Decision letter: α-synuclein strains that cause distinct pathologies differentially inhibit proteasome (2020)

Article Figures and data Abstract Introduction Results Discussion Materials and methods Data availability References Decision letter Author response Article and author information Metrics Abstract Abnormal α-synuclein aggregation has been implicated in several diseases and is known to spread in a prion-like manner. There is a relationship between protein aggregate structure (strain) and clinical phenotype in prion diseases, however, whether differences in the strains of α-synuclein aggregates account for the different pathologies remained unclear. Here, we generated two types of α-synuclein fibrils from identical monomer and investigated their seeding and propagation ability in mice and primary-cultured neurons. One α-synuclein fibril induced marked accumulation of phosphorylated α-synuclein and ubiquitinated protein aggregates, while the other did not, indicating the formation of α-synuclein two strains. Notably, the former α-synuclein strain inhibited proteasome activity and co-precipitated with 26S proteasome complex. Further examination indicated that structural differences in the C-terminal region of α-synuclein strains lead to different effects on proteasome activity. These results provide a possible molecular mechanism to account for the different pathologies induced by different α-synuclein strains. Introduction Misfolding and aggregation of normally soluble proteins are common pathological features of many neurodegenerative diseases, including Alzheimer's, Parkinson's, Creutzfeldt–Jacob and Huntington's diseases (Ross and Poirier, 2004). For example, Parkinson's disease (PD), dementia with Lewy bodies (DLB) and multiple system atrophy (MSA) are characterized by accumulation of misfolded α-synuclein in neuronal and/or glial cells, and therefore these diseases are termed α-synucleinopathies. In PD and DLB, α-synuclein pathologies are mainly observed in neurons in the form of Lewy bodies (LBs) and Lewy neurites (LNs) (Baba et al., 1998), while glial cytoplasmic inclusions (GCIs) are seen in oligodendrocytes in MSA (Wakabayashi et al., 1998). The abnormal α-synuclein observed in brains of patients is accumulated as fibrous or filamentous forms with cross-β structures (Araki et al., 2019; Spillantini et al., 1997), existing in phosphorylated and partially ubiquitinated states (Fujiwara et al., 2002; Hasegawa et al., 2002). These abnormal α-synuclein species exhibit seeding activity for prion-like conversion, being similar in this respect to the infectious forms of prion protein (PrP) causing Creutzfeldt-Jakob disease (CJD) and bovine spongiform encephalopathy (Goedert, 2015). Various other neurodegenerative disease-related proteins, including amyloid-β, tau and TDP-43, can also propagate through neural networks in a similar manner. α-Synuclein is a natively unfolded protein of 140 amino acid residues, normally found in both soluble and membrane-associated fractions and localized in synaptic termini. Although its physiological function has not been fully clarified, it appears to be involved in the regulation of SNARE complex and in dopamine production. Disease-linked missense mutations and multiplication of the SNCA gene encoding α-synuclein have been reported in familial forms of α-synucleinopathies, indicating that structural changes and overexpression of α-synuclein protein are involved in the development of α-synucleinopathies (Wong and Krainc, 2017). Recombinant soluble α-synuclein proteins purified from bacterial cells form amyloid-like fibrils that are morphologically and physicochemically similar to those observed in patients' brains (Araki et al., 2019; Goedert, 2015). These synthetic α-synuclein fibrils can act as seeds and induce seeded aggregation of α-synuclein in cultured cells or primary cultured neurons, as well as in animal brains. Intracerebral inoculation of synthetic α-synuclein fibrils induces phosphorylated and ubiquitinated α-synuclein pathologies even in wild-type (WT) mice (Luk et al., 2012; Masuda-Suzukake et al., 2013). It has also been reported that extracts from