatum of WT mice. Three months after injection, paraffin-embedded brain sections were prepared and immunostained with anti-phospho-␣S antibody (pSer-129/EP1536Y) (Fig. 2, A and B). In mice injected with WT seeds, Lewy body/Lewy neurite-like phospho-␣S aggregates were distributed bilaterally through the brain, including striatum (Str), substantia nigra (SN), amygdala (Amy), and anterior cingulate cortex (ACC). Only sparse phospho-␣S-positive structures were observed in mice injected with ⌬C10 seeds, ⌬C20 seeds, and ⌬C30 seeds, and these were restricted to Str, SN, and Amy in the injected hemisphere. On the contrary, abundant phospho-␣S aggregates were observed throughout the bilateral brain, including Str, SN, Amy, and ACC, in mice injected with ⌬N10 seeds and ⌬N30 seeds. To evaluate the accumulated ␣S biochemically, we analyzed sarkosyl-insoluble fractions (Sar-ppt) from whole mouse brain (Fig. 2C). The amounts of insoluble phospho-␣S in mice injected with ⌬N10 seeds and ⌬N30 seeds were larger than in mice injected with WT seeds, whereas only very faint bands were detected in mice injected with ⌬C10 seeds, ⌬C20 seeds, ⌬C30 seeds, and ⌬N20 seeds (Fig. 2D). These phospho-␣S bands were stained with anti-mouse ␣S-specific antibody (D37A6), but not with anti-human ␣S-specific antibody (LB509). We also compared the amount of insoluble mouse ␣S but did not find any difference among the mice because of the high basal signal detected in mock-treated mice. These results indicate that N-terminal 10-or 30-residue truncation of human ␣S enhances the cross-seeding activity with WT mouse ␣S in vivo, despite the low seeding activity in vitro (Fig. 1D). On the other hand, the cross-seeding activities are reduced by C-terminal truncations and N-terminal 20-residue truncation.

Effect of truncation on seed-dependent aggregation of ␣S in cultured cells
To further confirm these differences, we compared the crossseeding activities using a cultured cell model. SH-SY5Y cells transiently expressing mouse WT ␣S were transfected with human WT and truncated ␣S fibril seeds: ⌬C20, ⌬N10, ⌬N20, and ⌬N30 seeds. Two days after transfection, we checked the presence of insoluble ␣S aggregates in cells by biochemical fractionation and Western blot analysis (Fig. 3A). The levels of mouse ␣S in sarkosyl-insoluble fraction were increased in cells treated with ␣S fibril seeds, and the insoluble mouse ␣S bands were positive to anti-phospho-␣S antibody (pSer-129/ pSyn#64). The amount of insoluble phosphorylated ␣S in cells treated with ⌬N30 seeds was larger than those in cells treated with WT seeds, whereas the amounts in cells treated with ⌬N20 seeds and ⌬C20 seeds were relatively small (Fig. 3B). We also compared the amount of insoluble mouse ␣S and found the amount of insoluble mouse ␣S in cells treated with ⌬N30 seeds was larger than those in cells treated with WT seeds. Similar results were observed when SH-SY5Y cells overexpressing FLAG-human ␣S were treated with truncated fibril seeds (Fig.  S2). These results indicate that human truncated ␣S fibrils can convert soluble mouse and human FL ␣S into insoluble form, and some N-terminal truncations have more potent crossseeding activities in cultured cells.

