Higher molecular weight alpha-synuclein oligomers are increased in brain cytosol from dementia with Lewy bodies

Higher molecular weight alpha-synuclein oligomers are increased in brain cytosol from dementia with Lewy bodies | 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 Short Report Higher molecular weight alpha-synuclein oligomers are increased in brain cytosol from dementia with Lewy bodies Emil Gregersen, Mia R. Antorini, Lasse Reimer, Ludovica Zaccagnini, and 3 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7957852/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 28 Feb, 2026 Read the published version in npj Parkinson's Disease → Version 1 posted 11 You are reading this latest preprint version Abstract The size-distribution of α-synuclein oligomers in brain cytosol from dementia with Lewy bodies (DLB) and control cases was investigated by combining size-exclusion chromatography and an aggregate specific MJFR14-6-4-2 ELISA. The oligomers in DLB peaked at 340 kDa and extended up to 2000 kDa. In controls, oligomers were smaller. Immunoblotting revealed the existence of pools of oligomers with different sensitivity to denaturation. Biological sciences/Biochemistry Health sciences/Neurology Biological sciences/Neuroscience Figures Figure 1 Figure 2 Figure 3 Main text Aggregation of the presynaptic protein α-synuclein (a-syn) is tightly associated with Parkinson’s disease (PD) and dementia with Lewy Bodies (DLB) based on genetic and biochemical evidence 1 – 3 . The aggregates are present as insoluble amyloid type fibrillar species hyperphosphorylated on Ser129, typically associated to Lewy body (LB)-type inclusions, and soluble oligomers 4 , 5 . High resolution cryo-EM tomography studies have revealed detailed structural insight into disease-associated fibrillar strains 6 . However, our insight into the nature of the oligomers in the brain is lacking in comparison, despite oligomers having been hypothesized to be pathogenic for decades 7 – 9 , and in vitro generated oligomers having been studied in detail 10 , 11 . Their existence and increase in extracts of human brains affected by synucleinopathies have been demonstrated by different biochemical assays 12 – 14 , and in cytosol from DLB brains by ELISA 15 . Recent immunochemical studies take advantage of a-syn proximity ligation assays (PLA) to detect a-syn oligomers. Two protocols have been developed, both using pairs of either the monoclonal pan-a-syn antibody Syn211 or the aggregate specific MJFR14-6-4-2 antibody 16 – 20 . Using the MJFR14-6-4-2 PLA, it was demonstrated that oligomers are abundant in tissue from PD brains where they develop prior to the formation of LBs but are absent or in very low abundance in non-synucleinopathy control brain tissue. Moreover, they exist in brains without LBs from patients with mutations in the LRRK2 gene 18 , 20 . Furthermore, the size distribution of total a-syn species in the cytosol of DLB and control brains has been analyzed by size-exclusion chromatography (SEC), followed by a-syn ELISA of the individual cytosolic fractions 21 . This demonstrated a symmetric peak with a maximum corresponding to a molecular size of 150 kDa and negligible levels larger than 300 kDa. In this study, we hypothesized that insight into the size of MJFR14-6-4-2 positive a-syn aggregates in DLB brain cytosol would be informative as to the size of the candidates responsible for the MJFR14-6-4-2 PLA signal in DLB brain tissue. Quantifying the samples isolated from DLB and control brains SEC and MJFR14-6-4-2 ELISA 22 demonstrated that both control and DLB brains contain significant amounts of low- and intermediate size a-syn species (150–400 kDa), which were shifted towards higher-molecular weight fractions in DLB patients. Larger oligomers in the range of 500–1800 kDa appeared selectively increased in DLB. This suggests large cytosolic oligomers are candidates for the structures generating the MJFR14-6-4-2 PLA positive signals in synucleinopathies, although other species present in other brain fractions, e.g. bound to vesicle or organelles, may also contribute. The fractions 1–14 from the SEC of the cytosol isolated from frontal cortex grey matter of neurologically healthy controls and DLB patients were analysed by ELISA for total a-syn and MJFR14-6-4-2-positive aggregates, and by a-syn immunoblotting. These fractions covered the molecular size range from > 2000 kDa to approx. 30 kDa. The 8 neurologically healthy controls and 8 DLB patients were chosen from a larger cohort previously analyzed for the presence of total a-syn 21 . The total a-syn ELISA demonstrated similar elution profiles for both control and DLB cytosol with the vast majority of a-syn eluting as a symmetric peak with a maximum in fraction 11 (Fig. 1 A). This corresponds to a molecular size of approx. 200 kDa, and with no substantial signal in the higher-molecular-weight fractions 9 and below. The scale of the total a-syn on the elution profiles is normalized to the highest measured fraction within each individual patient profile in that set of fractions, being fraction 11 for both control and DLB patients. This molecular size profile is in line with the previous measurement of the larger cohort wherefrom the 8 controls and 8 DLB patients were chosen 21 . Brain homogenates from dementia with Lewy body (DLB) patients or neurological healthy controls (CTRL) were separated by size-exclusion chromatography (SEC). The fractions obtained were analyzed by ELISA for the content of total a-syn (A), and aggregated a-syn (B) using the polyclonal ASY-1 and monoclonal a-syn aggregate-specific MJF14-6-4-2 rabbit IgG as primary antibodies, respectively. Each set of fractions from individual patients were diluted until the signal was in the linear part of the standard curve (usually 156 pg/mL − 20 ng/mL). To compare SEC profiles, fraction measurements was normalized to the highest measurement within that individual set of fractions. Control samples are depicted in blue and DLB in red. The molecular size markers are based on the calibration presented in Sanderson 2020 21 where the samples were initially isolated. High molecular weight fractions (HMW) 2–7, intermediate molecular weight fractions 8–10 (IMW) and low molecular weight fractions (LMW) 11–13) are marked. The mean value of the CTRL (blue) and DLB (red) are displayed with standard deviations, n = 8. Statistical significance was determined by multiple unpaired t-test with Welch’s correction (*p < 0.05, **p < 0.01, ****p < 0.0001). Compared to the total a-syn ELISA, the aggregate-specific MJF14-6-4-2 ELISA detected a broader peak, with oligomeric a-syn spanning from fractions 8 to 12 in control patients (Fig. 1 B). This lower-molecular-weight oligomer peak was shifted to higher-molecular weight sizes in the DLB cytosol with a maximum in fraction 9 (approx. 340 kDa) compared to fraction 10 (approx. 250 kDa) for the controls. In addition to the peak in fractions 8–12, a high-molecular weight (HMW) pool in fractions 2–7 could be found in the DLB samples as a prominent HMW “shoulder” on the peak. This contrasted with the controls, where this HMW “shoulder” was smaller and tapered off in fractions 5–7. Hence, there exists a quantitative difference between the HMW pool of a-syn oligomers in DLB and controls where it accounts for 25.67% ± 4.87 compared to 10.19% ± 4.82 of the total aggregated a-syn signal (calculated based on area under the curve). This may indicate an increased aggregation or reduced catabolism of oligomers in the DLB patient frontal cortical grey matter, resulting in the cytosolic accumulation of larger oligomers with a molecular size of around 500–1800 kDa. As an orthogonal analysis to the quantitative ELISA, we subjected the fractions from the SEC to reducing SDS-PAGE and immunoblotting using a polyclonal a-syn antibody ASY-1 23,24 . This analysis holds potential to reveal qualitative differences in the SDS-sensitivity of oligomer pools of DLB patients and controls. Figure 2 A + B demonstrate representative immunoblots of cytosol fractions from patients Control #2 and DLB #7. The fractions can be categorized into three groups: A LMW pool in fractions 11–13, an intermediate molecular weight (IMW) pool in fractions 8–10, and a HMW pool in fractions 2–7. The LMW pool is dominated by SDS-sensitive species dissociating into 17 kDa monomers upon denaturation in the loading buffer for the SDS-PAGE. The IMW pool is dominated by species dissociating into SDS-resistant dimers and trimers. The LMW and IMW fractions do not differ noticeably in their ASY-1 immunoreactive patterns between controls and DLB patients, although it is noteworthy that the IMW pool represents the fractions wherein the ELISA-positive oligomers accumulated in DLB patients compared to controls. The HMW pool is weakly positive on the immunoblots but evidently occupied by 17 kDa monomeric a-syn species, thus indicating higher sensitivity to denaturation by SDS compared to the IMW pool (Fig. 2 B). To better appreciate this pool, we analyzed longer exposures of the immunoblots (Suppl. Figure 1). This allowed the demonstration of a DLB-associated signature of increased HMW oligomers based on the following criterion: The 17 kDa signal in fraction 3 is higher or equal to the signal in fraction 5 and/or 6. None of the 7 controls (#2–8) fulfilled this criterion, whereas 6 of the 8 DLB patients were positive (DLB #2, 4–8). This corroborates the ELISA data that demonstrated increased levels of aggregates in the HMW fraction and suggests that these oligomers are SDS-sensitive in contrast to the LMW oligomers that generates trimers and dimers by SDS treatment. Brain homogenates from dementia with Lewy body (DLB) patients or healthy controls (CTRL) were separated by SEC. The collected fractions were resolved by denaturing SDS-PAGE