Intracerebroventricular injection of Syn61–84 induces Parkinsonian pathology in mice

Intracerebroventricular injection of Syn61–84 induces Parkinsonian pathology in mice | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Intracerebroventricular injection of Syn61–84 induces Parkinsonian pathology in mice Rina Nakamura, Akira Matsuda, Motomi Konishi, Motoaki Saito, Toshifumi Akizawa This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-9354345/v1 This work is licensed under a CC BY 4.0 License Status: Under Revision Version 1 posted 11 You are reading this latest preprint version Abstract Aggregation of α-synuclein is a hallmark of Parkinson's disease (PD). The NAC domain plays an important role in fibrillogenesis. In this study, we focused on the NAC domain and synthesized peptides of various lengths to identify the nucleus of aggregation and evaluate the effect on motor function when administered intraventricularly to mice. First, aggregation was evaluated using a thioflavin T fluorescence assay, and Syn61–84 was identified as the core sequence responsible for aggregation. Next, we evaluated whether Syn61–84 induces Parkinson's disease-like pathology by administering it intraventricularly to mice. Behavioral analysis using the rotarod test revealed progressive motor impairment from day 7, with significant impairment by day 29. Immunohistochemical analysis showed increased activation of microglial cells (Iba1) and a significant decrease in dopaminergic neurons (TH) in the substantia nigra. These findings suggest that Syn61–84 causes PD-like pathology and motor dysfunction and may provide a model for studying α-synuclein aggregation and neurodegeneration and for screening α-synuclein-targeted therapeutics. α-synuclein Parkinson's disease Syn61–84 NAC domain Neurodegeneration Figures Figure 1 Figure 2 Figure 3 Introduction Parkinson's disease (PD) is a progressive neurodegenerative disease characterized by the loss of dopamine neurons in the substantia nigra and accumulation of α-synuclein (α-Syn) aggregates in the form of Lewy bodies [ 1 – 7 ]. To elucidate the pathology of PD and develop treatments, various animal models have been developed, such as neurotoxin-induced models [ 8 ] and transgenic mice overexpressing human α-Syn [ 9 ]. These models have several problems. Neurotoxic models cannot induce the deposition of α-Syn, the protein that causes PD, in the brain; therefore, these models cannot be used for research targeting α-Syn. In transgenic models, even if α-Syn deposition is observed, obvious motor dysfunction is not observed and it takes a longer duration for the symptoms to appear. In Alzheimer’s disease, a commonly used mouse model involves intracerebroventricular injection of Aβ25–35, a peptide identified as the core aggregation sequence of Aβ42, for drug screening and mechanistic studies [ 10 – 12 ]. Based on this precedent, we hypothesized that identifying the nucleus of aggregation through α-Syn targeting could become a simple screening method for developing PD drugs. In this study, we focused on the NAC domain, and synthesized peptides of various lengths to identify the nucleus of aggregation and evaluate the effect on motor function when intraventricularly administered to mice. Aggregation was evaluated using a thioflavin T fluorescence assay and Syn61–84 was identified as the core sequence responsible for aggregation. Next, we evaluated whether intraventricularly administered Syn61–84 induces PD-like pathology in mice. Materials and methods Peptide preparation Five fragment peptides derived from the NAC domain of α-synuclein were synthesized: Syn61–69, Syn61–84, Syn70–95, Syn70–84, and Syn85–95. The peptides were prepared as previously described [ 10 ]. They were synthesized using an automated peptide synthesizer (Model 433A, Applied Biosystems, Foster City, CA, USA, 0.1 mmol scale with preloaded resin) and purified using reverse-phase high-performance liquid chromatography. Each purified peptide was characterized using MALDI-TOF/TOF5800 (ABI SCIEX, Toronto, Canada). Peptide synthesis and aggregation assay Peptides were dissolved in PBS at 100 µM and incubated with Thioflavin-T (ThT) at 37°C for 24 h. The ThT signal was monitored by measuring fluorescence emission at 480 nm for 10 s when excited at 444 nm using a Cytation 5 (BioTek, Winooski, VT, USA). Concentration-dependent aggregation was evaluated at 100, 200, and 300 µM. Animals All procedures conformed with the U.K. Animals for Scientific Procedures and Directive 2010/63/EU of the European Parliament and the National Institutes of Health guide for the care and use of laboratory animals and ARRIVE guidelines 2.0 and were approved by the committee for the Care and Use of Laboratory Animals at Kochi University (permission number: R-00040). Fifteen male ICR mice (4 weeks old; Japan SLC, Shizuoka, Japan) were housed per cage and maintained at a controlled temperature (23 ± 1℃) and humidity (55 ± 2%), with a constant day-night rhythm (14/10-h light/dark cycle; lights on at 05:00) with free access to water and food. The experiment was conducted with 18 mice. Intracerebroventricular injection ICR mice (7 weeks old) were anesthetized with 1–3% isoflurane in a 75:25 mixture of nitrous oxide and oxygen. Mice were administered a stereotaxic injection of saline or 9 nmol Syn61–84 in the right lateral ventricle using a 10-µL Hamilton syringe [ 13 ]. Motor function assessment Mice underwent Rota-Rod (Muromachi Kikai Co. Ltd., Tokyo, Japan) training for two consecutive days prior to injection. On day 1, training was performed at a speed of 5 rpm. On day 2, mice were trained at 10 rpm, followed by a 1-h rest period, and then trained again at 20 rpm. To