Truncation induces fibril polymorphs of ␣S
As described above, N-terminal 10-or 30-residue truncation of human ␣S results in increased cross-seeding activity in vivo, suggesting that these truncations induce different fibrillar polymorphs. In this context, the protease digestion pattern reflect the structure of the cross-␤ sheet-rich fibril core region, and is commonly used to characterize the conformations of amyloid fibrils (27)(28)(29). Therefore, we compared the proteinase K (PK)resistant fractions of truncated human ␣S fibrils with those of WT mouse ␣S fibrils. WT mouse ␣S fibrils (WT Mo seed) showed ϳ10 and ϳ8 kDa PK-resistant fractions after PK digestion, whereas WT human ␣S fibrils (WT Hu seed) yielded ϳ12 and ϳ8 kDa fractions (Fig. 4A). The N-terminally truncated human ␣S fibrils showed a WT mouse-like digestion pattern, suggesting that these fibrils share a common fibril core structure with WT mouse fibrils. In contrast, the ⌬C20 fibrils showed a WT human-like digestion pattern. Next, we compared the digestion patterns of FL mouse ␣S aggregates (FLA Mo ) seeded with the ␣S fibrils. FL mouse ␣S aggregates obtained with WT Mo seed (FLA Mo -WT Mo ) showed ϳ10 and ϳ8 kDa resistant fractions after PK digestion, whereas aggregates with WT Hu seed (FLA Mo -WT Hu ) were digested into lowmolecular fractions (Fig. 4B). FL mouse ␣S aggregates obtained with N-terminal truncated fibril seeds (FLA Mo -⌬N10, -⌬N20, and -⌬N30) showed FLA Mo -WT Mo -like digestion patterns, whereas FLA Mo -⌬C20 showed a FLA Mo -WT Hu -like pattern. These results imply that the N-terminal truncations of human ␣S have similar conformations to WT mouse ␣S fibrils, and can work as seeds for mouse ␣S monomer to form ␣S fibrils with structural properties similar to those of WT mouse ␣S fibrils.

Truncation induces ␣-synuclein fibril polymorphs N-terminal 10-and 30-residue-truncated human ␣S fibrils have decreased conformational stability
As described above, the N-terminal truncations of human ␣S form mouse ␣S-like fibrils, but sequential N-terminal truncations show biphasic seeding effects in mouse brain: Fibrils of ⌬N10 and ⌬N30 have greater effects than fibrils of ⌬N20 (Fig.  2). In the prion field, there is a general consensus that seeding capacity is inversely related to fibril stability (30). Here, we examined the stability of the fibrils by treating WT and N-terminally truncated human ␣S fibrils with a chemical denaturant,

Truncation induces ␣-synuclein fibril polymorphs
sarkosyl, and separating the products into supernatant and pellet fractions by ultracentrifugation (Fig. 5). In the case of WT human ␣S, ϳ36% of the total protein was fractionated into the pellet (36 Ϯ 4% of total protein). The ⌬N10 and ⌬N30 fibrils, which could propagate efficiently in mouse brain, showed decreased conformational stability (15 Ϯ 5% and 19 Ϯ 6% of total protein in the pellet, respectively). On the other hand, the ⌬N20 fibrils, which exhibited inefficient seeding in vivo, showed increased stability (58 Ϯ 3% of total protein in the pellet). These results indicate that N-terminal 10-or 30-residue truncation enhances the cross-seeding activity in vivo as a result of decreased stability, implying that ␣S fibrils are propagated through a prion-like molecular mechanism.