gel followed by western blot analysis using the polyclonal antibody ASY-1. A protein ladder was used as molecular size marker. The blots represent CTRL (#2) and DLB (#7) and are representative for their groups (see suppl. Figure 1a-d). Please note that the α-syn bands in HMW fractions 3–5 in the DLB case is almost exclusively monomeric and not detectable in the CTRL case. By contrast, the α-syn bands in fractions 8–10 are dominated by dimeric and trimeric species. This demonstrates the HMW α-syn oligomer species in the DLB cases are sensitive to denaturation by SDS whereas the smaller oligomers in fractions 8–10 predominantly dissociate into trimers and dimers. A strength of our study is the combination of SEC and an a-syn aggregate specific ELISA on a subfraction of a well characterized cohort of control and DLB brains. Our data demonstrates that a-syn aggregates able to bind the aggregate-specific MJFR14-6-4-2 antibody exist in both control and DLB brain cytosol, but their average size increases in DLB. Meanwhile, the very large cytosolic oligomers 500–1800 kDa are exclusively present in DLB brains and are thus candidates for generating the PLA signal recently demonstrated to develop in brains affected by synucleinopathies (Fig. 3 ). We hypothesize that a pool of low-molecular weight (LMW) and intermediate-molecular weight (IMW) a-syn species are physiologically present in healthy brains (green box). These correspond to the oligomers detected in fractions 11–13 and 8–10, respectively, by ELISA. Meanwhile, the a-syn species found in DLB patients (red box) are shifted towards higher-molecular weight fractions in LMW and IMW fractions, and to high-molecular weight (HMW) species not found in control brains, corresponding to fractions 3–7 in our ELISA. According to our SDS-PAGE, the IMW pool is more SDS-stable with more SDS-resistant trimers and dimers compared to the LMW and HMW pools that denature into 17 kDa monomers. A limitation of the study is its exploratory nature by only studying a small cohort and only focusing on the cytosolic fraction. Our findings motivate further studies using similar approaches on all the subcellular fractions of brain tissue to bring more insight into the size distribution of oligomers. For example, a previous study demonstrated that aggregated a-syn species were associated to ER membranes in PD 25 . One goal would be to develop purification protocols that can allow high resolution cryo-EM tomography studies to solve the structure of these hitherto elusive pathogenic a-syn aggregates. Material and Methods Brain samples and Size Exclusion Chromatography Human brain tissue from post-mortem brains of 8 healthy controls and 8 patients with a neuropathological diagnosis of DLB was sampled from the cohort previously analyzed 21 . Human brain tissue was provided by Brigham and Women’s Hospital (Boston, MA, USA), Mayo Clinic (Jacksonville, FL, USA), Massachusetts General Hospital/Massachusetts Alzheimer’s Disease Research Center (Boston, MA, USA), Newcastle Brain Tissue Resource (Newcastle upon Tyne, UK) and Queen Square Brain Bank for Neurological Disorders (London, UK). Information about the brain samples used in this study is summarized in Table 1 . Consent was obtained from patients prior to death at each brain collection centre. All five brain banks approved of the proposal for the use of human tissue in this study, and the IRB and REC at the first and last authors’ institution deemed the planned use of this tissue to be appropriate and ethical (IRB 1999P001180/BWH, REC 18/LO/0721). Approximately 500 mg of frontal cortex tissue pieces was homogenized with 25 strokes using a manual Dounce homogenizer overhead stirrer (Wheaton, Millville, NJ, USA) in four volumes (weight:volume) Tris-buffered saline (TBS)/protease inhibitor (PI) (20 mM Tris–HCl, 500mM NaCl, pH 7.5 with complete PI tablet; Sigma- Aldrich, St. Louis, MO, USA). Homogenates were centrifuged for 5 minutes at 1000 x g at 4°C to remove highly insoluble structures and debris from the tissue. The supernatant was subjected to an additional centrifugation at 175.000 x g for 30 minutes. 1–2.5 mg total protein of the resulting supernatant was subjected to SEC over a Superose 6 Increase 10/300GL size-exclusion column (GE Healthcare) using an ÄKTA chromatography system (GE Healthcare). The separation was carried out in 50 mM ammonium acetate, pH 7.4, at a flow rate of 1.5 mL/min. Each collected fraction of 1 mL was frozen in liquid nitrogen and stored at -80 o C. Molecular size was estimated based on a gel filtration molecular marker kit ranging from 29–700 kDa range (Sigma-Aldrich, MGGF1000). Table 1 – Demographics and clinical characteristics of the DLB cohort and controls included in this study. CASE # SOURCE DIAGNOSIS AGE SEX DLB 1 20070105 Newcastle DLB 71 M DLB 2 Syn2 Mayo Clinic DLB 67 F DLB 3 Syn3 Mayo Clinic DLB 68 F DLB 4 1594 MGH DLB 81 F DLB 5 Syn6 Mayo Clinic DLB 54 M DLB 6 A01-213 Brigham and Women's DLB 83 M DLB 7 1650 MGH DLB 76 M DLB 8 1751 MGH DLB 87 F Ctrl 1 P48/07 Queen Square Cog. normal control F Ctrl 2 CON 2 Mayo Clinic PA 75 F Ctrl 3 CON 3 Mayo Clinic PA 81 F Ctrl 4 1901 MGH Control 54 M Ctrl 5 1887 MGH Control 60 M Ctrl 6 1821 MGH Control 92 M Ctrl 7 CON 7 Mayo Clinic SC/VaD 79 F Ctrl 8 CON 8 Mayo Clinic SC 70 M Frontal cortex grey matter was analysed from DLB patients and corresponding controls. F: female; M: male; MGH: Massachusetts General Hospital; NA: not applicable; PA: pathological aging; PMI: post-mortem interval; SC: senile changes; VaD: vascular dementia. Aggregated and total a-syn ELISA ELISAs detecting aggregated and total a-syn were performed as previously described 22 . As capture antibody, the a-syn aggregate ELISA utilized the aggregate-specific antibody MJF14-6-4-2 (Abcam, ab209538) 22 , 26 , while the total a-syn ELISA applied our in-house-made affinity purified rabbit polyclonal ASY-1 antibody 23 , 24 . (0.26 µg/mL). Both ELISAs utilized Syn-1 (BD Transduction Laboratories, 610787) as detection antibody. Fractions were diluted in 50 mM ammonium acetate, pH = 7.4, such that the strongest signal was above 5 ng/mL and did not exceed the linear part of the standard curve (usually between 156 pg/mL − 20 ng/mL). The standard curves were obtained using purified recombinant human monomeric and oligomeric a-syn as previously described. To allow comparison of individual patient SEC profiles of total and aggregated a-syn, each fraction measured within a patient’s SEC profile was normalized to the fraction measurement with the highest level of analyte within this individual patient sample set. This allowed all profiles to be presented with values between 0 and 1 despite variations in their concentrations of total and aggregated a-syn. SDS-PAGE and western blot 10 \(\:{\mu\:}\) l fraction sample was combined with 2 x SDS-loading buffer (20 mM Tris, pH 6.8, 2 mM EDTA, 2% SDS, 20% sucrose, 80 mM DTE) and boiled for 5 minutes at 96°C. Samples were subjected to SDS-PAGE using Bis-tris 8–16% gels (Genscript, M81615) together with a molecular size marker (ThermoFisher, 26616). Next, proteins were transferred to a PVDF membrane using an IBlot™ 2. The membrane was fixed in 4% PFA for 30 min at RT, boiled for 5 minutes in PBS, and blocked in blocking buffer (5% skimmed milk powder, 20 mM Tris base, 150 mM NaCl, 0.05% Tween 20, 0.02% NaN 3 ) for 1 hour at RT. Incubation of polyclonal ASY-1 rabbit IgG 23 , 24 . as primary antibody detecting total a-syn was carried out in blocking buffer overnight at 4°C. To validate the band pattern obtained by the ASY-1 IgG, filter was also probed with a mouse monoclonal Syn-1 antibody (610787) BD Transduction Laboratories, which yielded similar results (data not shown). Incubation with secondary antibody conjugated to horseradish peroxidase was performed in blocking buffer without NaN 3 for 1 hour at RT. Thereafter, the membrane was washed 3 x 5 minutes in tris-buffered saline (TBS), 0.05% Tween 20. Bound antibodies were visualized by ECL™ (GE Healthcare, RPN2209) and captured in a Fuji Las-3000 intelligent dark box (Fujifilm). Western blots were quantified using ImageJ2 (vers. 2.14.0). Statistics Fractions were analyzed in pairs, assuming Gaussian distribution without assuming a consistent standard deviation. Comparisons of multiple groups were analyzed by multiple unpaired t test with Welch’s correction. Statistical significance was defined as a p-value < 0.05. All statistical analyses were performed using GraphPad Prism (GraphPad Software 9.2.0). Declarations Acknowledgements We thank all patients and their caregivers who contributed to this study. Funding We thank our funders – the Lundbeck Foundation grants R223-2015-4222, R248-2016-2518 for Danish Research Institute of Translational Neuroscience-DANDRITE Nordic-EMBL Partnership for Molecular Medicine, Aarhus University, Denmark and R361-2020-2654 for the ERDYS project (PHJ). EG was supported by a fellowship from H. Lundbeck A/S and MRA by a scholarship from the Novo Nordic Foundation. The Chan Zuckerberg Neurodegeneration Challenge Network (CZI NDCN) Collaborative Pairs (T.B.), the UK Dementia Research Institute (which receives its funding from UK DRI Ltd, funded by the UK Medical Research Council, Alzheimer’s Society and Alzheimer’s Research UK) (T.B). We thank the US National Institute of Neurological Disorders and Stroke grants U54-NS110435, R01-NS109209, and R01-NS078165, R01-NS133979 (T.B.). Author contributions E.G., M.R.A., and P.H.J. wrote the main manuscript text and prepared figures. L.Z., D.S., and T.B. provided materials for the study. E.G. and L.R. were responsible for data collection. E.G. and M.R.A. were responsible for data analyses, including statistical analyses. All authors reviewed the manuscript. Competing interests D.J.S. is a director of Prothena Biosciences and an ad