brains of patients with α-synucleinopathies induce α-synuclein pathologies in cellular and animal models (Bernis et al., 2015; Watts et al., 2013). In addition, recent studies have suggested that α-synuclein strains with distinct conformations exist, which is a characteristic of prions (Bousset et al., 2013; Gribaudo et al., 2019; Guerrero-Ferreira et al., 2019; Peelaerts and Baekelandt, 2016; Peelaerts et al., 2015; Shahnawaz et al., 2020; Woerman et al., 2019). Synthetic α-synuclein fibrils formed under different physiological conditions in vitro show distinct seeding activities and cytotoxicity in cultured cells and rat brains. Furthermore, MSA brain extracts exhibit distinct infectivity compared to PD or control brain extracts in cultured cells or mice expressing mutant A53T or WT α-synuclein (Lau et al., 2020; Peng et al., 2018; Prusiner et al., 2015; Woerman et al., 2019; Woerman et al., 2015). These observations support the idea that α-synuclein shows prion-like behavior, because they can be accounted for by a typical hallmark of the prion phenomenon, that is, the presence of strains. In prion diseases, the variety of strains that can be differentiated in terms of the clinical signs, incubation period after inoculation, and the vacuolation lesion profiles in the brain of affected animals is due to structural differences of PrP aggregates, as identified by biochemical analyses including glycosylation profile, electrophoretic mobility, protease resistance, and sedimentation. These PrP strains are thought to correspond to different conformations of PrP aggregates, as demonstrated for the yeast prion [PSI+], which induces aggregates of Sup35p (Ohhashi et al., 2010). Thus, as the case of prion disease, differences of lesions among α-synucleinopathies are thought to be caused by conformational heterology of α-synuclein assemblies, probably amyloid-like fibrils (Lau et al., 2020; Schweighauser et al., 2020; Shahnawaz et al., 2020). However, little is known about how conformational differences of protein aggregates induce a variety of lesions, not only in prion disease, but also in other neurodegenerative diseases, such as α-synucleinopathies. In this study, we prepared two α-synuclein assemblies from identical wild-type α-synuclein monomer under different conditions, and established that they have distinct conformations, that is, we succeeded to generating two α-synuclein fibrils from the same monomer. We examined their seeding abilities to convert endogenous soluble α-synuclein monomers into phosphorylated aggregates in mice and primary-cultured neurons, indicating the formation of two α-synuclein strains. Notably, only one strain induced the accumulation of ubiquitinated proteins, as well as phosphorylated α-synuclein aggregates, indicating the ability of this strain to inhibit proteasome activity. Moreover, only this strain strongly inhibited proteasome activity and co-precipitated with purified 26S proteasome complex in vitro. Structural studies suggested that the C-terminal region plays a key role in the different properties of the two strains. Taken together, these results provide a possible molecular mechanism to account for the different lesions induced by distinct α-synuclein strains. Results Formation of amyloid-like fibrils from α-synuclein proteins in vitro We generated two distinct α-synuclein assemblies from the identical purified α-synuclein monomer using the method described in the previous report, with minor modifications (Figure 1A; Bousset et al., 2013; Lau et al., 2020). Specifically, we prepared recombinant α-synuclein monomer and agitated it in the presence or absence of salt at a physiological concentration. In the presence of physiological salt (30 mM Tris, pH 7.5, 150 mM KCl), the monomer formed a cloudy solution of assemblies with higher turbidity, while in the absence of salt (30 mM Tris, pH 7.5), a clear solution containing assemblies with lower turbidity was formed (Figure 1A and B). Almost all of the α-synuclein was present as aggregates rather than soluble oligomers under both conditions (Figure 1—figure supplement 1; Thibaudeau et al., 2018). Both assemblies showed