Inflammation drives synucleinopathy propagation

In ammation drives synucleinopathy propagation Tae-Kyung Kim Korea National Sport University Eun-Jin Bae Seoul National University College of Medicine https://orcid.org/0000-0002-4369-7405 Byung Chul Jung https://orcid.org/0000-0003-0732-0122 Minsun Choi Department of Biomedical Sciences and Medicine, Neuroscience Research Institute, Seoul National University College of Medicine, Seoul Soo-Jean Shin Department of Biomedical Sciences and Medicine, Neuroscience Research Institute, Seoul National University College of Medicine, Seoul Jeong Tae Kim Department of Biomedical Sciences and Medicine, Neuroscience Research Institute, Seoul National University College of Medicine, Seoul Min-kyo Jung University of Ulsan Ayse Ulusoy German Center for Neurodegenerative Diseases (DZNE) https://orcid.org/0000-0003-2840-3429 Mi-Young Song Jun Sung Lee Department of Biomedical Sciences and Medicine, Neuroscience Research Institute, Seoul National University College of Medicine, Seoul He-Jin Lee Konkuk University Donato Di Monte German Center for Neurodegenerative Diseases (DZNE) https://orcid.org/0000-0002-8296-836X Seung-Jae Lee (  sjlee66@snu.ac.kr ) Department of Biomedical Sciences and Medicine, Neuroscience Research Institute, Seoul National University College of Medicine, Seoul https://orcid.org/0000-0002-5155-5335 Article Keywords: synucleinopathy, neurodegenerative disease, α-synuclein (V40G), in ammation Page 1/28 DOI: https://doi.org/10.21203/rs.3.rs-774660/v1 License:   This work is licensed under a Creative Commons Attribution 4.0 International License. Read Full License Page 2/28 Abstract The clinical progression of neurodegenerative diseases correlates with the spread of proteinopathy in the brain. Understanding of the mechanism of the proteinopathy spread is far from complete. Here, we propose that in ammation is fundamental to proteinopathy spread. A sequence variant of α-synuclein (V40G) was much less capable of bril formation than wild-type α-synuclein (WT-syn) and, when mixed with WT-syn, interfered with its brillation. Yet when V40G was injected intracerebrally into mice, it induced aggregate spreading even more effectively than WT-syn. The aggregate spreading was preceded by sustained microgliosis and in ammatory responses, which were more robust with V40G than with WTsyn. Oral administration of an anti-in ammatory agent suppressed aggregate spreading, in ammation, and behavioral de cits in mice. Furthermore, exposure of cells to in ammatory cytokines increased the cell-to-cell propagation of α-synuclein. These results suggest that the in ammatory microenvironment is the major driver of the spread of synucleinopathy in the brain. Introduction Protein aggregation is the major pathological hallmark of several neurodegenerative diseases, with different types of aggregates and distribution patterns characterizing each disease. Large-scale pathological post-mortem examinations have indicated that protein aggregates in Alzheimer’s and Parkinson’s diseases spread from a few initial sites and progressively involves more and more brain regions in a highly speci c topographic sequence 1, 2. This spreading of pathological aggregates most likely occurs via direct cell-to-cell transfer of aggregation-prone pathogenic proteins, such as tau and αsynuclein 3, 4, 5, 6. Although the mechanism remains unclear, the aggregate spreading phenomenon has been veri ed in several model systems. The most commonly used animal model for studying aggregate spreading is mice injected intracerebrally with preformed brils (PFFs). A single injection of PFF causes aggregation of the corresponding protein (e.g., tau or α-synuclein) in different brain regions 7. Features underlying the transmission of prion proteins have led researchers to presume that aggregate spread in Alzheimer’s disease and Parkinson’s disease also occurs by a mechanism known as templated conformational seeding 8. However, this theory is apparently inconsistent with some experimental observations. For example, PFFs are rapidly cleared after injection and there is always an incubation time before aggregates reappear 7. Furthermore, injection of an α-synuclein variant lacking the critical region for brillation still resulted in α-synuclein aggregate spreading 9, 10, 11. Thus, mechanisms other than templated seeding may contribute to the spread of protein aggregates. Here, we directly tested whether templated seeding is su cient for explaining protein aggregate spreading by using a speci c sequence variant of α-synuclein (V40G) that cannot seed aggregation. Results Page 3/28 The α-synuclein V40G variant forms amyloid brils much less e ciently than WT and blocks bril formation in vitro To nd a sequence variant without seeding ability, we screened a pool of α-synuclein mutants in the hinge region (amino acids 38–44) for variants unable to form amyloid brils after 9 days of incubation and identi ed a variant with a substitution of glycine for valine 40 (V40G). Circular dichroism (CD) spectra indicated that “fresh” wild-type α-synuclein (WT-syn) and V40G monomeric proteins showed random coil con gurations (Fig. 1a). To determine the behavior of the proteins after a period of aging, we incubated them for 9 days. After the incubation, “aged” WT-syn acquired a β-sheet-rich conformation (Fig. 1a) with lamentous morphology (Fig. 1c, Supplementary Fig. 1a) and exhibited thio avin T (Thio T) uorescence (Fig. 1d), re ecting the structural transition to amyloid brils. In contrast, aged V40G contain much less βsheet contents than WT (Fig. 1a, b) and consistently exhibited minimal Thio T uorescence (Fig. 1d). Further dye binding assays with SybrGreen, X-34, and curcumin showed that V40G had different dyes binding properties from WT (Fig. 1e). Size exclusion chromatography showed that a large portion of V40G was multimeric after the 9-day incubation (Fig. 1f). Electron microscopy (EM) con rmed that V40G formed a variety of structures, including short bril-like structures and small multimers with heterogeneous morphologies (Fig. 1c, Supplementary Fig. 1b). In addition, V40G samples show some typical brils, which would explain the presence of a small amount of β-sheet signal in the CD spectrum (Supplementary Fig. 1b). Velocity ultracentrifugation con rmed that V40G formed less amount of the large sedimenting aggregates than WT, while monomer showed similar pattern (Fig. 1g, Supplementary Fig. 1c). Proteinase K digestion of the aged WT-syn and V40G produced different fragmentation patterns (Fig. 1h), which indicates that these aggregates have different conformations. Thus, V40G generates heterogeneous aggregates, with