hoc consultant to Roche and Eisai. Otherwise, authors declare no competing financial and/or non-financial interests. Data Availability Data is provided within the manuscript, and raw data for the western blots analyses can be found in the supplementary information files. References Singleton, A. B., Hardy, J. A. & Gasser, T. The Birth of the Modern Era of Parkinson's Disease Genetics. J Parkinsons Dis 7, S87-s93 (2017). https://doi.org:10.3233/jpd-179009 Spillantini, M. G. & Goedert, M. Neurodegeneration and the ordered assembly of α-synuclein. Cell Tissue Res 373, 137–148 (2018). https://doi.org:10.1007/s00441-017-2706-9 Peng, C., Gathagan, R. J. & Lee, V. M. Distinct α-Synuclein strains and implications for heterogeneity among α-Synucleinopathies. Neurobiol Dis 109, 209–218 (2018). https://doi.org:10.1016/j.nbd.2017.07.018 Spillantini, M. G., Crowther, R. A., Jakes, R., Hasegawa, M. & Goedert, M. α-Synuclein in filamentous inclusions of Lewy bodies from Parkinson’s disease and dementia with Lewy bodies. Proceedings of the National Academy of Sciences 95, 6469–6473 (1998). https://doi.org:10.1073/pnas.95.11.6469 Anderson, J. P. et al. Phosphorylation of Ser-129 is the dominant pathological modification of alpha-synuclein in familial and sporadic Lewy body disease. J Biol Chem 281, 29739–29752 (2006). https://doi.org:10.1074/jbc.M600933200 Yang, Y. et al. Structures of α-synuclein filaments from human brains with Lewy pathology. Nature 610, 791–795 (2022). https://doi.org:10.1038/s41586-022-05319-3 Conway, K. A. et al. Acceleration of oligomerization, not fibrillization, is a shared property of both alpha-synuclein mutations linked to early-onset Parkinson's disease: implications for pathogenesis and therapy. Proc Natl Acad Sci U S A 97, 571–576 (2000). https://doi.org:10.1073/pnas.97.2.571 Caughey, B. & Lansbury, P. T. Protofibrils, pores, fibrils, and neurodegeneration: separating the responsible protein aggregates from the innocent bystanders. Annu Rev Neurosci 26, 267–298 (2003). https://doi.org:10.1146/annurev.neuro.26.010302.081142 Alam, P., Bousset, L., Melki, R. & Otzen, D. E. α-synuclein oligomers and fibrils: a spectrum of species, a spectrum of toxicities. J Neurochem 150, 522–534 (2019). https://doi.org:10.1111/jnc.14808 Cremades, N., Chen, S. W. & Dobson, C. M. Structural Characteristics of α-Synuclein Oligomers. Int Rev Cell Mol Biol 329, 79–143 (2017). https://doi.org:10.1016/bs.ircmb.2016.08.010 Varela, J. A. et al. Optical Structural Analysis of Individual α-Synuclein Oligomers. Angew Chem Int Ed Engl 57, 4886–4890 (2018). https://doi.org:10.1002/anie.201710779 Dickson, D. W. et al. Widespread alterations of alpha-synuclein in multiple system atrophy. Am J Pathol 155, 1241–1251 (1999). https://doi.org:10.1016/s0002-9440(10)65226-1 Sharon, R. et al. The formation of highly soluble oligomers of alpha-synuclein is regulated by fatty acids and enhanced in Parkinson's disease. Neuron 37, 583–595 (2003). https://doi.org:10.1016/s0896-6273(03)00024-2 Gould, N. et al. Evidence of native α-synuclein conformers in the human brain. J Biol Chem 289, 7929–7934 (2014). https://doi.org:10.1074/jbc.C113.538249 Paleologou, K. E. et al. Detection of elevated levels of soluble alpha-synuclein oligomers in post-mortem brain extracts from patients with dementia with Lewy bodies. Brain 132, 1093–1101 (2009). https://doi.org:10.1093/brain/awn349 Roberts, R. F., Wade-Martins, R. & Alegre-Abarrategui, J. Direct visualization of alpha-synuclein oligomers reveals previously undetected pathology in Parkinson's disease brain. Brain 138, 1642–1657 (2015). https://doi.org:10.1093/brain/awv040 Jensen, N. M. et al. MJF-14 proximity ligation assay detects early non-inclusion alpha-synuclein pathology with enhanced specificity and sensitivity. NPJ Parkinsons Dis 10, 227 (2024). https://doi.org:10.1038/s41531-024-00841-9 Jensen, N. M. et al. Abundant non-inclusion α-synuclein pathology in Lewy body-negative LRRK2-mutant cases. Acta Neuropathol 149, 41 (2025). https://doi.org:10.1007/s00401-025-02871-w Sekiya, H. et al. Wide distribution of alpha-synuclein oligomers in multiple system atrophy brain detected by proximity ligation. Acta Neuropathol 137, 455–466 (2019). https://doi.org:10.1007/s00401-019-01961-w Sekiya, H. et al. Widespread distribution of α-synuclein oligomers in LRRK2-related Parkinson's disease. Acta Neuropathol 149, 42 (2025). https://doi.org:10.1007/s00401-025-02872-9 Sanderson, J. B. et al. Analysis of α-synuclein species enriched from cerebral cortex of humans with sporadic dementia with Lewy bodies. Brain Commun 2, fcaa010 (2020). https://doi.org:10.1093/braincomms/fcaa010 Lassen, L. B. et al. ELISA method to detect α-synuclein oligomers in cell and animal models. PLoS One 13, e0196056 (2018). https://doi.org:10.1371/journal.pone.0196056 Jensen, P. H. et al. Microtubule-associated protein 1B is a component of cortical Lewy bodies and binds alpha-synuclein filaments. J Biol Chem 275, 21500–21507 (2000). https://doi.org:10.1074/jbc.M000099200 Jensen, P. H., Li, J. Y., Dahlström, A. & Dotti, C. G. Axonal transport of synucleins is mediated by all rate components. Eur J Neurosci 11, 3369–3376 (1999). https://doi.org:10.1046/j.1460-9568.1999.00754.x Colla, E. et al. Accumulation of toxic α-synuclein oligomer within endoplasmic reticulum occurs in α-synucleinopathy in vivo. J Neurosci 32, 3301–3305 (2012). https://doi.org:10.1523/jneurosci.5368-11.2012 Liekniņa, I. et al. Structural basis of epitope recognition by anti-alpha-synuclein antibodies MJFR14-6-4-2. NPJ Parkinsons Dis 10, 206 (2024). https://doi.org:10.1038/s41531-024-00822-y Additional Declarations Competing interest reported. D.J.S. is a director of Prothena Biosciences and an ad hoc consultant to Roche and Eisai. Otherwise, authors declare no competing financial and/or non-financial interests. Supplementary Files Supplfig1ab.jpg Supplfig1cd.jpg Cite Share Download PDF Status: Published Journal Publication published 28 Feb, 2026 Read the published version in npj Parkinson's Disease → Version 1 posted Editorial decision: Revision requested 19 Dec, 2025 Reviews received at journal 08 Dec, 2025 Reviews received at journal 04 Dec, 2025 Reviewers agreed at journal 27 Nov, 2025 Reviewers agreed at journal 26 Nov, 2025 Reviews received at journal 17 Nov, 2025 Reviewers agreed at journal 10 Nov, 2025 Reviewers invited by journal 03 Nov, 2025 Editor assigned by journal 02 Nov, 2025 Submission checks completed at journal 02 Nov, 2025 First submitted to journal 26 Oct, 2025 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. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. 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-7957852","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Short Report","associatedPublications":[],"authors":[{"id":542702308,"identity":"9e8c5a12-de76-4a3c-8edb-19cadbd18ab8","order_by":0,"name":"Emil Gregersen","email":"","orcid":"","institution":"Dept. Biomedicine, DANDRITE, Aarhus University","correspondingAuthor":false,"prefix":"","firstName":"Emil","middleName":"","lastName":"Gregersen","suffix":""},{"id":542702309,"identity":"7d889a91-59b1-4531-8a8e-19b04e116843","order_by":1,"name":"Mia R. Antorini","email":"","orcid":"","institution":"Dept. Biomedicine, DANDRITE, Aarhus University","correspondingAuthor":false,"prefix":"","firstName":"Mia","middleName":"R.","lastName":"Antorini","suffix":""},{"id":542702310,"identity":"c81f5c61-7e98-4166-a185-538c80c269e9","order_by":2,"name":"Lasse Reimer","email":"","orcid":"","institution":"Dept. Biomedicine, DANDRITE, Aarhus University","correspondingAuthor":false,"prefix":"","firstName":"Lasse","middleName":"","lastName":"Reimer","suffix":""},{"id":542702313,"identity":"12c078f9-d3b9-4dab-a5d4-e0cad3e4a54f","order_by":3,"name":"Ludovica Zaccagnini","email":"","orcid":"","institution":"UK Dementia Research Institute, University College London","correspondingAuthor":false,"prefix":"","firstName":"Ludovica","middleName":"","lastName":"Zaccagnini","suffix":""},{"id":542702315,"identity":"6f852e41-3e7d-4dfb-8805-27011cf56dc1","order_by":4,"name":"Dennis Selkoe","email":"","orcid":"","institution":"Harvard Medical School","correspondingAuthor":false,"prefix":"","firstName":"Dennis","middleName":"","lastName":"Selkoe","suffix":""},{"id":542702316,"identity":"1591f9cb-c7ce-4d01-a727-2e821dfc471a","order_by":5,"name":"Tim Bartels","email":"","orcid":"","institution":"UK Dementia Research Institute, University College London","correspondingAuthor":false,"prefix":"","firstName":"Tim","middleName":"","lastName":"Bartels","suffix":""},{"id":542702317,"identity":"f03a8f46-a339-42b1-9149-b6e0829dccd4","order_by":6,"name":"Poul Henning Jensen","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAv0lEQVRIiWNgGAWjYDACCYbExwwGIFYCiGA2IEZLsjHJWtikISxitZhLNzyrLiiwieZnT2D8zMNgbUxQi+WcA2m3Zxik5c7secAszcOQbkZQi8GNhLTbPAaHczfcSGAAajlsQ5SWYpCW/TcSmH8TrYUZbItEAhvIFsIOs5yRkCwN8suMMw/bLOcYpBP2vrlETuLngj82uf3tyYdvvKmwNmwg6DAGngQok7GBgYGIiASqYT9AhLJRMApGwSgY0QAAWXY5AxUM2CoAAAAASUVORK5CYII=","orcid":"","institution":"Dept. Biomedicine, DANDRITE, Aarhus University","correspondingAuthor":true,"prefix":"","firstName":"Poul","middleName":"Henning","lastName":"Jensen","suffix":""}],"badges":[],"createdAt":"2025-10-27 12:17:17","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-7957852/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-7957852/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1038/s41531-026-01301-2","type":"published","date":"2026-02-28T15:58:51+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":96240515,"identity":"9e8b5754-ea52-465d-b889-f9f6804d2b8d","added_by":"auto","created_at":"2025-11-19 