fibrillar morphology, but the previously reported ribbon-like morphology was not observed (Figure 1C; Bousset et al., 2013). Therefore, we will refer to the former assemblies as α-synuclein fibrils (+) and the later assemblies as α-synuclein fibrils (-). Both fibrils were stained with Thioflavin T and Congo red, indicating their amyloid-like nature (Figure 1D and E). The differences of Thioflavin T fluorescence and Congo red binding of these α-synuclein fibrils suggested that these fibrils have a different structures each other. Next, we examined the in vitro seeding activity of these fibrils and found that α-synuclein fibrils (+) showed higher seeding activity than α-synuclein fibrils (-) in vitro (Figure 1F). These results indicated that we had successfully prepared two distinct types of α-synuclein fibrils from the same monomer. Figure 1 with 1 supplement see all Download asset Open asset Preparation and characterization of two α-synuclein strains. (A) Schematic representation of two α-synuclein (αS) strains (left) and the resulted two α-synuclein assemblies (right). (B) Turbidity of these assemblies. Analysis was performed using student t test. (mean ± S.E.M; n = 3) Formation of aggregates rather than soluble oligomer is shown in Figure 1—figure supplement 1. (C) Transfer electron microscopy (TEM) images of these assemblies. Scale bar, 200 nm. (D) Thioflavin T fluorescence of these assemblies and αS monomer. Analysis was performed using one-way ANOVA and Tukey post hoc test. (mean ± S.E.M; n = 3) (E) Congo red bindings of these strains and αS monomer. (mean ± S.E.M; n = 3) (F) In vitro seeding activity of these strains. α-synuclein monomers were incubated with α-synuclein fibril (-) (orange) or α-synuclein fibril (+) (gray). Kinetics of Thioflavin T fluorescence were shown. Analysis was performed using one-way ANOVA and Tukey post hoc test. **p<0.01, ***p<0.001, ****p<0.0001. Figure 1—source data 1 Quantification for graph in Figure 1B,D and E. https://cdn.elifesciences.org/articles/56825/elife-56825-fig1-data1-v2.xlsx Download elife-56825-fig1-data1-v2.xlsx Figure 1—source data 2 Three independent data in Figure 1F. https://cdn.elifesciences.org/articles/56825/elife-56825-fig1-data2-v2.xlsx Download elife-56825-fig1-data2-v2.xlsx Formation of phosphorylated α-synuclein pathology by injection of α-synuclein strains into mouse brain We next investigated whether there was a strain-dependent difference of prion-like propagation in mouse brain. We injected α-synuclein fibrils (+) and α-synuclein fibrils (-) into striatum of wild-type mice, and after one month, we examined the accumulation of phosphorylated α-synuclein deposits resembling those observed in patients' brains (Luk et al., 2012; Masuda-Suzukake et al., 2013). In contrast to the in vitro seeding activities, α-synuclein fibrils (-) induced Lewy body/Lewy neurite-like abnormal phosphorylated α-synuclein deposits through the mouse brain, including cortex, striatum and corpus callosum, whereas few phosphorylated α-synuclein deposits were induced by α-synuclein fibrils (+) (Figure 2A and Figure 2—figure supplement 1). We used three mice each for this experiment and confirmed the reproducibility (Figure 2—figure supplement 2). The deposits induced by α-synuclein fibrils (-) were also positive for ubiquitin staining in cortex, like Lewy bodies and Lewy neurites (Figure 2B). Compared with phosphorylated α-synuclein, which is the most representative marker in the α-synucleinopathies, ubiquitin staining is weaker, thus we could find only few ubiquitin positive deposits in striatum and corpus callosum (data not shown). Taken together, there was a strain-dependent difference of α-synuclein fibril formation from the identical α-synuclein monomer when the two strains were inoculated into the mouse brain, and α-synuclein fibrils (-) had higher prion-like seeding activity than α-synuclein fibrils (+) in mouse brain, contrary to the in vitro seeding results (Figure 1F). Figure 2 with 2 supplements see all Download asset Open asset Comparison of pathologies in WT mouse brains by inoculation of two α-synuclein strains. (A) Distribution of phosphorylated α-synuclein