less amount of brils than WT. To examine the seeding activities of WT-syn brils and V40G multimers, we performed the following experiments. First, protein misfolding cyclic ampli cation (PMCA), a method that ampli es brils through the seeding mechanism, resulted in much better ampli cation of WT-syn brils than of V40G multimers, suggesting that V40G multimers are much less ampli able through templated seeding than WT brils (Fig. 1i, j). Next, we mixed WT-syn monomers with 5% (w/w) of WT-syn brils or V40G multimers, and brillation was assayed using Thio T uorescence. The addition of WT-syn brils eliminated the lag time to brillation, clearly demonstrating its seeding effect (Fig. 1k). In contrast, the addition of V40G multimers completely inhibited brillation of the WT monomer (Fig. 1k). Therefore, human WT-syn can seed the brillation of mouse WT-syn, while human V40G multimers inhibit the aggregation of mouse WTsyn. These results suggest that the V40G variant produces heterogeneous forms of multimers that can block bril formation in the WT-syn protein in vitro. V40G injection causes robust synucleinopathy in mice To examine the abilities of WT-syn brils and V40G multimers to induce aggregate spreading in the brains of animals, we injected these proteins or vehicle (phosphate buffered saline; PBS) into the striatum of naïve C57BL/6 mice and examined the neuropathology 2, 4 and 10 weeks after injection (Fig. 2). Surgery Page 4/28 itself did not cause blood cell in ltration or serum extravasation (Supplementary Fig. 2a, c). The injected proteins were rapidly cleared from the brain. WT-α-syn brils were more stable than V40G multimers with half-life of 0.67 days vs. 0.45 days for the detergent-soluble proteins and 4.49 days vs 0.43 days for the detergent-insoluble proteins (Supplementary Fig. 3a-f). Spreading of phospho-α-synuclein (pS129), a marker of brillar aggregates in synucleinopathy, was apparent in several brain regions 10 weeks after injection of WT-syn brils (Fig. 2a, b). Unexpectedly, injection of V40G multimers, which blocked seeding in the in vitro assays, resulted in more robust pS129 signals throughout the brain than did WT-syn bril injection (Fig. 2a, b, Supplementary Fig. 4, Supplementary Table 1). Western analysis also showed that the detergent-insoluble α-synuclein was increased after the injection of V40G multimers (Fig. 2c, d). The pS129-positive aggregates were co-labeled with Thio avin S, suggesting that these aggregates were βsheet-rich (Fig. 2e). Filamentous structures of these aggregates were validated with immuno-EM with an α-synuclein antibody (Fig. 2f). Some immune-positive aggregates in the brain showed globular structures as well (Fig. 2f). These results suggest that templated seeding is not necessary for driving aggregate spreading and, thus, is not the only mechanism. The early phases of aggregate spreading, are characterized by a strong in ammatory response that correlates with the levels of aggregation To determine the molecular processes occurring during the early phases of aggregate spreading, we analyzed the transcriptome changes in the rhinal cortex, one of the major initial spreading sites, 4 weeks after an intrastriatal injection of WT-syn brils, V40G multimers, or vehicle (PBS). The heat map (Supplementary Fig. 5a, b), which shows the Euclidean distances between the samples, demonstrates that the gene expression patterns in V40G-injected mice were more similar to those in WT-syn-injected mice than to those in vehicle-injected mice. We ltered the genes by the magnitude of their difference in expression (fold change > 1.5) and p values (< 0.05) (Supplementary Fig. 5c, d, Supplementary Table 2) to identify a total of 418 differentially expressed genes (DEGs) in WT-syn-injected mice and 485 in V40Ginjected mice, compared to expression in vehicle-injected mice (Fig. 3a, b, Supplementary Table 2). Enrichment map based on Gene Ontology (GO) terms manifested that the immune system process is the key enriched term in both groups (Fig. 3c, d). The DEGs in WT-syn-injected mice were mostly related to immune responses, such as leukocyte proliferation and migration, chemokine production, and T cell activation, antigen processing and presentation, and in ammatory responses (Fig. 3c, Supplementary Fig. 5e). Consistent with the enriched GO analysis, enriched Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis of DEGs in WT-syn-injected mice identi ed mostly immune-related pathways, e.g., antigen processing and presentation, Toll-like receptor signaling, cytokine–cytokine receptor interaction, and complement and coagulation cascades (Supplementary Fig. 5f). GO analysis of DEGs from V40G-injected mice also showed changes in immune responses, including leukocyte-mediated immunity, T cell activation, interferon beta production, chemokine production, and cytokine production, cytokine responses, immune system processes, and in ammatory responses (Fig. 3d, Supplementary Fig. 5g). Likewise, three immune response-related KEGG pathways—the tumor necrosis factor (TNF) signaling pathway, complement and coagulation cascades, and cytokine–cytokine receptor interactions— were enriched in the same DEGs (Supplementary Fig. 5h). Among these DEGs, 110 were common Page 5/28 between WT-syn- and V40G-injected mice (Fig. 3b, Supplementary Table 3). We analyzed the enriched GO pathways to which the 110 common DEGs belonged, among which 84 genes were upregulated and 26 were downregulated (Fig. 3b, Supplementary Table 3). GO analysis of these common DEGs revealed immune-related pathways almost exclusively (Fig. 3e). Next, we compared the extent of immune-related GO enrichment between the WT-syn- and V40G-injected mice, with lower p values indicating more meaningful enrichment in the GO pathway. The majority of immune-related GO pathways were more signi cantly enriched in V40G-injected mice than in WT-syn-injected mice (Fig. 3f), suggesting that immune/in ammatory responses occur early in α-synuclein aggregate-injected mouse brains and that V40G multimers cause more robust immune-related changes than WT-syn brils. Consistent with this suggestion, when 40 immune-related DEGs were selected and their fold changes were compared, the majority of genes showed a more robust fold change in V40G-injected mice than in WT-syn-injected mice (Fig. 3g). To validate the in ammatory responses experimentally, we performed immunohistochemical analysis of interleukin (IL)-1β as an in ammatory marker in the same group of animals described in Fig. 4. IL-1β immunoreactivity was found in many regions throughout the brain 2 weeks after injection in all groups, including PBS-injected animals (Fig. 4a). However, this