minimize environmental stress and allow acclimatization, mice were transferred to the room containing the Rota-Rod apparatus 1 h prior to each test session. Motor performance was assessed three times per mouse on days 1, 4, 7, 11, 18, and 29 post-injection using the Rota-Rod apparatus at a constant speed of 20 rpm. Immunohistochemistry On day 29 post-injection, mice were deeply anesthetized with isoflurane and subjected to transcardially perfused, and brains were fixed in 4% paraformaldehyde. Coronal sections of the midbrain were subjected to immunofluorescence staining. After blocking with PBS buffer, 0.1% Tween 20, and 3% bovine serum albumin (BSA) for 1 h at 25°C, sections were incubated overnight at 4°C with a rabbit anti-Iba1 antibody (Abcam, Cambridge, UK; 1:500 dilution in PBS buffer, 0.1% Tween 20, and 3% BSA) or anti-TH (Sigma–Aldrich, St. Louis, MO, USA; 1:1500 dilution in PBS buffer, 0.1% Tween 20, and 3% BSA). Following three washes with PBS-T (0.1% Tween-20), sections were incubated for 1 h at room temperature in the dark with a donkey anti-rabbit IgG Alexa Fluor 488 (Abcam; 1:500 dilution in PBS buffer, 0.1% Tween 20, and 3% BSA) or goat anti-mouse IgG Alexa Fluor secondary antibody 488 (Abcam; 1:500 dilution in PBS buffer, 0.1% Tween 20 and 3% BSA). After additional washes with PBS-T and PBS containing 0.02% sodium azide, sections were mounted with Hard Set Mounting Medium with DAPI (Vector Laboratories Inc., Newark, CA, USA). Fluorescence images were captured using an all-in-one fluorescence microscope BZ-9000 (Keyence, Osaka, Japan) with a 20× objective lens. Quantification of TH- and Iba1-positive cells was performed using the BZ- II Analyzer Ver. 1.42. Co-localization of TH with DAPI and Iba1 with DAPI was assessed by overlaying fluorescence images and identifying regions. Automated cell counting was conducted under consistent imaging conditions across multiple fields of view. Statistical analysis All data are expressed as mean ± standard error of the mean (SEM). Statistical analyses were performed using GraphPad Prism (version 10.5.0; GraphPad Software, LLC, Boston, MA, USA). For motor performance data obtained from the Rota-Rod test, unpaired two-tailed t-tests were used to assess differences between saline and Syn61–84-treated groups. For immunohistochemical quantification (Iba1 and TH staining), the Mann–Whitney U test was used to compare non-parametric data between groups. The selection of statistical tests was based on the distribution characteristics of the data and the experimental design. Statistical significance was set at p < 0.05. Results α-Syn consists of 140 amino acid residues and the NAC region is considered important for aggregation [ 13 , 14 ]. Therefore, we attempted to identify the main aggregation mechanism. Five peptides of various lengths and regions (Syn-FPs) were synthesized: Syn61–69, Syn61–84, Syn70–84, Syn70–95, and Syn85–95 (Fig. 1 A and B). To evaluate the aggregation of Syn-FPs, they were incubated with Thioflavin-T and the fluorescence intensity was measured. The strongest fluorescence intensity was observed in the Syn61–84 reaction solution (Fig. 1 C), which slightly increased by approximately 30% of that of Syn70–95. In contrast, no increase in fluorescence intensity was observed for Syn61–69, Syn70–84, and Syn85–95 (Fig. 1 C; Supplementary Table 1A). Furthermore, concentration-dependent aggregation was assessed for Syn61–84 and Syn70–95, revealing that both peptides exhibited a marked increase in fluorescence intensity at 300 µM, with Syn61–84 exhibiting a three-fold higher fluorescence intensity than that of Syn70–95 (Fig. 1 D; Supplementary Table 1B and C). These results indicate that Syn61–84 exhibits the highest aggregation propensity among the tested peptides, suggesting that this region plays a core role in α-Syn aggregation. Next, we examined whether Syn61–84, which was identified as the main component of aggregation, could be injected into the ventricles of the mouse brain to develop PD (Fig. 2 A). The time spent on the Rota-Rod decreased in the Syn61–84 intracerebroventricular group compared with that in the saline intracerebroventricular (control) group, from day 7. Thereafter, the time spent on Rota-Rod continued to decrease in the Syn61–84 intracerebroventricular group and significantly decreased on days 18 and 29 compared with that in the control group (Fig. 2 B; Supplementary Table 2A). Therefore, intracerebroventricular administration of Syn61–84 caused a decrease in locomotor function, similar to that observed in genetically engineered PD model mice. After evaluation of motor function, immunostaining was performed for Iba1, a marker of microglia, and TH, a marker of dopaminergic neurons, mainly in the midbrain substantia nigra. Iba1 was significantly increased in the Syn61–84 intracerebroventricular group compared with that in the control group (Fig. 3 A, C; Supplementary Table 2B). Immunostaining with anti-TH antibodies showed clear dopaminergic neuronal shedding in the Syn61–84 intracerebroventricular administration. Furthermore, quantification of the number of TH-positive cells showed a significant decrease in the Syn61–84 intracerebroventricular group compared with the control group (Fig. 3 B, D; Supplementary Table 2B). Therefore, intracerebroventricular administration of Syn61–84 induced dopaminergic neuronal loss, which is one of the typical pathologies of PD. Discussion In this study, we revealed that the NAC domain peptide of α-synuclein, Syn61–84, is a nucleating sequence for aggregation and induces PD-like pathology in mice. Furthermore, we demonstrated that Syn61–84 exhibits concentration-dependent toxicity in SH-SY5Y neuroblastoma cells (Supplementary Fig. 1). Compared with conventional PD models, these models do not require genetic manipulation, rapidly inducing pathology and directly targeting α-synuclein aggregation. 