pathology in mouse brain. α-synuclein fibrils (-) or α-synuclein fibrils (+) were injected into the striatum of WT mouse brain and stained with phosphorylated α-synuclein antibody 1 month after injection. Regions surrounded by white rectangles in left panels are magnified and shown in right panels. Typical pathological α-synuclein deposits are indicated by the arrowheads. Sections were counterstained with hematoxylin. Representative images of cortex of mice injected with α-synuclein fibrils (-) (upper), α-synuclein fibrils (+) (middle) or not injected mice as negative controls (lower) are shown. Representative images of corpus callosum and striatum are shown in Figure 2—figure supplement 1. Representative images of cortex of other two mice injected with α-synuclein fibrils are also shown in Figure 2—figure supplement 2. (B) Distribution of ubiquitin pathology in mouse brain. α-synuclein fibrils (-) or α-synuclein fibrils (+) were injected into the striatum of WT mouse brain and stained with ubiquitin antibody 1 month after injection. Representative images of cortex of mice injected with α-synuclein fibrils (-) (upper), α-synuclein fibrils (+) (middle) or not injected mice as negative controls (lower) are shown. Regions surrounded by white rectangles in left panels are magnified and shown in right panels. Typical pathological ubiquitin positive deposits are indicated by the arrowheads. Sections were counterstained with hematoxylin. Scale bars, 50 µm. Formation of phosphorylated α-synuclein pathology induced by the two α-synuclein strains in cultured neurons To further study the difference in the formation of pathological α-synuclein aggregates in neurons, we compared the ability of the two α-synuclein strains to induce seed-dependent aggregation of α-synuclein in primary mouse cortical cells. When primary mouse cortical cells from non-transgenic mice were treated with α-synuclein fibrils for two weeks, we observed a dramatic increase of phosphorylated α-synuclein accumulation, which is also positive for ubiquitin, only in the case of α-synuclein fibrils (-), while little accumulation was seen with α-synuclein fibrils (+) (Figure 3A). These results are consistent with those observed in the mouse brain. To investigate whether these accumulations occur in neurons, we performed double staining for phosphorylated α-synuclein and neuronal markers or an astrocyte marker using cells treated with α-synuclein fibrils (-) (Figure 3—figure supplement 1). We found that phosphorylated α-synuclein accumulations were seen in neurons, not in astrocytes. Thus, α-synuclein fibrils (-) could induce phosphorylated α-synuclein accumulations in primary mouse cortical neurons. Figure 3 with 2 supplements see all Download asset Open asset Seeding activities α-synuclein strains in primary mouse cortical neurons. The two α-synuclein fibrils were transduced into primary mouse cortical cells. 14 days after fibril transduction, the accumulation of abnormal phosphorylated α-synuclein and ubiquitinated aggregates are detected by immunofluorescence microscopy and western blotting. (A) Phosphorylated α-synuclein (Phos-αS), ubiquitin (Ub) and nuclei (DAPI) were stained. Scale bar, 20 µm. Images of cells double stained with antibody against phosphorylated α-synuclein and neuronal or astrocyte markers are shown in Figure 3—figure supplement 1. Uptake of these α-synuclein fibrils into cells are shown in Figure 3—figure supplement 2. (B) Detection of sarkosyl insoluble phosphorylated α-synuclein (left), endogenous mouse α-synuclein (center) and ubiquitinated proteins (right) by western blotting. (C) Detection of sarkosyl soluble GAPDH as a loading control. (D) The quantification data of sarkosyl insoluble phosphorylated α-synuclein (left) and ubiquitinated proteins (right) shown in (B) (mean ± S.E.M; n = 3). Analysis was performed using one-way ANOVA and Tukey post hoc test. ****p<0.0001. Figure 3—source data 1 Quantification for graph in Figure 3D and Figure 3—figure supplement 2C. https://cdn.elifesciences.org/articles/56825/elife-56825-fig3-data1-v2.xlsx Download elife-56825-fig3-data1-v2.xlsx Next, we performed biochemical analysis