in ammatory marker disappeared rapidly in PBSinjected brains, whereas it remained present in brains injected with either WT-syn or V40G until at least 10 weeks after injection. V40G injection resulted in more sustained and robust IL-1β immunoreactivity than WT-syn injection, and the immunoreactivity correlated well with the extent of α-synuclein accumulation (Fig. 4a, Supplementary Table 4). Co-immunostaining of IL-1β with cellular markers, such as NeuN, GFAP, and Iba-1, showed that IL-1β expression occurred exclusively in Iba-1-positive microglia (Fig. 4b). We then analyzed the extents of microgliosis with Iba-1 immunohistochemistry and found that microgliosis was present in animals injected with either WT-syn brils or V40G multimers, but was more extensive and sustained after V40G injection (Fig. 4c-g). In contrast to microgliosis, the extent of astrogliosis did not correlate well with pS129 spreading (Supplementary Fig. 6). To verify the in ammatory responses of microglia after exposure to WT-α-syn brils and V40G multimers, we analyzed the in ammasome activation and cytokine production in cultured primary microglia. Both WT-α-syn brils and V40G multimers activated in ammasome, showing induction of NLRP3 (Fig. 5a, b) and ASC speck formation (Fig. 5c). NLRP3 induction was signi cantly higher with V40G-α-syn treatment than with WT-α-syn treatment (Fig. 5a, b). Production of cytokines, such as TNF-α, IL-1β, and IL-6, was stronger after V40G multimer treatment than after WT-α-syn bril treatment (Fig. 5d-h). The stronger responses for V40G multimers than for WT-α-syn brils were not attributed to the stability of the proteins; WT-α-syn brils were more stable than V40G multimers in microglia (Supplementary Fig. 3h-j). These results con rmed the transcriptome analysis, and indicate that in ammatory responses precede aggregate spreading and that the extent of in ammation correlates with that of α-synuclein aggregation. To directly test the effects of microglia and in ammatory cytokines on the propagation of α-synuclein, we used the dual-cell BiFC system, which is composed of two cell lines, V1S and SV2 12. In this system, when α-synuclein is transferred from cell to cell, yellow uorescent puncta appear in the cellular cytoplasm, Page 6/28 allowing quantitative assessment of α-synuclein propagation. By measuring the percentage of cells with uorescent puncta, we found that both co-culturing with microglia and exposing V1S and SV2 cell lines to TNF-α and IL-1β signi cantly increased the cell-to-cell propagation of α-synuclein (Fig. 5i-l). Aspirin reversed V40G-induced behavioral de cits and pathology To con rm the role of in ammation in the pathology of α-synuclein spreading, we administered aspirin, a common anti-in ammatory drug, to C57BL/6 mice for 17 weeks, starting 1 week after intrastriatal injection of WT brils, V40G multimers, or PBS and examined pathological changes and behavioral outcomes (Fig. 6, Supplementary Fig. 7a). Aspirin was delivered daily in the drinking water at approximate doses of 2 mg per kg and 40 mg per kg. When we examined the effects of aspirin administration on in ammation and aggregation at 18 weeks after injection, we found that the V40Ginduced increases in the levels of TNF-α and microgliosis were prevented, con rming the antiin ammatory e cacy of this drug in the brain (Fig. 6a-c). Aspirin also signi cantly decreased abnormal pS129 deposition and total α-synuclein in the injected animals (Fig. 6a, d and e). Likewise, loss of dopaminergic terminals in the striatum and dopaminergic cell bodies in the substantia nigra pars compacta were reversed by aspirin treatment (Fig. 6f, g, Supplementary Fig. 7b-e). Injection of WT-α-syn brils also caused dopaminergic cell loss, although to a lesser extent than V40G multimers (Supplementary Fig. 7e vs. Figure 6g). The cell loss caused by WT-α-syn brils were also reversed by aspirin administration (Supplementary Fig. 7e). All pathological changes described here, excepted for the dopaminergic cell count, were observed not only in the injected hemisphere but also in the contralateral hemisphere. These results emphasize the roles of proin ammatory factors in aggregate propagation and ensuing pathology. To examine the effects of aspirin on behavioral de cits, the mice were subjected to a battery of behavioral assessments at 12 and 18 weeks after injection. The total activity and anxiety-like behavior (measured in the open eld test) of V40G-injected mice did not differ from that of either naïve (noninjected) controls or PBS-injected controls. However, V40G-injected mice showed de cits in motor control (rotarod test), motor strength (four-limb hanging test), and sensory spatial memory (Y maze test) (Fig. 6h-m show data at 18 weeks). The behavioral de cits developed in a time-dependent manner, being less severe at 12 weeks than at 18 weeks after injection (Supplementary Fig. 7a, 8a-g). Aspirin 40 mg per kg completely reversed all the behavioral de cits, and some de cits were even reversed by 2 mg per kg. The normal behavior of noninjected or PBS-injected mice was not affected by aspirin treatment (Supplementary Fig. 8h-8k). Discussion Herein, we have identi ed a sequence variant of α-synuclein (V40G) that forms stable multimers that not only lack seeding ability but also block the brillation of WT-α-syn in vitro. In striking contrast to templated seeding model predictions, V40G multimers effectively propagated α-synuclein aggregates Page 7/28 after intrastriatal injections in mice. These ndings suggest that templated seeding is not solely responsible for the aggregate spreading. The synucleinopathy induced by injection of either WT-syn or V40G was preceded by in ammatory responses, and the degree of synucleinopathy correlated with the extent of in ammation. Furthermore, V40G-induced synucleinopathy was suppressed by the administration of an anti-in ammatory drug, consistent with an important role of in ammation in synucleinopathy spreading. Previous studies have shown a relationship between neuroin ammation and protein aggregation. Lipopolysaccharide (LPS)-induced brain in ammation induces α-synuclein aggregation in mice 13. The present work also showed that the proin ammatory cytokines TNF-α and IL-1β stimulated cell-to-cell propagation of α-synuclein in vitro. Neuron-released oligomeric α-synuclein has been shown to induce in ammatory responses from microglia through the activation of Toll-like receptor 2 (TLR2) 14. Therefore, as an alternative to the templated seeding model, we propose a model that emphasizes the role of the in ammatory microenvironment in aggregate propagation. In this model, protein aggregates would initially