2,16 However, this study has several limitations. First, because pharmacological intervention was not examined, it remains unclear whether existing PD therapeutics can ameliorate the pathology induced by Syn61–84. Second, although motor dysfunction and neuroinflammation were observed, we have not directly confirmed the deposition of α-synuclein in the brain after Syn61–84 administration. Further studies are required to verify the presence of α-synuclein aggregates and evaluate the therapeutic responsiveness of this model [ 7 ]. Despite these limitations, the Syn61–84-induced model may serve as a valuable tool for studying α-synuclein aggregation and screening potential therapeutic agents targeting PD-related neurodegeneration. Declarations Competing interests The authors report no competing interests. Supplementary material Supplementary material is available at online. Funding This work was partially supported by the Japan Society for the Promotion of Science (JSPS) Grants-in-Aid for Scientific Research (KAKENHI) Program (grant numbers: 19K22499, 25K09919, and 25K18714) and AMED (grant number: JP22ym0126810j0001). The funding source had no role in study design, collection, analysis, and interpretation of data, writing of the report, or in the decision to submit the article for publication. Author Contribution Conceptualization, T.A.; methodology, T.A. and R.N.; validation, R.N.; formal analysis, R.N., M.K., and A.M.; investigation, R.N., and A.M.; data curation, R.N., T.A., and M.S.; writing—original draft preparation, T.A. and R.N; writing—review and editing, T.A. and R.N.; visu-alization, R.N.; supervision, T.A.; project administration, T.A. All authors have read and agreed to the published version of the manuscript. Acknowledgement We thank the Division of Biological Research, Science Research Center, Kochi University for the use of research instruments. References Hirsch EC, Hunot S, Faucheux B et al (1999) Dopaminergic neurons degenerate by apoptosis in Parkinson's disease. Mov Disord 14:383–385 Kalia LV, Lang AE (2015) Parkinson's disease. Lancet 386:896–912 Koss DJ, Erskine D, Porter A et al (2022) Nuclear alpha-synuclein is present in the human brain and is modified in dementia with Lewy bodies. Acta Neuropathol Commun 10:98 Lippa CF, Fujiwara H, Mann DM et al (1998) Lewy bodies contain altered alpha-synuclein in brains of many familial Alzheimer's disease patients with mutations in presenilin and amyloid precursor protein genes. Am J Pathol 153:1365–1370 Mezey E, Dehejia AM, Harta G et al (1998) Alpha synuclein is present in Lewy bodies in sporadic Parkinson's disease. Mol Psychiatry 3:493–499 Peña-Bautista C, Kumar R, Baquero M et al (2023) Misfolded alpha-synuclein detection by RT-QuIC in dementia with Lewy bodies: a systematic review and meta-analysis. Front Mol Biosci 10:1193458 Volpicelli-Daley LA, Luk KC, Patel TP et al (2011) Exogenous alpha-synuclein fibrils induce Lewy body pathology leading to synaptic dysfunction and neuron death. Neuron 72:57–71 Jackson-Lewis V, Przedborski S (2007) Protocol for the MPTP mouse model of Parkinson's disease. Nat Protoc 2:141–151 Zhu B, Park J-M, Coffey SR et al (2024) Single-cell transcriptomic and proteomic analysis of Parkinson's disease brains. Sci Transl Med 16:eabo1997 Nakamura R, Konishi M, Higashi Y et al (2023) Five-mer peptides prevent short-term spatial memory deficits in Aβ25-35-induced Alzheimer’s model mouse by suppressing Aβ25–35 aggregation and resolving its aggregate form. Alzheimers Res Ther 15:83–83 D'Agostino G, Russo R, Avagliano C et al (2012) Palmitoylethanolamide protects against the amyloid-Β25-35-induced learning and memory impairment in mice, an experimental model of Alzheimer disease. Neuropsychopharmacology 37:1784–1792 Dahlgren KN, Manelli AM, Stine WB Jr et al (2002) Oligomeric and fibrillar species of amyloid-β peptides differentially affect neuronal viability. J Biol Chem 277:32046–32053 Dou T, Zhou L, Kurouski D (2021) Unravelling the structural organization of individual alpha-synuclein oligomers grown in the presence of phospholipids. J Phys Chem Lett 12:4407–4414 Rhoades E, Ramlall TF, Webb WW et al (2006) Quantification of alpha-synuclein binding to lipid vesicles using fluorescence correlation spectroscopy. Biophys J 90:4692–4700 Izco M, Blesa J, Verona G et al (2021) Glial activation precedes alpha-synuclein pathology in a mouse model of Parkinson's disease. Neurosci Res 170:330–340 Spillantini MG, Schmidt ML, Lee VM et al (1997) Alpha-synuclein in Lewy bodies. Nature 388:839–840 Nakamura R, Konishi M, Taniguchi M et al (2019) The discovery of shorter synthetic proteolytic peptides derived from Tob1 protein. Peptides 116:71–77 Additional Declarations No competing interests reported. Supplementary Files 260408SUP.pptx Cite Share Download PDF Status: Under Revision Version 1 posted Editorial decision: Revision requested 13 May, 2026 Reviews received at journal 11 May, 2026 Reviews received at journal 05 May, 2026 Reviews received at journal 03 May, 2026 Reviewers agreed at journal 27 Apr, 2026 Reviewers agreed at journal 27 Apr, 2026 Reviewers agreed at journal 26 Apr, 2026 Reviewers invited by journal 26 Apr, 2026 Editor assigned by journal 13 Apr, 2026 Submission checks completed at journal 13 Apr, 2026 First submitted to journal 08 Apr, 2026 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-9354345","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":633720421,"identity":"d552caad-0745-49ec-9df8-fca877de291a","order_by":0,"name":"Rina