07:09:01","extension":"jpg","order_by":0,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":1955561,"visible":true,"origin":"","legend":"","description":"","filename":"Fig1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7957852/v1/1fae2498786236f0d5788823.jpg"},{"id":95845760,"identity":"f5fe4b77-f359-4277-b4a3-69b6fa60f194","added_by":"auto","created_at":"2025-11-13 14:50:40","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":1691621,"visible":true,"origin":"","legend":"","description":"","filename":"MJFR14ELISApaperrevised103025.docx","url":"https://assets-eu.researchsquare.com/files/rs-7957852/v1/f8f1a09ffd15c71fa28624c2.docx"},{"id":95845763,"identity":"6f844e2a-48ee-4181-95f4-893b76eeff9d","added_by":"auto","created_at":"2025-11-13 14:50:40","extension":"docx","order_by":2,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":21381,"visible":true,"origin":"","legend":"","description":"","filename":"Table1.docx","url":"https://assets-eu.researchsquare.com/files/rs-7957852/v1/6865d1279ebfba6243bbc30c.docx"},{"id":95845769,"identity":"9f0d9545-3188-41eb-b2c9-a83d66f2a412","added_by":"auto","created_at":"2025-11-13 14:50:40","extension":"jpg","order_by":3,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":2654911,"visible":true,"origin":"","legend":"","description":"","filename":"Fig2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7957852/v1/c5cd6e5a7931135fc1b191f7.jpg"},{"id":96240498,"identity":"d68629dc-c376-46ef-bb33-f99167262dfc","added_by":"auto","created_at":"2025-11-19 07:08:58","extension":"jpg","order_by":4,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":1596179,"visible":true,"origin":"","legend":"","description":"","filename":"Fig3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7957852/v1/26b62a1cdcea127a6443eab2.jpg"},{"id":95845765,"identity":"4c372fae-6129-42dc-a3dd-2d55e472c54d","added_by":"auto","created_at":"2025-11-13 14:50:40","extension":"json","order_by":5,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":7426,"visible":true,"origin":"","legend":"","description":"","filename":"e5ed2632b7b343df9248b51a6953e39c.json","url":"https://assets-eu.researchsquare.com/files/rs-7957852/v1/e34ca688ec81d8ceb3c41cfe.json"},{"id":96240692,"identity":"054d3508-1487-42b2-ae66-a3b4a3c2add8","added_by":"auto","created_at":"2025-11-19 07:09:22","extension":"jpg","order_by":6,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":5905756,"visible":true,"origin":"","legend":"","description":"","filename":"Supplfig1ab.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7957852/v1/b2ebeacc2cb72cd3d106b689.jpg"},{"id":96240058,"identity":"36a72f22-4c17-4027-a597-e9238ce67b4b","added_by":"auto","created_at":"2025-11-19 07:08:18","extension":"jpg","order_by":7,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":4755959,"visible":true,"origin":"","legend":"","description":"","filename":"Supplfig1cd.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7957852/v1/e7d20e7856be14857bde7cb1.jpg"},{"id":95845776,"identity":"6c64a8d5-bc14-46d0-af9d-630f526f9dd2","added_by":"auto","created_at":"2025-11-13 14:50:40","extension":"xml","order_by":8,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":87045,"visible":true,"origin":"","legend":"","description":"","filename":"e5ed2632b7b343df9248b51a6953e39c1enriched.xml","url":"https://assets-eu.researchsquare.com/files/rs-7957852/v1/112ca1439bc95f63eb85840e.xml"},{"id":96240367,"identity":"76515a37-88bd-4ff3-ab18-bb1f292fb166","added_by":"auto","created_at":"2025-11-19 07:08:52","extension":"jpg","order_by":9,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":1955561,"visible":true,"origin":"","legend":"","description":"","filename":"Fig1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7957852/v1/0a54f4ebb51eb0f55f7b6a8e.jpg"},{"id":95845774,"identity":"4fed780e-b512-4288-8362-12c5776cb093","added_by":"auto","created_at":"2025-11-13 14:50:40","extension":"jpg","order_by":10,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":2654911,"visible":true,"origin":"","legend":"","description":"","filename":"Fig2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7957852/v1/b415ed69c682ae6ab4b1085e.jpg"},{"id":95845778,"identity":"735afb16-6456-4633-8794-0804f9ca3672","added_by":"auto","created_at":"2025-11-13 14:50:40","extension":"jpg","order_by":11,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":1596179,"visible":true,"origin":"","legend":"","description":"","filename":"Fig3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7957852/v1/2ed72e395ef21bdf3d4008e6.jpg"},{"id":96241439,"identity":"ad600433-3709-41aa-af48-9ec4806cee9a","added_by":"auto","created_at":"2025-11-19 07:10:43","extension":"jpeg","order_by":12,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":688068,"visible":true,"origin":"","legend":"","description":"","filename":"floatimage1.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-7957852/v1/c37b793b1a7c2d8e86a9bb3c.jpeg"},{"id":95845781,"identity":"eb92f255-ca2e-41d9-89b0-bc29cffad50a","added_by":"auto","created_at":"2025-11-13 14:50:40","extension":"jpeg","order_by":13,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":515761,"visible":true,"origin":"","legend":"","description":"","filename":"floatimage2.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-7957852/v1/5d1ef2d6453b29cc34746af9.jpeg"},{"id":96240654,"identity":"6954d061-f4dc-4542-8029-dc5ca275e713","added_by":"auto","created_at":"2025-11-19 07:09:17","extension":"jpeg","order_by":14,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":516110,"visible":true,"origin":"","legend":"","description":"","filename":"floatimage3.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-7957852/v1/9147477a2f1c0c171b628ec4.jpeg"},{"id":95845788,"identity":"69d1560e-5b8f-41b6-a2d6-d0428dff1e14","added_by":"auto","created_at":"2025-11-13 14:50:41","extension":"png","order_by":15,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":279610,"visible":true,"origin":"","legend":"","description":"","filename":"OnlineFig1.png","url":"https://assets-eu.researchsquare.com/files/rs-7957852/v1/7bb05482afc94849de475e1e.png"},{"id":95845779,"identity":"1772a294-84a3-4d63-b01f-daf34379ea7e","added_by":"auto","created_at":"2025-11-13 14:50:40","extension":"png","order_by":16,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":585149,"visible":true,"origin":"","legend":"","description":"","filename":"OnlineFig2.png","url":"https://assets-eu.researchsquare.com/files/rs-7957852/v1/d4f408be496af9b19874653d.png"},{"id":95845784,"identity":"b2fbb2e7-c6dd-456c-8841-4dbd74300a7a","added_by":"auto","created_at":"2025-11-13 14:50:40","extension":"png","order_by":17,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":241469,"visible":true,"origin":"","legend":"","description":"","filename":"OnlineFig3.png","url":"https://assets-eu.researchsquare.com/files/rs-7957852/v1/ba862f1d63242d4a95c8237a.png"},{"id":96240852,"identity":"67cd9bfd-846e-47ea-9122-8c529734a1b5","added_by":"auto","created_at":"2025-11-19 07:09:38","extension":"png","order_by":18,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":190442,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinefloatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-7957852/v1/6fe315187060f82418feee6e.png"},{"id":96240540,"identity":"0c7bc686-04d0-4425-b690-d0bca3d32de9","added_by":"auto","created_at":"2025-11-19 07:09:03","extension":"png","order_by":19,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":151147,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinefloatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-7957852/v1/3ae29de89ea64f5ff3fe25de.png"},{"id":95845786,"identity":"0a929bf7-2dc3-4ecd-a7f8-388747f22839","added_by":"auto","created_at":"2025-11-13 14:50:41","extension":"png","order_by":20,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":110890,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinefloatimage3.png","url":"https://assets-eu.researchsquare.com/files/rs-7957852/v1/26cb95bb27cccfa1ed10b993.png"},{"id":95845785,"identity":"0c70b13e-d5c4-497b-99a5-8603a0ec5efd","added_by":"auto","created_at":"2025-11-13 14:50:41","extension":"xml","order_by":21,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":86323,"visible":true,"origin":"","legend":"","description":"","filename":"e5ed2632b7b343df9248b51a6953e39c1structuring.xml","url":"https://assets-eu.researchsquare.com/files/rs-7957852/v1/023327b8d3ad5b8b4d48b90e.xml"},{"id":95845783,"identity":"82c95853-a657-47e2-abdd-1b62b7b66a75","added_by":"auto","created_at":"2025-11-13 14:50:40","extension":"html","order_by":22,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":95584,"visible":true,"origin":"","legend":"","description":"","filename":"earlyproof.html","url":"https://assets-eu.researchsquare.com/files/rs-7957852/v1/07fb4925b02677c34b5a8910.html"},{"id":96240645,"identity":"eafb6f5a-ac31-4fdc-8c23-3369e944e0fc","added_by":"auto","created_at":"2025-11-19 07:09:16","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":1955561,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eDetection of increased oligomerization in DLB cortex by α-syn aggregate-specific ELISA.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eBrain homogenates from dementia with Lewy body (DLB) patients or neurological healthy controls (CTRL) were separated by size-exclusion chromatography (SEC). The fractions obtained were analyzed by ELISA for the content of total a-syn (A), and aggregated a-syn (B) using the polyclonal ASY-1 and monoclonal a-syn aggregate-specific MJF14-6-4-2 rabbit IgG as primary antibodies, respectively. Each set of fractions from individual patients were diluted until the signal was in the linear part of the standard curve (usually 156 pg/mL - 20 ng/mL). To compare SEC profiles, fraction measurements was normalized to the highest measurement within that individual set of fractions. Control samples are depicted in blue and DLB in red. The molecular size markers are based on the calibration presented in Sanderson 2020\u003csup\u003e21\u003c/sup\u003e where the samples were initially isolated. High molecular weight fractions (HMW) 2-7, intermediate molecular weight fractions 8-10 (IMW) and low molecular weight fractions (LMW) 11-13) are marked. The mean value of the CTRL (blue) and DLB (red) are displayed with standard deviations, n=8. Statistical significance was determined by multiple unpaired t-test with Welch’s correction (*p\u0026lt;0.05, **p\u0026lt;0.01, ****p\u0026lt;0.0001).