induce chronic in ammation, which in turn would create a microenvironment that would favor protein aggregation in neurons, establishing a vicious cycle between protein aggregation and in ammation. The protein aggregates would then be transferred through anatomical neural connections and would establish another in ammatory microenvironment, expanding the vicious cycle between protein aggregation and in ammation to the new location. Consistent with this model, Olanow et al. recently reported that brain immune cell activation preceded α-synuclein aggregation in fetal mesencephalic neuronal transplants in patients with Parkinson’s disease 15. Our work is also in line with recent work in which in ammasomes were shown to play important roles in tau propagation 16. The incubation time, that occurs prior to aggregate spreading in experimental animals, may be the period that is required for the cycle between the in ammatory microenvironment and protein aggregation to be established. The coexistence of different pathological structures, e.g., Lewy bodies and neuro brillary tangles, in many human specimens might also be explained by the in ammatory microenvironment model, in which aggregation of one protein generates a microenvironment that favors not only the aggregation of homotypic aggregates but also that of other aggregation-prone proteins. Our current experimental system mimics the in ammatory microenvironment that triggers protein aggregation and other pathological changes, and we provide evidence that these phenotypes are led by microglia. These ndings are in line with the recent observations showing detrimental effects of immune cells and in ammation in synucleinopathy models 17, 18, 19, 20. However, the opposite effects of immune cells have also been reported 21, 22, 23. In the latter cases, immune cells adapted to the protective states, in which the scavenging activity was strong while the detrimental in ammation was suppressed. It has become evident that the cells involved in the brain innate immunity, including microglia and astrocytes, exist in many different states24, 25, 26, 27. These cells may transform from one state to another, sensing the microenvironment. The mechanism by which these different states are switched is still unknown and remains one of the most important and challenging questions in neurobiology. Page 8/28 Results of this study clearly underscore the key role played by in ammatory processes in α-synuclein spreading. They also raise important new questions and suggest new pathogenic mechanisms underlying the development of α-synuclein pathology. For example, during the development of proteinopathies, different aggregates with distinct conformations might generate different types of in ammatory microenvironments that would favor the formation of aggregates with corresponding structures. In conclusion, our results provide new insights into how protein aggregates propagate, and are likely to lead to new research unveiling core principles of disease progression in Parkinson’s disease. Furthermore, as a result of this study, new lines of research will open up to investigate the role of in ammation in propagation of other proteins whose aggregation is associated to neurodegenerative diseases. Materials And Methods Mutagenesis A mutation (V40G) was introduced into human wild-type α-synuclein (α-synuclein/pDual GC; Agilent Technologies, Santa Clara, CA, USA, #214503) using a QuikChange II XL Site-Directed Mutagenesis Kit (Agilent Technologies, #200521. The primers used were the following: 5′- GGT GTT CTC TAT GGC GGC TCC AAA ACC AAG − 3′ (sense), 5′- CTT GGT TTT GGA GCC GCC ATA GAG AAC ACC − 3′ (antisense). Protein puri cation and bril preparation The pDdulGC vector expressing mouse wild-type α-synuclein, human wild-type α-synuclein (WT), or V40G α-synuclein was transformed in Escherichia coli BL21(DE3) (RBC Korea, Seoul, Korea, #RH217). WT-syn and V40G were expressed and puri ed as previously described 28. For brillation, α-synuclein (200 µM in PBS) was incubated at 37°C for 9 days with constant shaking at 1,050 rpm in a ThermoMixer C (Eppendorf, Hamburg, Germany, #5382000015). When used as seeds, the brils were sonicated for 1 min (amplitude 30%) before undergoing the brillation reaction 28. Circular Dichroism (CD) spectroscopy All CD spectra of protein samples (0.5 mg/ml) were recorded using a Chirascan Plus spectropolarimeter (Applied Photophysics, Leatherhead, Surrey, UK) between 190 and 260 nm in 0.1 mm cells with a step resolution of 1.0 nm, bandwidth of 1.0 nm, and scan speed of 100 nm/min. All spectra were obtained on an average of 10 separate measurements. Transmission electron microscopy The aged WT-syn and V40G were adsorbed onto 200-mesh carbon-coated copper grids (Electron Microscopy Sciences, Hat eld, PA, USA, #CF200-Cu), then subjected to add 20 µl of 2% uranyl acetate (Electron Microscopy Sciences, #22400) for negative staining. The prepared grids were observed using a JEM1010 transmission electron microscope (JEOL, Akishima, Tokyo, Japan). Page 9/28 Fluorescent dye binding assay Recombinant α-synuclein samples were mixed either 50 µl of 10 µM Thio T (Sigma, #T3516) solution in glycine (Fisher Scienti c, Hampton, NH, USA, #BP3815)–NaOH (pH 8.5), 40 µl of 0.001% Sybr Green commercial stock solution (#S7563, Invitrogen, Carlsbad, CA, USA), 50 µM X-34 (Sigma, #SML1954), or 100 µl of 50 µM curcumin (Sigma, #C1386). After incubation at room temperature, uorescence measurements were performed on Synergy NEO plate reader (Biotek, Winooski, VT, USA). Excitation and emission wavelengths were set at 440 nm and 490 nm, respectively, for Thio T, at 485 nm and 520 nm for Sybr Green, at 380 nm and 520 nm for X-34, and at 440 nm and 519 nm for curcumin. Sedimentation assay Twenty microliters of human WT and V40G α-synuclein monomer and bril (200 µM in PBS) were each used for sedimentation assays. Twenty microliters of samples were mixed with 280 µl of DPBS (Gibco, Carlsbad, CA, USA, #A1285601) and placed on top of 30% sucrose, bottom of the tube being 5% sucrose. Samples were centrifuged at 38,000 rpm in a Beckman XL-90K ultracentrifuge using a SW-41Ti rotor (Beckman). Fractions were collected and mixed with Laemmli sample buffer. Proteinase K (PK) digestion α-Synuclein samples (5 µM) were incubated with a nal concentration of 10 µg/ml PK (Sigma, #P4850) for 1 h 20 min at 37°C. The reactions were stopped by the addition of Laemmli sample buffer, followed by heating at 95°C for 10 min. Protein misfolding cyclic ampli cation (PMCA) PMCA was performed as previously described 28. α-Synuclein monomers were prepared up to a nal concentration of 5 µM in conversion buffer (1% Triton