of detergent-insoluble α-synuclein prepared from these cells. Phosphorylated α-synuclein were accumulated in cells treated with both α-synuclein fibrils (-) and α-synuclein fibrils (+) (Figure 3B and C). However, α-synuclein fibrils (-) induced a greater accumulation of phosphorylated α-synuclein than did α-synuclein fibrils (+) (Figure 3D). The phosphorylated and aggregated α-synuclein in these cells was found to be endogenous mouse α-synuclein, indicating that the introduced human α-synuclein fibrils worked as seeds (Figure 3B). We also examined the accumulation of detergent-insoluble ubiquitinated proteins and found that not only ubiquitinated α-synuclein, but also other ubiquitinated proteins were accumulated in cells treated with α-synuclein fibrils (-). There was no significant increase of ubiquitinated protein accumulation in cells treated with α-synuclein fibrils (+) (Figure 3B and D). These results indicated that only α-synuclein fibrils (-) induced much accumulation of phosphorylated α-synuclein and ubiquitinated proteins in primary mouse cortical neurons, in accordance with the findings in mouse brain. Seed dependent aggregation of α-synuclein must depend on the efficiency of fibril uptake by cells. We confirmed whether the two α-synuclein fibrils were taken up into primary mouse cortical cells with similar efficiency. Cells were treated with the two α-synuclein fibrils or buffer. A day after treatment, we treated cells with trypsin for digestion of extracellular fibrils, collected them and examined the α-synuclein fibrils taken up into the cells by western blotting of sarkosyl insoluble fractions. We found no α-synuclein signal in pellet fraction of cells with buffer control indicating that detected α-synuclein signals were originated from added fibrils. We found α-synuclein fibrils (-), which induced much α-synuclein accumulation, were taken up into the cells lesser than α-synuclein fibrils (+), demonstrating that α-synuclein fibrils (-) efficiently caused the accumulation of α-synuclein and ubiquitinated proteins in spite of their inefficient uptake into the cells (Figure 3—figure supplement 2). Different interactions of α-Synuclein strains with 26S Proteasome The above results motivated us to examine proteasome activity in the presence of these two types of α-synuclein fibrils. We purified 26S proteasome complex from budding yeast expressing FLAG-tagged Rpn11p, a subunit of 19S regulatory complex (Figure 4—figure supplement 1; Saeki et al., 2005). The activity of the purified 26S proteasome was examined in the presence or absence of α-synuclein fibrils. The eukaryotic proteasome has three active subunits, β1, β 2 and β 5, each displaying a specific catalytic activity, trypsin-like, chymotrypsin-like and caspase-like activity, respectively. Thus, we examined the chymotrypsin-like (Figure 4A), caspase-like (Figure 4B) and trypsin-like (Figure 4C) activities of 26S proteasome in the presence of these α-synuclein fibrils using each fluorogenic peptide substrate in vitro. The catalytic activity of 26S proteasome was drastically impaired in the presence of α-synuclein fibrils (-), whereas α-synuclein fibrils (+) were ineffective (Figure 4A–C). These results accord with our mouse and primary-cultured neuron data, i.e., only α-synuclein fibrils (-) could induce much accumulation of phosphorylated α-synuclein and ubiquitinated proteins. Co-aggregation of functional proteins with misfolded protein aggregates may cause the impairment of their function. Therefore, we investigated the interaction of 26S proteasome with the fibrils. α-Synuclein fibrils were mixed with purified 26S proteasome, and the mixture was centrifuged. The supernatants and precipitates were analyzed by western blotting. We found that both α-synuclein fibrils (-) and α-synuclein fibrils (+) were fractionated to the pellet fractions (Figure 4D). However, only α-synuclein fibrils (-) co-precipitated with 26S proteasome, while α-synuclein fibrils (+) did not (Figure 4E). Taken together, these results indicate that only α-synuclein fibrils (-) interact with 26S proteasome and impair the proteasome activity. Figure 4 with 1 supplement see all Download asset Open asset Different interaction of α-synuclein strains with 26S proteasome. (A–C) Effects of the α-synuclein strains on the 26S proteasome activity. 