\u003c/p\u003e","description":"","filename":"Fig1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7957852/v1/c225b92d0cc3b48b7f3d697a.jpg"},{"id":95845771,"identity":"f557132a-0f6c-4d27-9342-4b7c30f995d4","added_by":"auto","created_at":"2025-11-13 14:50:40","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":2654911,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eLMW oligomers are more SDS-stable than HMW oligomers.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eBrain homogenates from dementia with Lewy body (DLB) patients or healthy controls (CTRL) were separated by SEC. The collected fractions were resolved by denaturing SDS-PAGE gel followed by western blot analysis using the polyclonal antibody ASY-1. A protein ladder was used as molecular size marker. The blots represent CTRL (#2) and DLB (#7) and are representative for their groups (see suppl. Figure 1a-d). Please note that the α-syn bands in HMW fractions 3-5 in the DLB case is almost exclusively monomeric and not detectable in the CTRL case. By contrast, the α-syn bands in fractions 8-10 are dominated by dimeric and trimeric species. This demonstrates the HMW α-syn oligomer species in the DLB cases are sensitive to denaturation by SDS whereas the smaller oligomers in fractions 8-10 predominantly dissociate into trimers and dimers.\u003c/p\u003e","description":"","filename":"Fig2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7957852/v1/e3898487c0f39817468f528c.jpg"},{"id":95845761,"identity":"dc1a1a38-1852-439c-8c74-4b51f0860979","added_by":"auto","created_at":"2025-11-13 14:50:40","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":1596179,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eHypothesis: cytosolic oligomer transitions in DLB.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe hypothesize that a pool of low-molecular weight (LMW) and intermediate-molecular weight (IMW) a-syn species are physiologically present in healthy brains (green box). These correspond to the oligomers detected in fractions 11-13 and 8-10, respectively, by ELISA. Meanwhile, the a-syn species found in DLB patients (red box) are shifted towards higher-molecular weight fractions in LMW and IMW fractions, and to high-molecular weight (HMW) species not found in control brains, corresponding to fractions 3-7 in our ELISA. According to our SDS-PAGE, the IMW pool is more SDS-stable with more SDS-resistant trimers and dimers compared to the LMW and HMW pools that denature into 17 kDa monomers.\u003c/p\u003e","description":"","filename":"Fig3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7957852/v1/21463c600389757a0b580d14.jpg"},{"id":103765838,"identity":"a5ba4bab-6eb9-44c5-8494-0c1cb337cc22","added_by":"auto","created_at":"2026-03-02 16:10:00","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":6821762,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7957852/v1/72378374-ae66-4079-9c1b-e6124ae5cafe.pdf"},{"id":95845768,"identity":"e5d8c4a0-d0d8-44e0-968d-540f5758833b","added_by":"auto","created_at":"2025-11-13 14:50:40","extension":"jpg","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":5905756,"visible":true,"origin":"","legend":"","description":"","filename":"Supplfig1ab.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7957852/v1/07cc24f3aafed978e0a6499b.jpg"},{"id":95845766,"identity":"cc4ae98c-4716-4982-a9f7-3b8ab10e6978","added_by":"auto","created_at":"2025-11-13 14:50:40","extension":"jpg","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":4755959,"visible":true,"origin":"","legend":"","description":"","filename":"Supplfig1cd.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7957852/v1/12a745731fdb79e02dfc80fd.jpg"}],"financialInterests":"Competing interest reported. D.J.S. is a director of Prothena Biosciences and an ad hoc consultant to Roche and Eisai. Otherwise, authors declare no competing financial and/or non-financial interests.","formattedTitle":"Higher molecular weight alpha-synuclein oligomers are increased in brain cytosol from dementia with Lewy bodies","fulltext":[{"header":"Main text","content":"\u003cp\u003eAggregation of the presynaptic protein α-synuclein (a-syn) is tightly associated with Parkinson\u0026rsquo;s disease (PD) and dementia with Lewy Bodies (DLB) based on genetic and biochemical evidence\u003csup\u003e\u003cspan additionalcitationids=\"CR2\" citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/sup\u003e. The aggregates are present as insoluble amyloid type fibrillar species hyperphosphorylated on Ser129, typically associated to Lewy body (LB)-type inclusions, and soluble oligomers\u003csup\u003e\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e,\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u003c/sup\u003e. High resolution cryo-EM tomography studies have revealed detailed structural insight into disease-associated fibrillar strains\u003csup\u003e\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u003c/sup\u003e. However, our insight into the nature of the oligomers in the brain is lacking in comparison, despite oligomers having been hypothesized to be pathogenic for decades\u003csup\u003e\u003cspan additionalcitationids=\"CR8\" citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u003c/sup\u003e, and in vitro generated oligomers having been studied in detail\u003csup\u003e\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e,\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u003c/sup\u003e. Their existence and increase in extracts of human brains affected by synucleinopathies have been demonstrated by different biochemical assays\u003csup\u003e\u003cspan additionalcitationids=\"CR13\" citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u003c/sup\u003e, and in cytosol from DLB brains by ELISA\u003csup\u003e\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003eRecent immunochemical studies take advantage of a-syn proximity ligation assays (PLA) to detect a-syn oligomers. Two protocols have been developed, both using pairs of either the monoclonal pan-a-syn antibody Syn211 or the aggregate specific MJFR14-6-4-2 antibody\u003csup\u003e\u003cspan additionalcitationids=\"CR17 CR18 CR19\" citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u003c/sup\u003e. Using the MJFR14-6-4-2 PLA, it was demonstrated that oligomers are abundant in tissue from PD brains where they develop prior to the formation of LBs but are absent or in very low abundance in non-synucleinopathy control brain tissue. Moreover, they exist in brains without LBs from patients with mutations in the LRRK2 gene\u003csup\u003e\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e,\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003eFurthermore, the size distribution of total a-syn species in the cytosol of DLB and control brains has been analyzed by size-exclusion chromatography (SEC), followed by a-syn ELISA of the individual cytosolic fractions\u003csup\u003e\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u003c/sup\u003e. This demonstrated a symmetric peak with a maximum corresponding to a molecular size of 150 kDa and negligible levels larger than 300 kDa.\u003c/p\u003e\u003cp\u003eIn this study, we hypothesized that insight into the size of MJFR14-6-4-2 positive a-syn aggregates in DLB brain cytosol would be informative as to the size of the candidates responsible for the MJFR14-6-4-2 PLA signal in DLB brain tissue. Quantifying the samples isolated from DLB and control brains SEC and MJFR14-6-4-2 ELISA\u003csup\u003e\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u003c/sup\u003e demonstrated that both control and DLB brains contain significant amounts of low- and intermediate size a-syn species (150\u0026ndash;400 kDa), which were shifted towards higher-molecular weight fractions in DLB patients. Larger oligomers in the range of 500\u0026ndash;1800 kDa appeared selectively increased in DLB. This suggests large cytosolic oligomers are candidates for the structures generating the MJFR14-6-4-2 PLA positive signals in synucleinopathies, although other species present in other brain fractions, e.g. bound to vesicle or organelles, may also contribute.\u003c/p\u003e\u003cp\u003eThe fractions 1\u0026ndash;14 from the SEC of the cytosol isolated from frontal cortex grey matter of neurologically healthy controls and DLB patients were analysed by ELISA for total a-syn and MJFR14-6-4-2-positive aggregates, and by a-syn immunoblotting. These fractions covered the molecular size range from \u0026gt;\u0026thinsp;2000 kDa to approx. 30 kDa. The 8 neurologically healthy controls and 8 DLB patients were chosen from a larger cohort previously analyzed for the presence of total a-syn\u003csup\u003e\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003eThe total a-syn ELISA demonstrated similar elution profiles for both control and DLB cytosol with the vast majority of a-syn eluting as a symmetric peak with a maximum in fraction 11 (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA). This corresponds to a molecular size of approx. 