X-100, 150 mM NaCl), and 50 µl of aliquot was transferred into PCR tube containing three Te on beads. Samples were subjected to 48 cycles of 20 s sonication (amplitude 1%) and 29 min 40 s incubation at 37°C for 24 h. Fifteen nanograms of aged WTsyn or V40G were added as exogenous seeds. Western blotting Western blotting was performed as previously described with a few modi cations 28, 29. In the case of PK digestion experiments, PK-treated samples were loaded onto 16% gels. Primary antibodies and their dilutions were: anti-α-synuclein monoclonal antibody (Syn-1, BD Biosciences, #610787; 1:1500), anti-αsynuclein antibody (LB509, Abcam, MA, USA; ab27766, Abcam, 1:1000), NLRP3 (EPR23094-1, Abcam, ab263899, 1:1000). The membrane was detected with ECL solution (GE Healthcare, # RPN2232). Image detection was performed using an Amersham Imager 600 (GE Healthcare) and Multi Gauge (v.3.0) software (Fuji lm, Akishima, Tokyo, Japan). Animals Page 10/28 Ten-week-old male wild-type C57BL/6 mice were purchased from The Jackson Laboratory (Bar Harbor, ME, USA). Three-month-old C57BL/6 mice overexpressing human α-synuclein under the murine Thy1 promoter (mThy1-α-syn tg, Line 61) were used for immune cell in ltration assay 30. Mice were housed and processed according to the standardized conditions at animal facility in the Seoul National University College of Medicine. All mice were maintained in the animal facility for habituation for at least one week before the start of the experiment. All mouse studies were conducted in compliance with the relevant ethics regulations and approved by the Seoul National University Ethics Committee (IACUC SNU-170428). Stereotaxic injection of WT brils and V40G multimers For intrastriatal injection of WT-syn brils and V40G multimers, 10-week-old male C57BL/6 mice were anesthetized with ketamine hydrochloride and xylazine hydrochloride (3.5:1, 2.5µl/g). PBS, WT-syn brils or V40G multimers (6 µg) in a volume of 2 µl were stereotaxically injected into the right striatum (anterior/posterior, 1.0 mm; medial/lateral, 1.5 mm; and dorsal/ventral, 3.0 mm) at a speed of 0.5 µl/min using a 30 G needle. Drug administration The administration of 2 mg/kg or 40 mg/kg acetylsalicylic acid (Aspirin; Sigma, #A5376) in drinking water was initiated 1 week after intrastriatal injection of PBS, WT-syn, or V40G. Monitoring the amounts of aspirin consumed, once every 2 weeks for 18 weeks, showed a consumption of 4.3–5.2 ml/day, and there was no signi cant difference in consumption between groups. The concentration of aspirin dissolved in drinking water provided for each animal was given at 12.5 µg/ml (2 mg/kg) or 250 µg/ml (40 mg/kg). Behavioral assessments We subjected mice to total activity, motor control, motor strength, sensory spatial memory and emotional behavior tests 1 week prior to sacri ce. Behavioral tests were performed using a computerized video recording and tracking system (Ethovision XT version 14, Noldus, Wageningen, Netherlands). Open eld test To assess activity, locomotion, and anxiety, we subjected mice to the open eld test as previously described 31. The testing room was indirectly illuminated to 15–20 lux. The open eld test apparatus was a square arena (40 × 40 × 40 cm) with white Plexiglas walls and oor. We placed the mice individually in the center, allowing them to freely navigate the arena for 10 min while their activity was video recorded. Rotarod test Motor coordination and balance were assessed with an accelerating rotarod system (Rotamex 5, Columbus Ins., Columbus, OH, USA). Mice were placed on the rotarod spindle (3.0 cm × 9.5 cm), which accelerated from 4 to 35 rpm over 300 s, and the latency to fall was measured. After one practice using this protocol, each mouse was tested twice, and the average latency was taken. Page 11/28 Four-limb hanging test The four-limb hanging test was used to measure the muscle strength of the four limbs. A grid apparatus (5 × 5 cm) was designed to allow the mice to grab the wire-mesh grid, and then the grid was inverted 180 degrees. The latency for the mice to fall from the grid was measured, with a maximum of 600 s. Y maze test To measure the prefrontal cortex- and hippocampus-dependent spatial memory de cits 32, we tested the spontaneous alternation behavior (SAB) and continuous alternation (CA) of mice using a standard protocol 33. The Y maze apparatus consisted of three arms (each 33 cm long) made of white plastic, joined at the center to form a Y shape. We placed the mice individually into one of the arms and allowed them to freely explore the three arms for 5 min while their activity was video recorded. Sample collection At 2, 4, 10 or 19 weeks after intrastriatal injection, mice were anaesthetized with ketamine hydrochloride and xylazine hydrochloride (3.5:1, 2.5µl/g) then perfused transcardially with saline followed by ice-cold 4% PFA. Brains were dissected out and xed in phosphate-buffered 4% PFA for at least 48 h at 4°C for neuropathological analysis. For biochemical analysis, brain samples were dissected and stored at -80°C at 0, 2, 7, 14 days and 5, 19 weeks after intrastriatal injection. Four weeks after intrastriatal injection, the brain samples were dissected into the rhinal cortex before freezing on dry ice and stored at -80°C for RNA analysis,. Evans blue assay Two weeks after intrastriatal injection, mice were anaesthetized with ketamine/xylazine mixture, and the cardiac perfusion was performed using 50 ml PBS (pH 7.2) followed by 50 ml of the cocktail containing 1% Evans blue (Sigma–Aldrich) dissolved in 4% PFA. Brains were dissected out and xed in phosphatebuffered 4% PFA for 4 h, cryoprotected in 30% sucrose overnight at 4°C, and then frozen in OCT medium on dry ice. Twenty-µm-thick brain cryosections were washed with PBST and mounted on uorescent mounting medium containing DAPI (H1200, Vector Laboratorie, CA, USA). Then, visualized using uorescence microscope (Olympus IX53) by excitation with 543-nm laser beams and visualized as red uorescence. Immunohistochemistry and neuropathological analysis The procedures for immunohistochemical experiments have been described in detail elsewhere 34. Fortyµm thick free- oating brain sections were reacted with primary antibodies at 4 °C overnight prior to incubation with secondary antibodies (Bio-Rad, #170–6515, #170–6516, #5204 − 2504) diluted 1:200 in PBST, and detected using avidin-biotin-peroxidase complex (ABC Elite kit, Vector Laboratories, Burlingame, CA, USA, #PK6200). Next, 3,3-diaminobenzidine (DAB)-stained sections were imaged using a ZEISS AX10 microscope and an Aperio AT2 microscope. The levels of immunoreactivity against total human α-synuclein (Syn1; BD