26S proteasome was purified from budding yeast as shown in Figure 4—figure supplement 1. 26S proteasome activities in the presence of the two α-synuclein fibrils were measured. Chymotrypsin like activity was measured by LLVY-MCA hydrolysis(A). Caspase like activity was measured by LLE-MCA hydrolysis (B). Trypsin like activity was measured by LRR-MCA hydrolysis (mean ± S.E.M; n = 3). (D and E) Co-precipitation of 26S proteasome with the α-synuclein strains. 26S proteasome was mixed with the two α-synuclein fibrils and centrifuged. Resulted supernatants (sup) and precipitates (ppt) were analyzed by western blotting against α-synuclein (D, upper) and Rpn11-HA (E, upper). Quantification of the α-synuclein (D, lower) and Rpn11-HA (E, lower) in ppt fractions were shown. (mean ± S.E.M; n = 3) (F) The core regions of the α-synuclein strains. These α-synuclein fibrils were mildly digested by proteinase K and centrifuged. The resulted pellet fractions were denatured by guanidine and analyzed by MALDI-TOF-MS. Peptide peaks identified by mass analysis and the predicted peptide regions corresponding to each peak are shown. (G) Dot blot analysis of the α-synuclein strains. These α-synuclein fibrils were spotted on nitrocellulose membranes and detected by various antibodies against α-synuclein (upper). Reactivity against the antibody raised against 115–122 region of α-synuclein was quantified (mean ± S.E.M; n = 3) (lower). Analysis was performed using one-way ANOVA and Tukey post hoc test. *p<0.05, ***p<0.001, ****p<0.0001 (A, B, C and E). Analysis was performed using student t test. *p<0.05 (D and G). Figure 4—source data 1 Quantification for graph in Figure 4A,B and C. https://cdn.elifesciences.org/articles/56825/elife-56825-fig4-data1-v2.xlsx Download elife-56825-fig4-data1-v2.xlsx Figure 4—source data 2 Quantification for graph in Figure 4D,E and G. https://cdn.elifesciences.org/articles/56825/elife-56825-fig4-data2-v2.xlsx Download elife-56825-fig4-data2-v2.xlsx If α-synuclein fibrils (-) bind other proteins, such as 26S proteasome, while α-synuclein fibrils (+) do not, the structures of the two types of fibrils should be distinguishable. Indeed, we did observe slight differences between these two types of fibrils (Figure 1). Amyloid-like fibrils are composed of core regions consisting of β-sheet-rich rigid structure, and exposed regions, which might interact with other molecules. The core regions tend to be resistant to protease attack, whereas exposed regions tend to be easily digested by protease. To identify the core regions and exposed regions of these α-synuclein fibrils, we carried out limited proteolysis with proteinase K, followed by mass spectrometric analysis (Figure 4F; Suzuki et al., 2012). We found that α-synuclein fibrils (-) have a smaller core region (amino acid residues 39–96 and 40–94; m/z 5656 and 5268, respectively) corresponding to the NAC region (amino acid residues 61–95), which had previously been reported as the core region of α-synuclein fibrils (Guerrero-Ferreira et al., 2018; Li et al., 2018). In contrast, α-synuclein fibrils (+) had a larger core region, extending to the C-terminal regions (residues 31–109, 28–118 and 25–132; m/z 7860, 9115 and 10907, respectively), indicating that α-synuclein fibrils (-) have amyloid structure with a more exposed C-terminal region than α-synuclein fibrils (+). To confirm these results, we performed dot-blot analysis using various antibodies against different regions of α-synuclein protein. Native α-synuclein fibrils were spotted on nitrocellulose membranes and detected by applying various α-synuclein antibodies. Antibodies raised against the N-terminal region of α-synuclein (residues 1–10) and the NAC region (91-99) almost equally recognized both types of α-synuclein fibrils, indicating that they have similar structure in the N-terminal and NAC regions (Figure 4G). However, as expected from the above data, the antibodies raised against C-terminal regions (amino acid 115–122 and 