Nakamura","email":"","orcid":"","institution":"Kōchi University","correspondingAuthor":false,"prefix":"","firstName":"Rina","middleName":"","lastName":"Nakamura","suffix":""},{"id":633720422,"identity":"57a66814-8cd9-4119-b20d-3c515f2db6f2","order_by":1,"name":"Akira Matsuda","email":"","orcid":"","institution":"Hiroshima International University","correspondingAuthor":false,"prefix":"","firstName":"Akira","middleName":"","lastName":"Matsuda","suffix":""},{"id":633720423,"identity":"69e61e83-8ba8-4143-9e84-443e57f7cc28","order_by":2,"name":"Motomi Konishi","email":"","orcid":"","institution":"Setsunan University","correspondingAuthor":false,"prefix":"","firstName":"Motomi","middleName":"","lastName":"Konishi","suffix":""},{"id":633720424,"identity":"5d3aec75-7cc1-4ec5-8e09-0854be526d0b","order_by":3,"name":"Motoaki Saito","email":"","orcid":"","institution":"Kōchi University","correspondingAuthor":false,"prefix":"","firstName":"Motoaki","middleName":"","lastName":"Saito","suffix":""},{"id":633720425,"identity":"aa962709-769b-4f36-ae9d-e79d0a53369f","order_by":4,"name":"Toshifumi Akizawa","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA60lEQVRIiWNgGAWjYBACxgYwJcHAxsB8GMg4wAOVYCNCCxtbMnFaEICNxxikhbBC5mkHWDd8zLGw55Pv+WzwoeaODINEAuOHHwx8eTgdNjuB7ebMbRLMbGy8mxNnHHvGA9TCLNnDwFaMW0v+t9u82yTYQFoO8zYc5rG/kcAgDXRnYgMeW0BaeNjYeB6DtYBs+U2MFqA1PMzJUC1sBG0B+cWAjS3N2HDGMaAWnodtlj0GuP1iCNRy4+O2Onv55sOPJT7UHLZnYE8+fONHxTGcIWaIxXpQ9BocS8ClRR6XRA1OLaNgFIyCUTDiAAAZCkq7Qq/ITgAAAABJRU5ErkJggg==","orcid":"","institution":"Kōchi University","correspondingAuthor":true,"prefix":"","firstName":"Toshifumi","middleName":"","lastName":"Akizawa","suffix":""}],"badges":[],"createdAt":"2026-04-08 09:22:38","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-9354345/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-9354345/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":108805084,"identity":"d6362d9b-d54e-4197-ae46-50ac8d542ab3","added_by":"auto","created_at":"2026-05-08 15:24:45","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":54206,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eIdentification of the nucleus of aggregation using fragment peptides derived from the NAC region domain structure of α-Syn. (a)\u003c/strong\u003e Domain structure of α-Syn. \u003cstrong\u003e(b)\u003c/strong\u003e Amino acid sequences of α-Syn NAC domain and five types of α-Syn fragment peptides. \u003cstrong\u003e(c)\u003c/strong\u003e Thioflavin T-fluorescence profile of α-Syn fragment peptides. \u003cstrong\u003e(d)\u003c/strong\u003e Concentration-dependent aggregation of Syn61–84 and Syn70–95 by ThT. Data are represented as mean + SEM, n =3.\u003c/p\u003e","description":"","filename":"floatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-9354345/v1/b9808204c7d67e3217c388ba.png"},{"id":108804906,"identity":"e3d1319b-8c09-4e61-a985-9de0083b3c88","added_by":"auto","created_at":"2026-05-08 15:24:12","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":18138,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eRotarod test after i.c.v. injection. (a)\u003c/strong\u003e Experimental scheme. \u003cstrong\u003e(b)\u003c/strong\u003eRotarod test after i.c.v. injection of Saline or Syn61–84. Data are represented as mean ± SEM, *p\u0026lt;0.05, ****p \u0026lt; 0.0001 vs. the corresponding Saline group; unpaired two-tailed t-tests, n =9. Day 1, p=0.876; Day 4, p=0.891; Day 7, p=0.037; Day 11, p=0.133; Day 18, p\u0026lt;0.0001; Day 29, p\u0026lt;0.0001.\u003c/p\u003e","description":"","filename":"floatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-9354345/v1/40f68d24980c60f911e82fcd.png"},{"id":108528581,"identity":"8191c62d-1e9c-4e9c-aa9b-40741272ec39","added_by":"auto","created_at":"2026-05-05 15:36:54","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":770517,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eNeuroinflammatory and neurodegenerative changes in Syn61–84 i.c.v. mice.\u003c/strong\u003e Representative microphotographs of the analyzed groups with Iba1 microglia (green) and DAPI+ (blue) staining \u003cstrong\u003e(a)\u003c/strong\u003e. Representative microphotographs of the analyzed groups with TH+ (green) and DAPI+ (blue) staining \u003cstrong\u003e(b)\u003c/strong\u003e. Number of Iba-1 positive (Iba+) microglia cells and DAPI+ cells in the substantia nigra of Saline and Syn61–84 i.c.v. mice on day 29 \u003cstrong\u003e(c)\u003c/strong\u003e. Number of TH positive (TH+) microglia cells and DAPI+ cells in the substantia nigra of Saline and Syn61–84 i.c.v. mice on day 29 \u003cstrong\u003e(d)\u003c/strong\u003e. Data are represented as mean + SEM, **p\u0026lt;0.01, ****p \u0026lt; 0.0001 vs. corresponding Saline group; Mann–Whitney U test, n = 20. Iba1\u003csup\u003e+\u003c/sup\u003eDAPI\u003csup\u003e+\u003c/sup\u003e, p\u0026lt;0.0001; TH\u003csup\u003e+\u003c/sup\u003eDAPI\u003csup\u003e+\u003c/sup\u003e, p=0.001.