C-terminal 20-residue truncation induces distinctive prion-like properties
Although ⌬C20 fibrils showed drastically enhanced seeding activity in vitro, they induced only sparse ␣S pathology in vivo (Figs. 1D and 2). To investigate whether the apparent inconsistency is because of their structural characteristics, we analyzed the structures of the seeded FL ␣S aggregates (FLA)-⌬C20 in vitro and in cultured cells. TEM images showed that FLA seeded with ⌬C20 seeds (FLA-⌬C20) had a twisted morphology that differed from the others, which showed straight, unbranched fibrillar structures with a diameter of ϳ10 nm ( Fig.  6A; Fig. S3A). To explore the difference of the underlying structures, we quantified the ThT-binding properties of the seeded FL ␣S aggregates. FLA-⌬C20 showed higher binding affinity (211 Ϯ 8% of FLA-WT) (Fig. S3B). The rate of FL WT ␣S aggregation in the presence of FLA-⌬C20 was faster (t1 ⁄ 2 ϭ 5 Ϯ 0 h) than that of FLA-WT Hu (t1 ⁄ 2 ϭ 28 Ϯ 2 h) (Fig. 6B). We also examined the seeding activities of the FL ␣S aggregates in cultured cells. FLA-⌬C20 showed poor seeding activity in cul-  Truncation induces ␣-synuclein fibril polymorphs tured cells (Fig. 6, C and D). These results indicate that ⌬C20 fibrils induce distinct structures of FL ␣S aggregates and these aggregates have similar but different seeding properties with ⌬C20 fibrils in vitro and in cells (Figs. 1 and 3).
The C-terminal region (residues 96 -140) of ␣S is highly enriched in negatively charged residues (aspartate and glutamate) (51) and proline residues (52). Long-range interactions of the C-terminal region (residues 110 -130) with the central hydrophobic NAC region (residues 85-95) (53,54) are important to stabilize the natively unfolded structure (55)(56)(57). When the interaction between the C-terminal and the NAC region is weakened by low pH, addition of polycations, or changes in ionic strength, exposure of the NAC region increases, and more tightly packed ␣S fibrils are formed (50,58). Tyrosine residues located at positions 125, 133, and 136 in the C-terminal region are crucial to maintain the N-and C-terminal intermolecular interactions and are related to aggregation (59). In addition to deletion of charged residues, complete removal of these tyrosine residues can result in structural polymorphisms. In the present study, the C-terminal 20-residue truncated fibrils showed enhanced seeding activity in vitro, and FLA seeded with ⌬C20 seeds (FLA-⌬C20) exhibited twisted morphologies distinct from that of the original ⌬C20 seeds and others, which showed straight structure. In our opinion, such twisted morphologies were likely caused by the negatively charged residues, which exist in FL monomers, but not in ⌬C20 seeds. These  . C-terminal 20-residue truncation of human ␣S also produces distinctive prion-like properties. A, negative staining TEM imaging of human FL ␣S aggregates seeded with FL (left) or ⌬C20 (right) seeds. Scale bars, 100 nm. Human FL ␣S aggregates seeded with ⌬C20 seeds showed twisted fibrillar structure. B, aggregation kinetics of human FL ␣S monomers under quiescent conditions in the absence or presence of 1 mol % human FL ␣S aggregates seeded with WT or ⌬C20 seeds, monitored by measuring ThT fluorescence. Data are averages of three samples. C, seed-dependent aggregation of ␣S induced by human FL ␣S aggregates seeded with WT or ⌬C20 seeds in the cultured cell model. SH-SY5Y cells expressing FLAG-␣S were transfected with ␣S monomer (FL Mono), human FL ␣S aggregates seeded with WT (FLA-WT), or ⌬C20 (FLA-⌬C20). Cell extracts were prepared with A68 buffer containing 1% sarkosyl and centrifuged. The supernatant (sup) and the pellet (ppt) fractions were subjected to Western blot analysis using anti-pSer-129 (pSyn#64), anti-FLAG, and anti-tubulin antibodies. D, the intensities of phosphorylated ␣S in (C) are quantified. ***, p Ͻ 0.001.