200 kDa, and with no substantial signal in the higher-molecular-weight fractions 9 and below. The scale of the total a-syn on the elution profiles is normalized to the highest measured fraction within each individual patient profile in that set of fractions, being fraction 11 for both control and DLB patients. This molecular size profile is in line with the previous measurement of the larger cohort wherefrom the 8 controls and 8 DLB patients were chosen\u003csup\u003e\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eBrain homogenates from dementia with Lewy body (DLB) patients or neurological healthy controls (CTRL) were separated by size-exclusion chromatography (SEC). The fractions obtained were analyzed by ELISA for the content of total a-syn (A), and aggregated a-syn (B) using the polyclonal ASY-1 and monoclonal a-syn aggregate-specific MJF14-6-4-2 rabbit IgG as primary antibodies, respectively. Each set of fractions from individual patients were diluted until the signal was in the linear part of the standard curve (usually 156 pg/mL \u0026minus;\u0026thinsp;20 ng/mL). To compare SEC profiles, fraction measurements was normalized to the highest measurement within that individual set of fractions. Control samples are depicted in blue and DLB in red. The molecular size markers are based on the calibration presented in Sanderson 2020\u003csup\u003e21\u003c/sup\u003e where the samples were initially isolated. High molecular weight fractions (HMW) 2\u0026ndash;7, intermediate molecular weight fractions 8\u0026ndash;10 (IMW) and low molecular weight fractions (LMW) 11\u0026ndash;13) are marked. The mean value of the CTRL (blue) and DLB (red) are displayed with standard deviations, n\u0026thinsp;=\u0026thinsp;8. Statistical significance was determined by multiple unpaired t-test with Welch\u0026rsquo;s correction (*p\u0026thinsp;\u0026lt;\u0026thinsp;0.05, **p\u0026thinsp;\u0026lt;\u0026thinsp;0.01, ****p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001).\u003c/p\u003e\u003cp\u003eCompared to the total a-syn ELISA, the aggregate-specific MJF14-6-4-2 ELISA detected a broader peak, with oligomeric a-syn spanning from fractions 8 to 12 in control patients (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB). This lower-molecular-weight oligomer peak was shifted to higher-molecular weight sizes in the DLB cytosol with a maximum in fraction 9 (approx. 340 kDa) compared to fraction 10 (approx. 250 kDa) for the controls. In addition to the peak in fractions 8\u0026ndash;12, a high-molecular weight (HMW) pool in fractions 2\u0026ndash;7 could be found in the DLB samples as a prominent HMW \u0026ldquo;shoulder\u0026rdquo; on the peak. This contrasted with the controls, where this HMW \u0026ldquo;shoulder\u0026rdquo; was smaller and tapered off in fractions 5\u0026ndash;7. Hence, there exists a quantitative difference between the HMW pool of a-syn oligomers in DLB and controls where it accounts for 25.67% \u0026plusmn; 4.87 compared to 10.19% \u0026plusmn; 4.82 of the total aggregated a-syn signal (calculated based on area under the curve). This may indicate an increased aggregation or reduced catabolism of oligomers in the DLB patient frontal cortical grey matter, resulting in the cytosolic accumulation of larger oligomers with a molecular size of around 500\u0026ndash;1800 kDa.\u003c/p\u003e\u003cp\u003eAs an orthogonal analysis to the quantitative ELISA, we subjected the fractions from the SEC to reducing SDS-PAGE and immunoblotting using a polyclonal a-syn antibody ASY-1\u003csup\u003e23,24\u003c/sup\u003e. This analysis holds potential to reveal qualitative differences in the SDS-sensitivity of oligomer pools of DLB patients and controls. Figure\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA\u0026thinsp;+\u0026thinsp;B demonstrate representative immunoblots of cytosol fractions from patients Control #2 and DLB #7. The fractions can be categorized into three groups: A LMW pool in fractions 11\u0026ndash;13, an intermediate molecular weight (IMW) pool in fractions 8\u0026ndash;10, and a HMW pool in fractions 2\u0026ndash;7. The LMW pool is dominated by SDS-sensitive species dissociating into 17 kDa monomers upon denaturation in the loading buffer for the SDS-PAGE. The IMW pool is dominated by species dissociating into SDS-resistant dimers and trimers. The LMW and IMW fractions do not differ noticeably in their ASY-1 immunoreactive patterns between controls and DLB patients, although it is noteworthy that the IMW pool represents the fractions wherein the ELISA-positive oligomers accumulated in DLB patients compared to controls. The HMW pool is weakly positive on the immunoblots but evidently occupied by 17 kDa monomeric a-syn species, thus indicating higher sensitivity to denaturation by SDS compared to the IMW pool (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB). To better appreciate this pool, we analyzed longer exposures of the immunoblots (Suppl. Figure\u0026nbsp;1). This allowed the demonstration of a DLB-associated signature of increased HMW oligomers based on the following criterion: The 17 kDa signal in fraction 3 is higher or equal to the signal in fraction 5 and/or 6. None of the 7 controls (#2\u0026ndash;8) fulfilled this criterion, whereas 6 of the 8 DLB patients were positive (DLB #2, 4\u0026ndash;8). This corroborates the ELISA data that demonstrated increased levels of aggregates in the HMW fraction and suggests that these oligomers are SDS-sensitive in contrast to the LMW oligomers that generates trimers and dimers by SDS treatment.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eBrain homogenates from dementia with Lewy body (DLB) patients or healthy controls (CTRL) were separated by SEC. The collected fractions were resolved by denaturing SDS-PAGE gel followed by western blot analysis using the polyclonal antibody ASY-1. A protein ladder was used as molecular size marker. The blots represent CTRL (#2) and DLB (#7) and are representative for their groups (see suppl. Figure\u0026nbsp;1a-d). Please note that the α-syn bands in HMW fractions 3\u0026ndash;5 in the DLB case is almost exclusively monomeric and not detectable in the CTRL case. By contrast, the α-syn bands in fractions 8\u0026ndash;10 are dominated by dimeric and trimeric species. This demonstrates the HMW α-syn oligomer species in the DLB cases are sensitive to denaturation by SDS whereas the smaller oligomers in fractions 8\u0026ndash;10 predominantly dissociate into trimers and dimers.\u003c/p\u003e\u003cp\u003eA strength of our study is the combination of SEC and an a-syn aggregate specific ELISA on a subfraction of a well characterized cohort of control and DLB brains. Our data demonstrates that a-syn aggregates able to bind the aggregate-specific MJFR14-6-4-2 antibody exist in both control and DLB brain cytosol, but their average size increases in DLB. Meanwhile, the very large cytosolic oligomers 500\u0026ndash;1800 kDa are exclusively present in DLB brains and are thus candidates for generating the PLA signal recently demonstrated to develop in brains affected by synucleinopathies (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eWe hypothesize that a pool of low-molecular weight (LMW) and intermediate-molecular weight (IMW) a-syn species are physiologically present in healthy brains (green box). These correspond to the oligomers detected in fractions 11\u0026ndash;13 and 8\u0026ndash;10, respectively, by ELISA. Meanwhile, the a-syn species found in DLB patients (red box) are shifted towards higher-molecular weight fractions in LMW and IMW fractions, and to high-molecular weight (HMW) species not found in control brains, corresponding to fractions 3\u0026ndash;7 in our ELISA. According to our SDS-PAGE, the IMW pool is more SDS-stable with more SDS-resistant trimers and dimers compared to the LMW and HMW pools that denature into 17 kDa monomers.\u003c/p\u003e\u003cp\u003eA limitation of the study is its exploratory nature by only studying a small cohort and only focusing on the cytosolic fraction. Our findings motivate further studies using similar approaches on all the subcellular fractions of brain tissue to bring more insight into the size distribution of oligomers. For example, a previous study demonstrated that aggregated a-syn species were associated to ER membranes in PD\u003csup\u003e25\u003c/sup\u003e. One goal would be to develop purification protocols that can allow high resolution cryo-EM tomography studies to solve the structure of these hitherto elusive pathogenic a-syn aggregates.\u003c/p\u003e"},{"header":"Material and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\n \u003ch2\u003eBrain samples and Size Exclusion Chromatography\u003c/h2\u003e\n \u003cp\u003eHuman brain tissue from \u003cem\u003epost-mortem\u003c/em\u003e brains of 8 healthy controls and 8 patients with a neuropathological diagnosis of DLB was sampled from the cohort previously analyzed \u003csup\u003e\u003cspan class=\"CitationRef\"\u003e21\u003c/span\u003e\u003c/sup\u003e. Human brain tissue was provided by Brigham and Women\u0026rsquo;s Hospital (Boston, MA, USA), Mayo Clinic (Jacksonville, FL, USA), Massachusetts General Hospital/Massachusetts Alzheimer\u0026rsquo;s Disease Research Center (Boston, MA, USA), Newcastle Brain Tissue Resource (Newcastle upon Tyne, UK) and Queen Square Brain Bank for Neurological Disorders (London, UK). Information about the brain samples used in this study is summarized in Table \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e. Consent was obtained from patients prior to death at each brain collection centre. All five brain banks approved of the proposal for the use of human tissue in this study, and the IRB and REC at the first and last authors\u0026rsquo; institution deemed the planned use of this tissue to be appropriate and ethical (IRB 1999P001180/BWH, REC 18/LO/0721).\u003c/p\u003e\n \u003cp\u003eApproximately 500 mg of frontal cortex tissue pieces was homogenized with 25 strokes using a manual Dounce homogenizer overhead stirrer (Wheaton, Millville, NJ, USA) in four volumes (weight:volume) Tris-buffered saline (TBS)/protease inhibitor (PI) (20 mM Tris\u0026ndash;HCl, 500mM NaCl, pH 7.5 with complete PI tablet; Sigma- Aldrich, St. Louis, MO, USA). Homogenates were centrifuged for 5 minutes at 1000 x g at 4\u0026deg;C to remove highly insoluble structures and debris from the tissue. The supernatant was subjected to an additional centrifugation at 175.000 x g for 30 minutes. 