\u003c/p\u003e","description":"","filename":"floatimage3.png","url":"https://assets-eu.researchsquare.com/files/rs-9354345/v1/ca181b5f6be5fd09aebe6ce4.png"},{"id":108809887,"identity":"bfd091e7-e8d6-4165-9759-53a3fb7f0a75","added_by":"auto","created_at":"2026-05-08 15:56:07","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":869333,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-9354345/v1/baa64908-d629-42f9-a635-77430536adaa.pdf"},{"id":108528579,"identity":"beee88c8-516b-4123-aec0-9143320c987c","added_by":"auto","created_at":"2026-05-05 15:36:54","extension":"pptx","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":156872,"visible":true,"origin":"","legend":"","description":"","filename":"260408SUP.pptx","url":"https://assets-eu.researchsquare.com/files/rs-9354345/v1/313ef6e62ff8c1d41a86b564.pptx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Intracerebroventricular injection of Syn61–84 induces Parkinsonian pathology in mice","fulltext":[{"header":"Introduction","content":"\u003cp\u003eParkinson's disease (PD) is a progressive neurodegenerative disease characterized by the loss of dopamine neurons in the substantia nigra and accumulation of α-synuclein (α-Syn) aggregates in the form of Lewy bodies [\u003cspan additionalcitationids=\"CR2 CR3 CR4 CR5 CR6\" citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. To elucidate the pathology of PD and develop treatments, various animal models have been developed, such as neurotoxin-induced models [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e] and transgenic mice overexpressing human α-Syn [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. These models have several problems. Neurotoxic models cannot induce the deposition of α-Syn, the protein that causes PD, in the brain; therefore, these models cannot be used for research targeting α-Syn. In transgenic models, even if α-Syn deposition is observed, obvious motor dysfunction is not observed and it takes a longer duration for the symptoms to appear. In Alzheimer\u0026rsquo;s disease, a commonly used mouse model involves intracerebroventricular injection of Aβ25\u0026ndash;35, a peptide identified as the core aggregation sequence of Aβ42, for drug screening and mechanistic studies [\u003cspan additionalcitationids=\"CR11\" citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. Based on this precedent, we hypothesized that identifying the nucleus of aggregation through α-Syn targeting could become a simple screening method for developing PD drugs.\u003c/p\u003e \u003cp\u003eIn this study, we focused on the NAC domain, and synthesized peptides of various lengths to identify the nucleus of aggregation and evaluate the effect on motor function when intraventricularly administered to mice. Aggregation was evaluated using a thioflavin T fluorescence assay and Syn61\u0026ndash;84 was identified as the core sequence responsible for aggregation. Next, we evaluated whether intraventricularly administered Syn61\u0026ndash;84 induces PD-like pathology in mice.\u003c/p\u003e"},{"header":"Materials and methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003ePeptide preparation\u003c/h2\u003e \u003cp\u003eFive fragment peptides derived from the NAC domain of α-synuclein were synthesized: Syn61\u0026ndash;69, Syn61\u0026ndash;84, Syn70\u0026ndash;95, Syn70\u0026ndash;84, and Syn85\u0026ndash;95. The peptides were prepared as previously described [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. They were synthesized using an automated peptide synthesizer (Model 433A, Applied Biosystems, Foster City, CA, USA, 0.1 mmol scale with preloaded resin) and purified using reverse-phase high-performance liquid chromatography. Each purified peptide was characterized using MALDI-TOF/TOF5800 (ABI SCIEX, Toronto, Canada).\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003ePeptide synthesis and aggregation assay\u003c/h3\u003e\n\u003cp\u003ePeptides were dissolved in PBS at 100 \u0026micro;M and incubated with Thioflavin-T (ThT) at 37\u0026deg;C for 24 h. The ThT signal was monitored by measuring fluorescence emission at 480 nm for 10 s when excited at 444 nm using a Cytation 5 (BioTek, Winooski, VT, USA). Concentration-dependent aggregation was evaluated at 100, 200, and 300 \u0026micro;M.\u003c/p\u003e\n\u003ch3\u003eAnimals\u003c/h3\u003e\n\u003cp\u003eAll procedures conformed with the U.K. Animals for Scientific Procedures and Directive 2010/63/EU of the European Parliament and the National Institutes of Health guide for the care and use of laboratory animals and ARRIVE guidelines 2.0 and were approved by the committee for the Care and Use of Laboratory Animals at Kochi University (permission number: R-00040). Fifteen male ICR mice (4 weeks old; Japan SLC, Shizuoka, Japan) were housed per cage and maintained at a controlled temperature (23\u0026thinsp;\u0026plusmn;\u0026thinsp;1℃) and humidity (55\u0026thinsp;\u0026plusmn;\u0026thinsp;2%), with a constant day-night rhythm (14/10-h light/dark cycle; lights on at 05:00) with free access to water and food. The experiment was conducted with 18 mice.\u003c/p\u003e\n\u003ch3\u003eIntracerebroventricular injection\u003c/h3\u003e\n\u003cp\u003eICR mice (7 weeks old) were anesthetized with 1\u0026ndash;3% isoflurane in a 75:25 mixture of nitrous oxide and oxygen. Mice were administered a stereotaxic injection of saline or 9 nmol Syn61\u0026ndash;84 in the right lateral ventricle using a 10-\u0026micro;L Hamilton syringe [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e].\u003c/p\u003e\n\u003ch3\u003eMotor function assessment\u003c/h3\u003e\n\u003cp\u003eMice underwent Rota-Rod (Muromachi Kikai Co. Ltd., Tokyo, Japan) training for two consecutive days prior to injection. On day 1, training was performed at a speed of 5 rpm. On day 2, mice were trained at 10 rpm, followed by a 1-h rest period, and then trained again at 20 rpm. To minimize environmental stress and allow acclimatization, mice were transferred to the room containing the Rota-Rod apparatus 1 h prior to each test session. Motor performance was assessed three times per mouse on days 1, 4, 7, 11, 18, and 29 post-injection using the Rota-Rod apparatus at a constant speed of 20 rpm.