Biosciences, 1:500), phospho-α-synuclein (pS129; Abcam, #ab59264; Page 12/28 Biolegend, CA, USA, #825701, 1:500), Iba-1 (Wako, Osaka, Japan, #019-19741, 1:200), glial brillary acidic protein (GFAP; Abcam, #ab7260, 1:500), tyrosine hydroxylase (TH; Abcam, #ab112, 1:2000), IL-1β (Abcam, #ab9722, 1:200), TNF-α (Abcam, #ab6671, 1:200), CD4 (BD Bioscience, #553727, 1:200) and CD8a (BD Bioscience, #553027, 1:200) were determined by optical density analysis using ImageJ (NIH) and corrected against background signal levels. Phospho-α-synuclein-positive cells in each animal were quanti ed as the percentage of positive cells in a eld of view based on cell body recognition using the ImageJ program (NIH). Immuno uorescence and Thio avin S staining Free- oating brain sections were blocked with 4% BSA in PBST and then reacted with primary antibodies phospho-α-synuclein (pS129; Abcam, #ab51253, 1:500), IL-1β (Abcam, #ab9722, 1:200), NeuN (Millipore, #MAB377, 1:500), GFAP (Abcam, #ab10062, 1:500) and Iba-1 (Novus Biologicals, CO, USA, #NB100-1028, 1:200) at 4°C light blocking overnight. The sections were washed with PBST, incubated with uorescent dye Alexa488-, or Rhodamine red-X-conjugated secondary antibodies (Jackson Immunoresearch Laboratories, PA, USA, #115-545-062, #705-545-147, #111-295-144) diluted 1:200 in PBST, and mounted with uorescence mounting medium (Vector Laboratories). The phospho-α-synuclein immunoreactive sections were treated with graded EtOH for hydration and incubated in ltered 1% aqueous Thio avin-S (Sigma, T1892) for 8 min. After washing with 80% and 95% EtOH, coverslip was placed in aqueous mounting medium and slides were dried in the dark overnight. The stained samples were observed under a Carl ZEISS-LSM 700 confocal laser-scanning microscope. Immuno-EM Mouse brain slices were xed using 2.5% glutaraldehyde and 2% paraformaldehyde in sodium cacodylate buffer (pH 7.2) at 4°C. Samples were xed again by using 1% osmium tetraoxide for 30 min at 4°C. The xed samples were dehydrated using an ethanol series (50%, 60%, 70%, 80%, 90%, and 100% ethanol) for 20 min and were transferred to LR White (Electron Microscopy Science, Hat eld, PA, USA). The samples were impregnated with and embedded in the same resin mixture, sectioned (60-nm-thick sections) with an ultramicrotome (Leica Ultracut UCT; Leica Microsystems, Vienna, Austria), and placed on nickel grids. αsynuclein bril in the samples was labeled with immunogold by using phospho-α-synuclein antibody (pS129; Abcam, #ab51253, 1:50) and 9- to 11-nm colloidal gold-conjugated goat anti-mouse IgG secondary antibodies (Sigma, St. Louis, MO, USA). The sections were double-stained with 2% uranyl acetate for 10 min and lead citrate for 5 min and were viewed under the transmission electron microscope at 120 kV (Tecnai G2, ThermoFisher, Waltham, MA, USA). Nigral tissue preparation and neuronal cell counting Forty-µm thick free- oating brain sections were processed for immunohistochemistry as previously described 35. Rabbit anti-TH antibody (Millipore, Burlington, MA, USA, #AB152; 1:1000) was used as primary antibody prior to incubation in biotinylated goat anti-rabbit secondary antibody solution (Vector laboratories, #BA1000; 1:200). The sections were treated with avidin-biotin-peroxidase (ABC Elite kit, Vector Laboratories) complex. The color reaction was developed by DAB. Sections were then mounted on Page 13/28 microscope slides, counterstained with cresyl violet (FD Neurotechnologies) and cover slipped using DPX mounting media (Sigma, #06522). Unbiased stereological counts were performed by an investigator blinded to the experimental groups using the optical fractionator and the Stereo Investigator 2019 software (MBF, version 1.3). The ipsilateral substantia nigra pars compacta was delineated on every 4th midbrain section in the rostro-caudal axis between Bregma − 2.7 mm and − 3.6 mm. Large and densely packed tyrosine hydroxylase-immunoreactive neurons characterized this midbrain region, allowing its delineation using a low-power objective lens (4X UPlanFL N) on an Olympus BX53 microscope equipped with an automated stage (MBF, mac6000) and a Heidenhein Z-axis decoder. The delineation excluded other tyrosine hydroxylase-positive cells in neighboring areas, namely the substantia nigra pars reticulata and pars lateralis (ventral and lateral to the compacta, respectively) and the ventral tegmental area (medial and dorsomedial). Counts were performed at higher magni cation (100X UPlanS Apo) using a 1µm guard zone on the top and bottom of each section. The coe cient of error was calculated according to Gundersen and Jensen 36; all values were < 0.10. Brain tissue and cell extraction Samples were homogenized with lysis buffer (1% Triton X-100, 1%(v/v) protease inhibitor cocktail (Sigma-Aldrich, P8340) in PBS. Lysates were incubated on ice for 10 min and centrifuge at 16,000g for 10 min. After collecting supernatant (Triton X-100 soluble fraction), Triton X-100 insoluble fraction was resuspended with 1X Laemmli sample buffer and sonicated brie y. RNA processing RNA was extracted using TRIzol (Invitrogen, #15596018) and quanti ed using an ND-2000 Spectrophotometer (Thermo Fisher Scienti c). Libraries were prepared using the QuantSeq 3’ mRNA-Seq Library Prep Kit (Lexogen, Inc., Greenland, NH, USA) and sequenced with 75-bp single-end reads on a NextSeq 500 (Illumina, Inc., San Diego, CA, USA). RNA sequencing (RNAseq) data analysis Analysis of the RNAseq data was performed using TopHat2 (version 2.1.1) and the Cu inks suite (version 2.1.1). The data were processed and quanti ed using HTSEq. Differential gene expression analyses were performed using DESeq2 (version 1.24.0) with the Wald test and their respective default lters 37. p < 0.05 was used as the threshold for differentially expressed genes (DEGs). Enrichment analyses of GO terms were analyzed using a Cytoscape plug-in ClueGO based on related terms and statistical signi cance 38. The enriched Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses of the DEGs were conducted on DAVID datasets (version 6.8) 39. Primary microglia culture Primary microglial cells were obtained from the cerebral cortices of 1-day-old neonatal C57BL/6 mice as previously described 40. Approval for the experiments was granted by the Institutional Animal Care and Use Committee in Seoul National University (SNU-171207-2-5). To induce in ammatory cytokines in microglia cells, microglia cells were treated with 200 nM of aged WT-synuclein or V40G-synuclein for 8 h. Page 14/28 To assess in ammasome activation and secretion of in ammatory cytokines, 200 nM of aged WTsynuclein or V40G-synuclein was treated to microglia for 