1\u0026ndash;2.5 mg total protein of the resulting supernatant was subjected to SEC over a Superose 6 Increase 10/300GL size-exclusion column (GE Healthcare) using an \u0026Auml;KTA chromatography system (GE Healthcare). The separation was carried out in 50 mM ammonium acetate, pH 7.4, at a flow rate of 1.5 mL/min. Each collected fraction of 1 mL was frozen in liquid nitrogen and stored at -80\u003csup\u003eo\u003c/sup\u003eC. Molecular size was estimated based on a gel filtration molecular marker kit ranging from 29\u0026ndash;700 kDa range (Sigma-Aldrich, MGGF1000).\u003c/p\u003e\n \u003cdiv class=\"gridtable\"\u003e\n \u003ctable id=\"Tab1\" border=\"1\"\u003e\n \u003ccaption language=\"En\"\u003e\n \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e\n \u003cdiv class=\"CaptionContent\"\u003e\n \u003cp\u003e\u0026ndash; Demographics and clinical characteristics of the DLB cohort and controls included in this study.\u003c/p\u003e\n \u003c/div\u003e\n \u003c/caption\u003e\n \u003cthead\u003e\n \u003ctr\u003e\n \u003cth align=\"left\"\u003e\u0026nbsp;\u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eCASE #\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eSOURCE\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eDIAGNOSIS\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eAGE\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eSEX\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003c/thead\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eDLB 1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e20070105\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eNewcastle\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eDLB\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e71\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eM\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eDLB 2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eSyn2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eMayo Clinic\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eDLB\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e67\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eF\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eDLB 3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eSyn3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eMayo Clinic\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eDLB\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e68\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eF\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eDLB 4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1594\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eMGH\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eDLB\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e81\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eF\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eDLB 5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eSyn6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eMayo Clinic\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eDLB\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e54\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eM\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eDLB 6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eA01-213\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eBrigham and Women\u0026apos;s\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eDLB\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e83\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eM\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eDLB 7\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1650\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eMGH\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eDLB\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e76\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eM\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eDLB 8\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1751\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eMGH\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eDLB\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e87\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eF\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eCtrl 1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eP48/07\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eQueen Square\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eCog. normal control\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eF\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eCtrl 2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eCON 2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eMayo Clinic\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003ePA\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e75\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eF\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eCtrl 3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eCON 3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eMayo Clinic\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003ePA\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e81\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eF\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eCtrl 4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1901\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eMGH\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eControl\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e54\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eM\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eCtrl 5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1887\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eMGH\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eControl\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e60\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eM\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eCtrl 6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1821\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eMGH\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eControl\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e92\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eM\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eCtrl 7\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eCON 7\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eMayo Clinic\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eSC/VaD\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e79\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eF\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eCtrl 8\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eCON 8\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eMayo Clinic\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eSC\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e70\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eM\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n \u003c/div\u003e\n \u003cp\u003eFrontal cortex grey matter was analysed from DLB patients and corresponding controls.\u003c/p\u003e\n \u003cp\u003eF: female; M: male; MGH: Massachusetts General Hospital; NA: not applicable; PA: pathological aging; PMI: post-mortem interval; SC: senile changes; VaD: vascular dementia.\u003c/p\u003e\n\u003c/div\u003e\n\u003ch3\u003eAggregated and total a-syn ELISA\u003c/h3\u003e\n\u003cp\u003eELISAs detecting aggregated and total a-syn were performed as previously described\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e22\u003c/span\u003e\u003c/sup\u003e. As capture antibody, the a-syn aggregate ELISA utilized the aggregate-specific antibody MJF14-6-4-2 (Abcam, ab209538)\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e22\u003c/span\u003e,\u003cspan class=\"CitationRef\"\u003e26\u003c/span\u003e\u003c/sup\u003e, while the total a-syn ELISA applied our in-house-made affinity purified rabbit polyclonal ASY-1 antibody\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e23\u003c/span\u003e,\u003cspan class=\"CitationRef\"\u003e24\u003c/span\u003e\u003c/sup\u003e. (0.26 \u0026micro;g/mL). Both ELISAs utilized Syn-1 (BD Transduction Laboratories, 610787) as detection antibody. Fractions were diluted in 50 mM ammonium acetate, pH\u0026thinsp;=\u0026thinsp;7.4, such that the strongest signal was above 5 ng/mL and did not exceed the linear part of the standard curve (usually between 156 pg/mL \u0026minus;\u0026thinsp;20 ng/mL). The standard curves were obtained using purified recombinant human monomeric and oligomeric a-syn as previously described. To allow comparison of individual patient SEC profiles of total and aggregated a-syn, each fraction measured within a patient\u0026rsquo;s SEC profile was normalized to the fraction measurement with the highest level of analyte within this individual patient sample set. This allowed all profiles to be presented with values between 0 and 1 despite variations in their concentrations of total and aggregated a-syn.