\u003c/p\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eImmunohistochemistry\u003c/h2\u003e \u003cp\u003eOn day 29 post-injection, mice were deeply anesthetized with isoflurane and subjected to transcardially perfused, and brains were fixed in 4% paraformaldehyde. Coronal sections of the midbrain were subjected to immunofluorescence staining. After blocking with PBS buffer, 0.1% Tween 20, and 3% bovine serum albumin (BSA) for 1 h at 25\u0026deg;C, sections were incubated overnight at 4\u0026deg;C with a rabbit anti-Iba1 antibody (Abcam, Cambridge, UK; 1:500 dilution in PBS buffer, 0.1% Tween 20, and 3% BSA) or anti-TH (Sigma\u0026ndash;Aldrich, St. Louis, MO, USA; 1:1500 dilution in PBS buffer, 0.1% Tween 20, and 3% BSA). Following three washes with PBS-T (0.1% Tween-20), sections were incubated for 1 h at room temperature in the dark with a donkey anti-rabbit IgG Alexa Fluor 488 (Abcam; 1:500 dilution in PBS buffer, 0.1% Tween 20, and 3% BSA) or goat anti-mouse IgG Alexa Fluor secondary antibody 488 (Abcam; 1:500 dilution in PBS buffer, 0.1% Tween 20 and 3% BSA). After additional washes with PBS-T and PBS containing 0.02% sodium azide, sections were mounted with Hard Set Mounting Medium with DAPI (Vector Laboratories Inc., Newark, CA, USA). Fluorescence images were captured using an all-in-one fluorescence microscope BZ-9000 (Keyence, Osaka, Japan) with a 20\u0026times; objective lens. Quantification of TH- and Iba1-positive cells was performed using the BZ- II Analyzer Ver. 1.42. Co-localization of TH with DAPI and Iba1 with DAPI was assessed by overlaying fluorescence images and identifying regions. Automated cell counting was conducted under consistent imaging conditions across multiple fields of view.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003eStatistical analysis\u003c/h2\u003e \u003cp\u003eAll data are expressed as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard error of the mean (SEM). Statistical analyses were performed using GraphPad Prism (version 10.5.0; GraphPad Software, LLC, Boston, MA, USA). For motor performance data obtained from the Rota-Rod test, unpaired two-tailed t-tests were used to assess differences between saline and Syn61\u0026ndash;84-treated groups. For immunohistochemical quantification (Iba1 and TH staining), the Mann\u0026ndash;Whitney U test was used to compare non-parametric data between groups. The selection of statistical tests was based on the distribution characteristics of the data and the experimental design. Statistical significance was set at p\u0026thinsp;\u0026lt;\u0026thinsp;0.05.\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cp\u003eα-Syn consists of 140 amino acid residues and the NAC region is considered important for aggregation [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e, \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. Therefore, we attempted to identify the main aggregation mechanism. Five peptides of various lengths and regions (Syn-FPs) were synthesized: Syn61\u0026ndash;69, Syn61\u0026ndash;84, Syn70\u0026ndash;84, Syn70\u0026ndash;95, and Syn85\u0026ndash;95 (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA and B). To evaluate the aggregation of Syn-FPs, they were incubated with Thioflavin-T and the fluorescence intensity was measured. The strongest fluorescence intensity was observed in the Syn61\u0026ndash;84 reaction solution (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC), which slightly increased by approximately 30% of that of Syn70\u0026ndash;95. In contrast, no increase in fluorescence intensity was observed for Syn61\u0026ndash;69, Syn70\u0026ndash;84, and Syn85\u0026ndash;95 (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC; Supplementary Table\u0026nbsp;1A). Furthermore, concentration-dependent aggregation was assessed for Syn61\u0026ndash;84 and Syn70\u0026ndash;95, revealing that both peptides exhibited a marked increase in fluorescence intensity at 300 \u0026micro;M, with Syn61\u0026ndash;84 exhibiting a three-fold higher fluorescence intensity than that of Syn70\u0026ndash;95 (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eD; Supplementary Table\u0026nbsp;1B and C). These results indicate that Syn61\u0026ndash;84 exhibits the highest aggregation propensity among the tested peptides, suggesting that this region plays a core role in α-Syn aggregation.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eNext, we examined whether Syn61\u0026ndash;84, which was identified as the main component of aggregation, could be injected into the ventricles of the mouse brain to develop PD (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA). The time spent on the Rota-Rod decreased in the Syn61\u0026ndash;84 intracerebroventricular group compared with that in the saline intracerebroventricular (control) group, from day 7. Thereafter, the time spent on Rota-Rod continued to decrease in the Syn61\u0026ndash;84 intracerebroventricular group and significantly decreased on days 18 and 29 compared with that in the control group (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB; Supplementary Table\u0026nbsp;2A). Therefore, intracerebroventricular administration of Syn61\u0026ndash;84 caused a decrease in locomotor function, similar to that observed in genetically engineered PD model mice.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eAfter evaluation of motor function, immunostaining was performed for Iba1, a marker of microglia, and TH, a marker of dopaminergic neurons, mainly in the midbrain substantia nigra. Iba1 was significantly increased in the Syn61\u0026ndash;84 intracerebroventricular group compared with that in the control group (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA, C; Supplementary Table\u0026nbsp;2B). Immunostaining with anti-TH antibodies showed clear dopaminergic neuronal shedding in the Syn61\u0026ndash;84 intracerebroventricular administration. Furthermore, quantification of the number of TH-positive cells showed a significant decrease in the Syn61\u0026ndash;84 intracerebroventricular group compared with the control group (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB, D; Supplementary Table\u0026nbsp;2B). Therefore, intracerebroventricular administration of Syn61\u0026ndash;84 induced dopaminergic neuronal loss, which is one of the typical pathologies of PD.