18 h. Measurement of Cytokine Secretion Microglial TNF-α and IL-1β secretion were measured in cell supernatants using the Mouse TNF-alpha Quantikine ELISA Kit (SMTA00B, R&D Systems, Minneapolis, MN, USA) and Mouse IL-1 beta/IL-1F2 Quantikine ELISA Kit (SMLB00C, R&D Systems) according to the manufacturer’s protocols. Immuno uorescence staining The procedure for immuno uorescence staining was performed as previously described 41. Cells were incubated with anti-ASC rabbit monoclonal antibody (D2W8U; Cell Signaling Tech, MA, USA, #67824S; 1:100 dilution) diluted in blocking solution. After incubation with uorescent dye-conjugated secondary antibodies diluted in blocking solution, nuclei were stained with DAPI (D1306, Invitrogen). Images were obtained using Zeiss LSM 700 confocal laser scanning microscope. Reverse transcription quantitative PCR Total RNA was extracted using the RNeasy Mini Kit (Qiagen, Hilden, NRW, Germany, #74106) and reversetranscribed using the iScript cDNA synthesis kit (Bio-Rad, #1708891). Target genes were ampli ed using iTaq Universal SYBR Green Supermix (Bio-Rad, #172–5121) with speci c primers. Primer sequences were as follows: mouse TNF-α; 5′- CCT CTT CTC ATT CCT GCT TGT TGG-3′ (forward), 5′- GGT GGT TTG TGA GTG TGA GGG-3′ (reverse), mouse IL-1β; 5′- ATC CCA AGC AAT ACC CAA AGA AGA A-3′ (forward), 5′- GTG AAG TCA ATT ATG TCC TGA CCA C-3′ (reverse), mouse GAPDH; 5′- AGA AGG TGG TGA AGC AGG CAT C-3′ (forward), 5′- CGA AGG TGG AAG AGT GGG AGT TG-3′ (reverse). Relative mRNA levels were calculated according to the 2− ΔΔCT method. All ΔCτ values were normalized to glyceraldehyde-3-phosphate dehydrogenase. Degradation kinetics of aged WT-synuclein or V40Gsynuclein in microglia Primary mouse microglia cells were treated with either 200 nM of aged WT-synuclein or V40G-synuclein for 30 min at 37°C. After washing with ice-cold PBS, cells were incubated with fresh growth media at 37°C, and harvested at the indicated times. Cell-to-cell propagation assay For co-culture with microglia, V1S and SV2 stable cells12 were plated on a poly-D-lysine-coated coverslip. Next day, microglia cells were added to V1S and SV2 coculture. Cells were cultured for 2 additional days. For treatment of recombinant in ammatory cytokines, mixture of V1S and SV2 cells was cultured for 3 days. Recombinant human TNF-α (Prospec, Ness-Ziona, Israel, #CYT223; 50 ng/ml) or IL-1β (Prospec, #CYT208, 50 ng/ml) was treated to V1S and SV2 co-cultured cells at the nal concentration of 50 ng/ml for the last 24 h. Cells were xed in 4% paraformaldehyde in PBS prior to nuclear staining with TOPRO-3 iodide (Invitrogen). Images were acquired by confocal microscopy (Zeiss LSM700, 63X). Page 15/28 Statistical analysis All experiments were performed blind-coded and at least in duplicate. Differences were considered signi cant at p < 0.05 and were calculated using paired, two-tailed Student’s t-tests, one-way ANOVA with Tukey’s post-hoc test, and two-way ANOVA with Bonferroni’s post hoc test using GraphPad Prism 7.04 and 9.0.2 (GraphPad Software Inc., La Jolla, CA, USA). The values in the gures are expressed as the mean ± standard error of the mean (s.e.m.). Data availability Raw sequencing reads for rhinal cortex have been deposited at the National Center for Biotechnology Information under BioProjects PRJNA605306, respectively. The sequences of the mice injected with PBS are deposited in GenBank under accession SRR11060104, SRR11060103 and SRR11060102. The sequences of the mice injected with WT-syn are deposited in GenBank under accession SRR11060101, SRR11060100 and SRR11060099. The sequences of the mice injected with V40G are deposited in GenBank under accession SRR11060098, SRR11060097 and SRR11060096. The Source Data are provided with this paper. Other data are available from the corresponding author upon reasonable request. Declarations Acknowledgements This work was supported by the National Research Foundation (NRF) grant funded by the Korean Government (MEST) (NRF-2018R1A5A2025964, NRF-2021R1A2C3012681 to S.-J.L. and NRF2019R1I1A1A01063394 to T.-K.K.), the Korea Healthcare Technology R&D Project, Ministry of Health & Welfare, Republic of Korea (HI19C0256 to S.-J.L.), KBRI basic research program through Korea Brain Research Institute funded by Ministry of Science and ICT(21-BR-01-11 to MKJ), Innovative Medicines Initiative 2 (IMI-2 821522; PD-MitoQUANT to DADM) and EU Joint Programme-Neurodegenerative Disease (JPND 01ED2005B to DADM). Author Contributions T-KK designed, performed, and analyzed all the animal experiments. E-JB analyzed the RNA-seq data and designed, performed, and analyzed the microglia experiments. BCJ designed, performed, and analyzed the protein samples. MC performed and analyzed the dual-cell BiFC experiment. SJS puri ed the proteins and performed the ultracentrifugation experiments. JTK performed the dye binding experiments. MKJ performed the electron microscopy. H-JL performed the ultracentrifugation experiments with SJS. AU and DADM performed and analyzed the stereology cell counting of dopaminergic neurons. M-YS performed and analyzed the initial mutagenesis screening of the hinge region. JSL provided protein samples for analysis at the initial stage of the study. S-JL conceived and led the study, designed and analyzed all the Page 16/28 data, wrote the initial draft of the manuscript. T-KK, BCJ, E-JB, MC, SJS, JTK, MGJ, AU, and DADM wrote methods of the manuscript. All the authors reviewed and commented on the manuscript. Competing interests S-JL is a co-founder and co-CEO of Neuramedy Co. Ltd. JSL is employed by Neuramedy Co. Ltd. References 1. Brundin P, Melki R, Kopito R. Prion-like transmission of protein aggregates in neurodegenerative diseases. 