Truncation induces ␣-synuclein fibril polymorphs
residues might interact with the core region of ⌬C20 to form the twisted morphologies. As a result, C-terminal 20-residue truncated fibrils induced full-length ␣S polymorphisms.
The N-terminal region (residues 1-60) of human ␣S contains four imperfect 11-residue repeats with a conserved motif (XKTKEGVXXXX), which interacts with the lipid membrane, with conformational changes to the ␣-helical structure (26,56). Although the N-terminal region directly interacts with the C-terminal region, but not the central NAC region, (57), N-terminal truncation also increases the ratio of ␤-sheet structure within ␣S fibrils (56). Recent studies have shown that familial PD-related mutations located in the N-terminal region influence the relative stability of the fibrils and their conformational preferences, rather than the global fibril structures (60, 61).
Truncation of the N-terminal 10 or 30 residues markedly enhanced cross-seeding activity in mouse brain. Structural compatibility between the seed and the host protein is important for cross-seeding activity in vivo (62). We found that N-terminal truncation switches the conformational preference of human ␣S fibrils, making them with structural properties similar to those of WT mouse ␣S fibrils, whereas C-terminal truncation does not have such an effect. Furthermore, we also found that N-terminal 10-or 30-residue truncation reduces conformational stability in a manner that is inversely correlated with in vivo propagation efficacy. Propagation of prion proteins depends on the stability of the pathogenic aggregates; less-stable prions replicate faster than more-stable prions (30). This would be another reason why these N-terminally truncated fibrils reduce the species barrier in WT mice. These results indicate that N-terminal truncation influences the prion-like seeding activity of ␣S.
As previously reported (62), we also found that propagation activities in vitro, in cells, and in vivo were not always consistent. In cultured cells, seeding reaction occurs in a more complex environment, such as molecular crowding, effects of chaperones and protein degradation machineries, other proteins interacting with seeds or monomers, and so on, compared with the simple reaction buffers used for in vitro assay (63). Moreover, seeds should be taken from outside of cells and escaped from endosomal vesicles if taken by endosome system in cultured cells. In mice, the cell to cell transmissibility might be influenced by secretion and uptake of seeds (64). In this study, we found both ⌬C20 seeds and FLA-⌬C20 seeds had markedly different seeding activities in vitro versus in cultured cells and mice. These seeds induced rapid aggregation in vitro, but not in cells and in mice. Contrary to these seeds, N-terminally truncated seeds such as ⌬N30 induced severe pathology in cells and mice but had reduced seeding activity in vitro. These differences in propagation characteristics may result from different susceptibility of fibrils to external factors described above. It was reported that even ␣S fibrils that could not propagate effectively in WT mice might trigger ␣S pathology and neurodegeneration when proteolytic systems become less effective with aging (65) or in the presence of a genetic factor, such as heterozygous mutation in the lysosomal glucocerebrosidase gene (GBA1) (66,67). Therefore, there is a possibility that ⌬C20 seeds induce severe ␣S pathology in aged mice or lysosomal mutation mice. Consequently, it is important to evaluate the propagation properties of ␣S fibrils using multiple models.
In summary, our findings indicate that differently truncated human ␣S can induce various polymorphisms of FL ␣S fibrils via a seeding mechanism. The properties of N-terminal truncations of human ␣S imply that the pathogenicity of ␣S involves a prion-like molecular mechanism. We believe these findings provide new insights into both the pathogenicity and phenotypic diversity of sporadic ␣-synucleinopathies.

Antibodies
Antibodies used in the present study were anti-phospho-␣S antibodies to pSer-129, including pSyn#64 (3) and EP1536Y (Abcam), and other anti-␣S antibodies, including LB509 (a gift from Dr. Iwatsubo) and D37A6 (Cell Signaling Technology). Biotin-labeled secondary antibodies were purchased from Vector Laboratories for use in the avidin-biotin complex method.
All constructs were verified by DNA sequencing. Recombinant proteins were expressed in E. coli BL21 (DE3) as described previously (70). Bacterial pellets were sonicated in extraction buffer A (50 mM Tris-HCl, pH 7.5, 1 mM EGTA, 1 mM DTT) on ice. The homogenates were centrifuged at 16,000 ϫ g for 15 min, and the supernatants were boiled for 5 min. Then, the The N-terminal truncation of human ␣S changes the structure of the fibrils to a form similar to that of WT mouse ␣S fibrils. Furthermore, the N-terminally 10-or 30-residue-truncated human ␣S fibrils have lower conformational stability than WT human ␣S fibrils. As a result, these fibrils propagate effectively in WT mice. The C-terminally 20-residuetruncated fibrils show enhanced seeding activity in vitro, but reduced seeding activity in cell model and mouse model. There is no direct correlation between in vitro and in vivo propagation activities.
Truncation induces ␣-synuclein fibril polymorphs supernatants (heat-stable fraction) of 15-min centrifugation at 16,000 ϫ g were subjected to anion-exchange (for WT, ⌬C10, ⌬C20, ⌬N10, ⌬20, and ⌬N30) or cation-exchange (for ⌬C30) chromatography on columns (Q Sepharose or SP Sepharose Fast Flow, respectively; GE Healthcare Japan) equilibrated with extraction buffer A. The columns were washed with extraction buffer A and eluted with a 100 -500 mM NaCl gradient. The eluates were concentrated by precipitation with 50% saturated ammonium sulfate. The pellets were re-suspended in and dialyzed against 30 mM Tris-HCl, pH 7.5. The protein concentrations were determined by reversed phase-HPLC (RP-HPLC), using an Aquapore RP300 column (71). Purified ␣S samples were analyzed by 13.5% SDS-PAGE and stained with CBB. Gel images were recorded with a Gel Doc TM EZ Imager (Bio-Rad).