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eIn this study, we revealed that the NAC domain peptide of α-synuclein, Syn61\u0026ndash;84, is a nucleating sequence for aggregation and induces PD-like pathology in mice. Furthermore, we demonstrated that Syn61\u0026ndash;84 exhibits concentration-dependent toxicity in SH-SY5Y neuroblastoma cells (Supplementary Fig.\u0026nbsp;1). Compared with conventional PD models, these models do not require genetic manipulation, rapidly inducing pathology and directly targeting α-synuclein aggregation.\u003csup\u003e2,16\u003c/sup\u003e\u003c/p\u003e \u003cp\u003eHowever, this study has several limitations. First, because pharmacological intervention was not examined, it remains unclear whether existing PD therapeutics can ameliorate the pathology induced by Syn61\u0026ndash;84. Second, although motor dysfunction and neuroinflammation were observed, we have not directly confirmed the deposition of α-synuclein in the brain after Syn61\u0026ndash;84 administration. Further studies are required to verify the presence of α-synuclein aggregates and evaluate the therapeutic responsiveness of this model [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eDespite these limitations, the Syn61\u0026ndash;84-induced model may serve as a valuable tool for studying α-synuclein aggregation and screening potential therapeutic agents targeting PD-related neurodegeneration.\u003c/p\u003e"},{"header":"Declarations","content":"\u003ch2\u003eCompeting interests\u003c/h2\u003e\n\u003cp\u003eThe authors report no competing interests.\u003c/p\u003e\n\u003ch2\u003eSupplementary material\u003c/h2\u003e\n\u003cp\u003eSupplementary material is available at online.\u003c/p\u003e\n\u003ch2\u003eFunding\u003c/h2\u003e\n\u003cp\u003eThis work was partially supported by the Japan Society for the Promotion of Science (JSPS) Grants-in-Aid for Scientific Research (KAKENHI) Program (grant numbers: 19K22499, 25K09919, and 25K18714) and AMED (grant number: JP22ym0126810j0001). The funding source had no role in study design, collection, analysis, and interpretation of data, writing of the report, or in the decision to submit the article for publication.\u003c/p\u003e\n\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\n\u003cp\u003eConceptualization, T.A.; methodology, T.A. and R.N.; validation, R.N.; formal analysis, R.N., M.K., and A.M.; investigation, R.N., and A.M.; data curation, R.N., T.A., and M.S.; writing\u0026mdash;original draft preparation, T.A. and R.N; writing\u0026mdash;review and editing, T.A. and R.N.; visu-alization, R.N.; supervision, T.A.; project administration, T.A. All authors have read and agreed to the published version of the manuscript.\u003c/p\u003e\n\u003ch2\u003eAcknowledgement\u003c/h2\u003e\n\u003cp\u003eWe thank the Division of Biological Research, Science Research Center, Kochi University for the use of research instruments.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eHirsch EC, Hunot S, Faucheux B et al (1999) Dopaminergic neurons degenerate by apoptosis in Parkinson's disease. Mov Disord 14:383\u0026ndash;385\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKalia LV, Lang AE (2015) Parkinson's disease. Lancet 386:896\u0026ndash;912\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKoss DJ, Erskine D, Porter A et al (2022) Nuclear alpha-synuclein is present in the human brain and is modified in dementia with Lewy bodies. Acta Neuropathol Commun 10:98\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLippa CF, Fujiwara H, Mann DM et al (1998) Lewy bodies contain altered alpha-synuclein in brains of many familial Alzheimer's disease patients with mutations in presenilin and amyloid precursor protein genes. Am J Pathol 153:1365\u0026ndash;1370\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMezey E, Dehejia AM, Harta G et al (1998) Alpha synuclein is present in Lewy bodies in sporadic Parkinson's disease. Mol Psychiatry 3:493\u0026ndash;499\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ePe\u0026ntilde;a-Bautista C, Kumar R, Baquero M et al (2023) Misfolded alpha-synuclein detection by RT-QuIC in dementia with Lewy bodies: a systematic review and meta-analysis. Front Mol Biosci 10:1193458\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eVolpicelli-Daley LA, Luk KC, Patel TP et al (2011) Exogenous alpha-synuclein fibrils induce Lewy body pathology leading to synaptic dysfunction and neuron death. Neuron 72:57\u0026ndash;71\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eJackson-Lewis V, Przedborski S (2007) Protocol for the MPTP mouse model of Parkinson's disease. Nat Protoc 2:141\u0026ndash;151\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZhu B, Park J-M, Coffey SR et al (2024) Single-cell transcriptomic and proteomic analysis of Parkinson's disease brains. Sci Transl Med 16:eabo1997\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eNakamura R, Konishi M, Higashi Y et al (2023) Five-mer peptides prevent short-term spatial memory deficits in Aβ25-35-induced Alzheimer\u0026rsquo;s model mouse by suppressing Aβ25\u0026ndash;35 aggregation and resolving its aggregate form. Alzheimers Res Ther 15:83\u0026ndash;83\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eD'Agostino G, Russo R, Avagliano C et al (2012) Palmitoylethanolamide protects against the amyloid-Β25-35-induced learning and memory impairment in mice, an