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Scale bars, 0.4 μm. d Thio T binding kinetics of WT-syn or V40G recombinant αsynuclein over 9 days. e Dye binding assays of aged WT-syn and V40G. f Size exclusion chromatography (top) and western blotting (bottom) of aged V40G. g Ultracentrifugation assay. Western blotting (top) and quanti cation (bottom). h Western blots of WT-syn and V40G without (left) or with PK digestion (right). Note that the size ranges of the left and right blots are different. i Western blotting of PMCA end-products without PK digestion. Aged WT-syn or V40G was used as seed for the PMCA reaction. j Western blotting of PMCA end-products with PK digestion. k Thio T binding kinetics of recombinant α-synuclein with aged WT-syn or aged V40G as seeds. In all panels, “fresh” indicates pure monomers, and “aged” indicates the protein samples incubated for 9 days at 37 °C with constant agitation. Signi cance was assessed by oneway ANOVA with Tukey’s post hoc comparison between groups (b) or by two-tailed unpaired Student’s ttest (e), *P<0.05, **P<0.01, ***P<0.0001. All data are presented as the mean ± SEM. Page 20/28 Figure 2 Spreading of synucleinopathy after intracerebral injections of the WT-syn bril and V40G multimer αsynuclein. a Representative images of mouse brain sections 10 weeks after injection of WT-syn brils and V40G multimers stained with phospho-α-synuclein (pS129). Scale bar, 50 μm. b Heat map of regions affected by α-synuclein pathology at 2, 4, and 10 weeks after seed injection (asterisks indicate the injection site; n = 6 per group). c, d Western blotting of brain tissue extracts obtained 10 weeks after Page 21/28 injection. The ratio of Tx-100 insoluble to soluble was quanti ed in d. Data are expressed as the mean ± SEM, one-way ANOVA with Tukey’s post hoc test, two-sided, *P<0.05. e Co-immuno uorescence images. Thio avin-positive pS129 aggregates are indicated with arrowheads. t=10 weeks. Scale bar, 100 μm. f Immunoelectron microscopy with a pS129 antibody. pS129-positive aggregates with globular and lamentous structures are indicated with arrowheads. t=10 weeks. Scale bar, 0.5 μm. Figure 3 Page 22/28 Differential gene expression related to in ammatory response by injection of α-synuclein. a Heat map representing expression levels (log2 read count number) of DEGs upregulated (fold change >1.5) or downregulated (fold change <0.5) after injection of WT-syn brils or V40G multimers vs. PBS (n = 3 per group). b Venn diagram of DEGs. c, d Simpli ed networks of signi cantly enriched GO terms. The network was made from DEGs in WT-Syn injected mice (c), and V40G injected mice (d). Each term is statistically signi cant (Benjamini-Hochberg correction <0.05). The nodes (colored circles) represent signi cantly enriched parent GO terms. The edges (lines between the nodes) show that there are overlapping genes within terms. The different sizes of the nodes represented the number of enriched genes. e The top 11 enriched GO terms for the 110 common DEGs in both WT-syn and V40G injected mice. f GO enrichment analysis of immune-related common DEGs in both WT-syn and V40G injected mice. g Heat map of the log2 fold changes of 40 common DEGs related to immune and in ammatory responses. Page 23/28 Figure 4 The in ammatory response strongly modulates α-synuclein spreading. a Heat map of regions affected by IL-1β pathology at 2, 4, and 10 weeks after seed injection (asterisks indicate the injection site) (n = 6 per group). b Co-immuno uorescence images of the striatum. IL-1β was produced in Iba-1-positive microglia, but not in neurons or astrocytes. t=4 weeks. Scale bar, 10 μm. c Representative images of regions in which α-synuclein propagation had been observed, showing reactive microglia; tissue stained with antiPage 24/28 Iba-1 antibody (microgliosis marker). Scale bar, 20 μm. d-g Optical density of areas covered by Iba-1 immunoreactivity. d Striatum (PBS: n=6, 6, 6 at 2, 4, and 10 weeks, respectively; WT-syn: n=4, 5, 7; V40G: n=6, 7, 7). e Motor cortex (PBS: n=5, 6, 6; WT-syn: n=4, 7, 7; V40G: n=6, 7, 7). f Rhinal cortex (PBS: n=5, 5, 6; WT-syn: n=4, 7, 7; V40G: n=6, 7, 7). g Amygdala (PBS: n=6, 6, 6; WT-syn: n=4, 7, 7; V40G: n=6, 7, 7). Data are expressed as the mean ± SEM, one-way ANOVA with Tukey’s post hoc test, two-sided, *P<0.05, **P<0.01, ***P<0.0001. Figure 5 Page 25/28 Microglial activation promotes α-synuclein propagation. a, b Induction of NLRP3 in microglia. NLRP3 intensity was normalized to the value of β-actin (n=3). c ASC speck formation in microglia. Arrowheads indicate ASC specks. Scale bar: 20 µm. d-f Relative expression of TNF-α (d), IL-1β (e), and IL-6 (f) in microglia. Quantitative PCR data were normalized to the average value of those in PBS treated group (n=3). g, h Secretion of in ammatory cytokines in microglia. The amounts of secreted TNF-α (g) and IL-1β (h) were quanti ed by ELISA. n=3 in g, n=6 in h. i, j Effects of microglia on cell-to-cell propagation of αsynuclein. i BiFC-positive cells are indicated with arrowheads. Scale bar: 20 µm. j Quanti cation of BiFCpositive cells (n=3, 200 cells per experiment). k, l Effects of TNF-α and IL-1β treatment on cell-to-cell propagation of α-synuclein. V1S and SV2 cells were cocultured and then treated with either TNF-α or IL-1β (50 ng/ml) for 24 h. k BiFC-positive cells are indicated with arrowheads. Scale bar, 20 µm. l Quanti cation of BiFC-positive cells (n=3, 300 cells per experiment). Statistical signi cance was determined by one-way ANOVA with Tukey’s post hoc comparison between groups (b, d-h) or by two-tailed unpaired Student’s ttest (j, l), *P<0.05, **P<0.01, ***P<0.0001. All data are presented as the mean ± SEM. Page 26/28 Figure 6 Suppression of synucleinopathy, dopaminergic terminal loss and motor functions by an antiin ammatory drug. a Representative images of mouse brain sections after injection of V40G multimers followed by 17 weeks of oral aspirin (ASP) administration and stained for Iba-1, TNF-α, pS129 and totalα-synuclein. Scale bar, 50 μm. b-e Optical density of several brain areas covered by Iba-1, TNF-α, pS129 and total-α-synuclein immunoreactivity (n=8–10). f Optical density of the ipsilateral striatal region Page 27/28 covered by TH immunoreactivity. (Noninjected (NI), n=10; PBS, n=8; V40G, n=10; V40G+ASP 2, n=9; V40G+ASP 40, n=9). g Stereological cell counts of TH-immunoreactive dopaminergic neurons in the ipsilateral substantia nigra pars compacta of mice (NI, n=5; PBS, n=5; V40G, n=6; V40G+ASP 2, n=5; V40G+ASP 40, n=6). h, i Open eld test: distance moved (cm) and time in the center (s) were measures of locomotion and anxiety, respectively (NI, n=10; PBS, n=8; V40G, n=9; V40G+ASP 2, n=10; V40G+ASP 40, n=10). j, k Rotarod and four-limb hanging tests: latency to fall (s) from each were measures of motor balance and strength, respectively (NI, n=10; PBS, n=8; V40G, n=10; V40G+ASP 2, n=10; V40G+ASP 40, n=10). l, m Y maze: spontaneous alternation (%) and continuous alternation (n) were indicators of sensory spatial memory (NI, n=10; PBS, n=8; V40G, n=10; V40G+ASP 2, n=10; V40G+ASP 40, n=10). Data are expressed as the mean ± SEM, one-way ANOVA with Tukey’s post hoc test, two-sided, *P<0.05, **P<0.01, ***P<0.0001. n Graphic model for the role of microglial in ammation in synucleinopathy propagation. See details in the Discussion. Supplementary Files This is a list of supplementary les associated with this preprint. Click to download. DescriptionofAdditionalSupplementaryFiles.pdf Supplementaryinformation.pdf Supplementarytable2.xlsx Supplementarytable3.xlsx nreditorialpolicychecklist2.pdf nrreportingsummary2.pdf Page 28/28