131–140) bound more strongly to α-synuclein fibrils (-) than to α-synuclein fibrils (+), supporting the idea that the C-terminal region of α-synuclein fibrils (-) is more exposed than that of α-synuclein fibrils (+). Thus, we considered that the C-terminal region of α-synuclein fibrils (-) might interact with 26S proteasome and impair its activities. Indeed, we previously reported that C-terminally truncated α-synuclein fibrils induced pathology in mouse brain less potently than did full-length α-synuclein fibrils, even though the C-terminally truncated α-synuclein fibrils had higher seeding activity in vitro (Terada et al., 2018). To confirm this, we next examined the seeding activity of C-terminally truncated fibrils formed in the absence of salt in primary-cultured neurons. We prepared C-terminally truncated α-synuclein monomer (residues 1–120) and agitated it in the absence of salt. The resulting assemblies showed fibrillar morphology and thioflavin T binding (data not and we refer to them as fibrils (-). We treated primary mouse cortical cells with fibrils (-) and examined the accumulation of phosphorylated α-synuclein and ubiquitinated proteins. we observed little accumulation of these proteins (Figure and B). Next, we examined the activities of 26S proteasome in the presence of fibrils (-) as shown in Figure The catalytic activity of 26S proteasome was not impaired in the presence of fibrils (-) (Figure We also investigated the interaction of 26S proteasome with fibrils (-) as shown in Figure and and found that fibrils (-) did not co-precipitated with 26S proteasome (Figure and E). all these results, we can that the C-terminal region of α-synuclein is exposed only in α-synuclein fibrils (-) and this region with 26S proteasome and its activity. Figure Download asset Open asset Effects of C-terminal truncated α-synuclein fibrils on proteasome in neurons and in vitro. (A–C) Seeding activities of C-terminal truncated α-synuclein fibrils in primary mouse cortical neurons. C-terminal truncated α-synuclein fibrils were transduced into primary mouse cortical cells. 14 days after fibril transduction, the accumulation of abnormal phosphorylated α-synuclein and ubiquitinated proteins are detected by immunofluorescence microscopy and western blotting. (A) Phosphorylated α-synuclein (Phos-αS), ubiquitin (Ub) and nuclei (DAPI) were stained. Scale bar, 20 µm. (B) Detection of sarkosyl insoluble phosphorylated α-synuclein (left) and endogenous mouse α-synuclein (right) by western blotting. (C) Detection of sarkosyl soluble GAPDH as a loading control. (D) Effects of the fibrils on the 26S proteasome activity. 26S proteasome activities in the presence of the fibrils were measured. Chymotrypsin like activity (left), like activity (center) and trypsin like activity (right) are shown. (mean ± S.E.M; n = 3). and Co-precipitation of 26S proteasome with the fibrils. 26S were mixed with the fibrils and centrifuged. Resulted supernatants (sup) and precipitates (ppt) were analyzed by western blotting against α-synuclein (E) and Rpn11-HA Quantification of the Rpn11-HA in ppt fractions were shown (mean ± S.E.M; n = 3) Analysis was performed using student t test. Figure data 1 Quantification for graph in Figure and Download Discussion to the prion differences in disease and lesions are caused by differences in the of strains. Therefore, α-synuclein is differences in the structure of α-synuclein aggregates should cause the differences in the lesions observed in various analyses have shown that of extracts from brains of patients with PD and MSA into mice and cells induces different pathologies et al., 2015; et al., 2018; Woerman et al., 2019; Woerman et al., 2015). results were with brain extracts of patients with in which tau is accumulated et al., et al., 2019). Moreover, it has been that aggregates have a different structure on the disease, and different lesions when the patients' brain extracts are introduced into cells or animals et al., 2019; et al., 2013; et al., 2018; et al., 2012). for α-synuclein, it has been reported that aggregates different structures are formed in vitro (Bousset et al., 2013; Shahnawaz et al.,