Preparation of ␣S fibril seeds
␣S fibrils were prepared as follows. Purified recombinant ␣S proteins were dissolved in 30 mM Tris-HCl, pH 7.5, containing 150 mM KCl and 0.1% NaN 3 , to a final concentration of 150 M protein. The proteins were incubated at 37°C under linear shaking at 200 rpm for 7 days. The assembled ␣S was collected by ultracentrifugation at 135,000 ϫ g for 20 min at 25°C. The resultant pellets were re-suspended and sonicated in 30 mM, Tris-HCl, pH 7.5, on ice with an ultrasonic homogenizer (VP-5S, TAITEC) and ultracentrifuged again. The pellet was re-suspended and sonicated again in 30 mM Tris-HCl, pH 7.5. The proteins were dissolved in 6 M guanidine hydrochloride and their concentrations were determined by HPLC as described above.

Thioflavin T-binding assay
The degree of fibrillation was measured in terms of thioflavin T fluorescence intensity, which is increased when ThT binds to amyloid-like fibrils. The ␣S proteins (7.5 M) were incubated with 20 M ThT in 30 mM Tris-HCl buffer, pH 7.5, for 30 min at 37°C. Fluorometry was performed using a microplate reader (Varioskan ® Flash, excitation 442 nm, emission 485 nm; Thermo Scientific) and normalized to the ThT intensity of WT ␣S fibrils.

Proteinase K digestion of ␣S fibrils
␣S fibrils (1 mg/ml) in 30 mM Tris-HCl, pH 7.5, were incubated for 30 min at 37°C in the presence of various concentrations of proteinase K (1, 10, and 100 g/ml; Promega). The reaction was stopped by boiling the mixture for 10 min. The samples were analyzed by 15-20% Tris-Tricine-SDS-PAGE; the gels were stained with CBB (28). Gel images were recorded with a Gel Doc TM EZ Imager (Bio-Rad).

Conformational stability assay of ␣S fibrils
␣S fibrils (1 mg/ml) were incubated in 30 mM Tris-HCl, pH 7.5, containing 1% sarkosyl (w/w) for 5 min at room temperature. After ultracentrifugation at 135,000 ϫ g for 20 min at 25°C, the supernatant was collected as sarkosyl-soluble fraction, and the pellet was re-suspended in 1ϫ SDS-PAGE buffer. The samples were analyzed by 15% Tris-Tricine-SDS-PAGE; the gels were stained with CBB (28). Gel images were recorded with a Gel Doc TM EZ Imager (Bio-Rad). The band intensities were quantified using ImageJ software. Conformational stability was determined as the ratio of ␣S in the pellet to total ␣S (supernatant ϩ pellet).

Immunocytochemistry
SH-SY5Y cells grown on collagen-coated coverslips were fixed with 4% paraformaldehyde and stained with the indicated primary antibodies at 1:1,000 dilution. After incubation for 1 h, cells were washed and treated with secondary antibodies (antirabbit IgG-conjugated Alexa Fluor 568 and anti-mouse IgGconjugated Alexa Fluor 488; Invitrogen) and Hoechst 33324 (Sigma-Aldrich) to counterstain nuclear DNA. The cells were mounted and analyzed using a BZ-X710 (Keyence) and BZ-X analyzer (Keyence).