experimental model of Alzheimer disease. Neuropsychopharmacology 37:1784\u0026ndash;1792\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDahlgren KN, Manelli AM, Stine WB Jr et al (2002) Oligomeric and fibrillar species of amyloid-β peptides differentially affect neuronal viability. J Biol Chem 277:32046\u0026ndash;32053\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDou T, Zhou L, Kurouski D (2021) Unravelling the structural organization of individual alpha-synuclein oligomers grown in the presence of phospholipids. J Phys Chem Lett 12:4407\u0026ndash;4414\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eRhoades E, Ramlall TF, Webb WW et al (2006) Quantification of alpha-synuclein binding to lipid vesicles using fluorescence correlation spectroscopy. Biophys J 90:4692\u0026ndash;4700\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eIzco M, Blesa J, Verona G et al (2021) Glial activation precedes alpha-synuclein pathology in a mouse model of Parkinson's disease. Neurosci Res 170:330\u0026ndash;340\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSpillantini MG, Schmidt ML, Lee VM et al (1997) Alpha-synuclein in Lewy bodies. Nature 388:839\u0026ndash;840\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eNakamura R, Konishi M, Taniguchi M et al (2019) The discovery of shorter synthetic proteolytic peptides derived from Tob1 protein. Peptides 116:71\u0026ndash;77\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"molecular-neurobiology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"moln","sideBox":"Learn more about [Molecular Neurobiology](https://www.springer.com/journal/12035)","snPcode":"12035","submissionUrl":"https://submission.nature.com/new-submission/12035/3","title":"Molecular Neurobiology","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"α-synuclein, Parkinson's disease, Syn61–84, NAC domain, Neurodegeneration","lastPublishedDoi":"10.21203/rs.3.rs-9354345/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-9354345/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eAggregation of α-synuclein is a hallmark of Parkinson's disease (PD). The NAC domain plays an important role in fibrillogenesis. In this study, we focused on the NAC domain and synthesized peptides of various lengths to identify the nucleus of aggregation and evaluate the effect on motor function when administered intraventricularly to mice. First, aggregation was evaluated using a thioflavin T fluorescence assay, and Syn61\u0026ndash;84 was identified as the core sequence responsible for aggregation. Next, we evaluated whether Syn61\u0026ndash;84 induces Parkinson's disease-like pathology by administering it intraventricularly to mice. Behavioral analysis using the rotarod test revealed progressive motor impairment from day 7, with significant impairment by day 29. Immunohistochemical analysis showed increased activation of microglial cells (Iba1) and a significant decrease in dopaminergic neurons (TH) in the substantia nigra. These findings suggest that Syn61\u0026ndash;84 causes PD-like pathology and motor dysfunction and may provide a model for studying α-synuclein aggregation and neurodegeneration and for screening α-synuclein-targeted therapeutics.\u003c/p\u003e","manuscriptTitle":"Intracerebroventricular injection of Syn61–84 induces Parkinsonian pathology in mice","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-05-05 15:36:49","doi":"10.21203/rs.3.rs-9354345/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2026-05-13T05:54:13+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-05-11T08:04:10+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-05-06T00:40:47+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-05-03T20:42:05+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"312851023119712148569717110928124736339","date":"2026-04-27T23:14:24+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"126187006739558940435160724050087421160","date":"2026-04-27T05:17:53+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"36743120747739703785897130412037791645","date":"2026-04-27T03:18:17+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2026-04-27T03:03:21+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2026-04-14T03:58:41+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2026-04-14T03:57:47+00:00","index":"","fulltext":""},{"type":"submitted","content":"Molecular Neurobiology","date":"2026-04-08T08:44:26+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"molecular-neurobiology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"moln","sideBox":"Learn more about [Molecular Neurobiology](https://www.springer.com/journal/12035)","snPcode":"12035","submissionUrl":"https://submission.nature.com/new-submission/12035/3","title":"Molecular Neurobiology","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"9491f693-cbf8-468d-b698-6328a84922ef","owner":[],"postedDate":"May 5th, 2026","published":true,"recentEditorialEvents":[{"type":"decision","content":"Revision requested","date":"2026-05-13T05:54:13+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-05-11T08:04:10+00:00","index":46,"fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-05-06T00:40:47+00:00","index":45,"fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-05-03T20:42:05+00:00","index":43,"fulltext":""}],"rejectedJournal":[],"revision":"","amendment":"","status":"in-revision","subjectAreas":[],"tags":[],"updatedAt":"2026-05-13T06:10:48+00:00","versionOfRecord":[],"versionCreatedAt":"2026-05-05 15:36:49","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-9354345","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-9354345","identity":"rs-9354345","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}