Biochemical analysis of cultured cells
After incubation for 2 days, cells were harvested by centrifugation (1,800 ϫ g, 5 min) and washed with PBS. The cellular proteins were extracted with 300 l of buffer A68 (10 mM Tris-HCl, pH 7.5, 1 mM EGTA, 10% sucrose, 0.8 M NaCl) containing final 1% sarkosyl (w/w) by sonication. After ultracentrifugation at 135,000 ϫ g for 20 min at 25°C, the supernatant was collected as sarkosyl-soluble fraction, and the protein concentration was Truncation induces ␣-synuclein fibril polymorphs determined by BCA assay. The pellet was washed once with 300 l of buffer A68 containing 1% sarkosyl, and solubilized in 100 l of SDS-sample buffer. Both sarkosyl-soluble and -insoluble fractions were analyzed by immunoblotting with appropriate antibodies as indicated (73). The band intensities of insoluble phospho-␣S and mouse-␣S were quantified using ImageJ software. The relative amount of precipitate mouse-␣S was calculated by insoluble mouse-␣S divided by insoluble plus soluble mouse-␣S. Data are expressed as -fold induction relative to that in the WT.

␣S inoculation into mice
Ten-week-old male C57BL/6J mice were purchased from CLEA Japan, Inc. All experimental protocols were performed according to the recommendations of the Animal Care and Use Committee of Tokyo Metropolitan Institute of Medical Science. ␣S proteins (150 M, 5 l) were injected into the right striatum (anterior-posterior, 0.5 mm; medial-lateral, Ϫ2.0 mm; dorsal-ventral, Ϫ3.5 mm). Inoculation into mouse brain was performed as described previously (10).

Immunohistochemistry of mouse brains
Three months after inoculation, mice were deeply anesthetized with pentobarbital injection and sacrificed. The brain was perfused with 0.1 M PBS, followed by 10% formalin neutral buffer solution. After fixation, the dissected brain was embedded in paraffin and 5 m-thick serial sections were prepared. For antigen retrieval, sections were treated with 100% formic acid (Wako) for 5 min and washed under running tap water, then brought to a boil in 0.1 M sodium citrate buffer (pH 6.0) for 10 min, followed by cooling for 1 h. Immunohistochemistry with mAb EP1536Y (1:2,000) directed against ␣S phosphorylated at Ser-129 was performed as described previously (10). Sections were counterstained with hematoxylin. ␣S pathologies were analyzed with a BZ-X710 (Keyence) and quantified using a BZ-X analyzer (Keyence).

Biochemical analysis of mouse brains
Frozen whole mouse brains were homogenized in 20 volumes of buffer A68 containing final 2% sarkosyl (w/v), and incubated at 37°C for 30 min. The brain homogenates were centrifuged at 9,460 ϫ g for 20 min at 25°C, and then the supernatants were collected and ultracentrifuged at 135,000 ϫ g for 20 min at 25°C. The pellets were washed with 30 mM Tris-HCl (pH 7.5) and ultracentrifuged as before. The pellet was once washed with 300 l of buffer A68 containing 1% sarkosyl, and solubilized in 100 l of SDS-sample buffer. Both sarkosyl-soluble and -insoluble fractions were analyzed by Western blotting with appropriate antibodies as indicated (10). The band intensities of insoluble phospho-␣S and mouse-␣S were quantified using ImageJ software. The relative amount of precipitate mouse-␣S was calculated by insoluble mouse-␣S divided by insoluble plus soluble mouse-␣S. Data are expressed as -fold induction relative to that in the WT.

Statistical analysis
Graphs were created, and statistics were calculated in Prism ver. 5 (GraphPad Software, San Diego, CA). Multiple group comparisons were made by one-way analysis of variance (ANOVA) followed by Tukey's post hoc test. Data are presented as mean Ϯ S.E. unless otherwise noted. Significance was determined at *, p Ͻ 0.05, **, p Ͻ 0.01, and ***, p Ͻ 0.001.