\u003c/p\u003e\n\u003ch3\u003eSDS-PAGE and western blot\u003c/h3\u003e\n\u003cp\u003e10 \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:{\\mu\\:}\\)\u003c/span\u003e\u003c/span\u003el fraction sample was combined with 2 x SDS-loading buffer (20 mM Tris, pH 6.8, 2 mM EDTA, 2% SDS, 20% sucrose, 80 mM DTE) and boiled for 5 minutes at 96\u0026deg;C. Samples were subjected to SDS-PAGE using Bis-tris 8\u0026ndash;16% gels (Genscript, M81615) together with a molecular size marker (ThermoFisher, 26616). Next, proteins were transferred to a PVDF membrane using an IBlot\u0026trade; 2. The membrane was fixed in 4% PFA for 30 min at RT, boiled for 5 minutes in PBS, and blocked in blocking buffer (5% skimmed milk powder, 20 mM Tris base, 150 mM NaCl, 0.05% Tween 20, 0.02% NaN\u003csub\u003e3\u003c/sub\u003e) for 1 hour at RT. Incubation of polyclonal ASY-1 rabbit IgG\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e23\u003c/span\u003e,\u003cspan class=\"CitationRef\"\u003e24\u003c/span\u003e\u003c/sup\u003e. as primary antibody detecting total a-syn was carried out in blocking buffer overnight at 4\u0026deg;C. To validate the band pattern obtained by the ASY-1 IgG, filter was also probed with a mouse monoclonal Syn-1 antibody (610787) BD Transduction Laboratories, which yielded similar results (data not shown). Incubation with secondary antibody conjugated to horseradish peroxidase was performed in blocking buffer without NaN\u003csub\u003e3\u003c/sub\u003e for 1 hour at RT. Thereafter, the membrane was washed 3 x 5 minutes in tris-buffered saline (TBS), 0.05% Tween 20. Bound antibodies were visualized by ECL\u0026trade; (GE Healthcare, RPN2209) and captured in a Fuji Las-3000 intelligent dark box (Fujifilm). Western blots were quantified using ImageJ2 (vers. 2.14.0).\u003c/p\u003e\n\u003ch3\u003eStatistics\u003c/h3\u003e\n\u003cp\u003eFractions were analyzed in pairs, assuming Gaussian distribution without assuming a consistent standard deviation. Comparisons of multiple groups were analyzed by multiple unpaired t test with Welch\u0026rsquo;s correction. Statistical significance was defined as a p-value\u0026thinsp;\u0026lt;\u0026thinsp;0.05. All statistical analyses were performed using GraphPad Prism (GraphPad Software 9.2.0).\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe thank all patients and their caregivers who contributed to this study.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe thank our funders – the Lundbeck Foundation grants R223-2015-4222, R248-2016-2518 for Danish Research Institute of Translational Neuroscience-DANDRITE Nordic-EMBL Partnership for Molecular Medicine, Aarhus University, Denmark and R361-2020-2654 for the ERDYS project (PHJ). EG was supported by a fellowship from H. Lundbeck A/S and MRA by a scholarship from the Novo Nordic Foundation. The Chan Zuckerberg Neurodegeneration Challenge Network (CZI NDCN) Collaborative Pairs (T.B.), the UK Dementia Research Institute (which receives its funding from UK DRI Ltd, funded by the UK Medical Research Council, Alzheimer’s Society and Alzheimer’s Research UK) (T.B). We thank the US National Institute of Neurological Disorders and Stroke grants U54-NS110435, R01-NS109209, and R01-NS078165, R01-NS133979 (T.B.).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eE.G., M.R.A., and P.H.J. \u0026nbsp;wrote the main manuscript text and prepared figures. L.Z., D.S., and T.B. provided materials for the study. E.G. and L.R. were responsible for data collection. E.G. and M.R.A. \u0026nbsp;were responsible for data analyses, including statistical analyses. All authors reviewed the manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eD.J.S. is a director of Prothena Biosciences and an ad hoc consultant to Roche and Eisai. Otherwise, authors declare no competing financial and/or non-financial interests.\u003c/p\u003e\u003ch2\u003eData Availability\u003c/h2\u003e\u003cp\u003eData is provided within the manuscript, and raw data for the western blots analyses can be found in the supplementary information files.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eSingleton, A. B., Hardy, J. A. \u0026amp; Gasser, T. The Birth of the Modern Era of Parkinson's Disease Genetics. \u003cem\u003eJ Parkinsons Dis\u003c/em\u003e 7, S87-s93 (2017). \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org:10.3233/jpd-179009\u003c/span\u003e\u003cspan address=\"https://doi.org:10.3233/jpd-179009\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eSpillantini, M. G. \u0026amp; Goedert, M. Neurodegeneration and the ordered assembly of α-synuclein. \u003cem\u003eCell Tissue Res\u003c/em\u003e 373, 137\u0026ndash;148 (2018). \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org:10.1007/s00441-017-2706-9\u003c/span\u003e\u003cspan address=\"https://doi.org:10.1007/s00441-017-2706-9\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003ePeng, C., Gathagan, R. J. \u0026amp; Lee, V. M. Distinct α-Synuclein strains and implications for heterogeneity among α-Synucleinopathies. \u003cem\u003eNeurobiol Dis\u003c/em\u003e 109, 209\u0026ndash;218 (2018). \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org:10.1016/j.nbd.2017.07.018\u003c/span\u003e\u003cspan address=\"https://doi.org:10.1016/j.nbd.2017.07.018\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eSpillantini, M. G., Crowther, R. A., Jakes, R., Hasegawa, M. \u0026amp; Goedert, M. α-Synuclein in filamentous inclusions of Lewy bodies from Parkinson\u0026rsquo;s disease and dementia with Lewy bodies. \u003cem\u003eProceedings of the National Academy of Sciences\u003c/em\u003e 95, 6469\u0026ndash;6473 (1998). \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org:10.1073/pnas.95.11.6469\u003c/span\u003e\u003cspan address=\"https://doi.org:10.1073/pnas.95.11.6469\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eAnderson, J. P. \u003cem\u003eet al.\u003c/em\u003e Phosphorylation of Ser-129 is the dominant pathological modification of alpha-synuclein in familial and sporadic Lewy body disease. \u003cem\u003eJ Biol Chem\u003c/em\u003e 281, 29739\u0026ndash;29752 (2006). \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org:10.1074/jbc.M600933200\u003c/span\u003e\u003cspan address=\"https://doi.org:10.1074/jbc.M600933200\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eYang, Y. \u003cem\u003eet al.\u003c/em\u003e Structures of α-synuclein filaments from human brains with Lewy pathology. \u003cem\u003eNature\u003c/em\u003e 610, 791\u0026ndash;795 (2022). \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org:10.1038/s41586-022-05319-3\u003c/span\u003e\u003cspan address=\"https://doi.org:10.1038/s41586-022-05319-3\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eConway, K. A. \u003cem\u003eet al.\u003c/em\u003e Acceleration of oligomerization, not fibrillization, is a shared property of both alpha-synuclein mutations linked to early-onset Parkinson's disease: implications for pathogenesis and therapy. \u003cem\u003eProc Natl Acad Sci U S A\u003c/em\u003e 97, 571\u0026ndash;576 (2000). \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org:10.1073/pnas.97.2.571\u003c/span\u003e\u003cspan address=\"https://doi.org:10.1073/pnas.97.2.571\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eCaughey, B. \u0026amp; Lansbury, P. T. Protofibrils, pores, fibrils, and neurodegeneration: separating the responsible protein aggregates from the innocent bystanders. \u003cem\u003eAnnu Rev Neurosci\u003c/em\u003e 26, 267\u0026ndash;298 (2003). \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org:10.1146/annurev.neuro.26.010302.081142\u003c/span\u003e\u003cspan address=\"https://doi.org:10.1146/annurev.neuro.26.010302.081142\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eAlam, P., Bousset, L., Melki, R. \u0026amp; Otzen, D. E. α-synuclein oligomers and fibrils: a spectrum of species, a spectrum of toxicities. \u003cem\u003eJ Neurochem\u003c/em\u003e 150, 522\u0026ndash;534 (2019). \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org:10.1111/jnc.14808\u003c/span\u003e\u003cspan address=\"https://doi.org:10.1111/jnc.14808\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eCremades, N., Chen, S. W. \u0026amp; Dobson, C. M. Structural Characteristics of α-Synuclein Oligomers. \u003cem\u003eInt Rev Cell Mol Biol\u003c/em\u003e 329, 79\u0026ndash;143 (2017). \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org:10.1016/bs.ircmb.2016.08.010\u003c/span\u003e\u003cspan address=\"https://doi.org:10.1016/bs.ircmb.2016.08.010\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eVarela, J. A. \u003cem\u003eet al.\u003c/em\u003e Optical Structural Analysis of Individual α-Synuclein Oligomers. \u003cem\u003eAngew Chem Int Ed Engl\u003c/em\u003e 57, 4886\u0026ndash;4890 (2018). \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org:10.1002/anie.201710779\u003c/span\u003e\u003cspan address=\"https://doi.org:10.1002/anie.201710779\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eDickson, D. W. \u003cem\u003eet al.\u003c/em\u003e Widespread alterations of alpha-synuclein in multiple system atrophy. \u003cem\u003eAm J Pathol\u003c/em\u003e 155, 1241\u0026ndash;1251 (1999). \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org:10.1016/s0002-9440(10)65226-1\u003c/span\u003e\u003cspan address=\"https://doi.org:10.1016/s0002-9440(10)65226-1\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eSharon, R. \u003cem\u003eet al.\u003c/em\u003e The formation of highly soluble oligomers of alpha-synuclein is regulated by fatty acids and enhanced in Parkinson's disease. \u003cem\u003eNeuron\u003c/em\u003e 37, 583\u0026ndash;595 (2003). \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org:10.1016/s0896-6273(03)00024-2\u003c/span\u003e\u003cspan address=\"https://doi.org:10.1016/s0896-6273(03)00024-2\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eGould, N. \u003cem\u003eet al.\u003c/em\u003e Evidence of native α-synuclein conformers in the human brain. \u003cem\u003eJ Biol Chem\u003c/em\u003e 289, 7929\u0026ndash;7934 (2014). \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org:10.1074/jbc.C113.538249\u003c/span\u003e\u003cspan address=\"https://doi.org:10.1074/jbc.C113.538249\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003ePaleologou, K. E. \u003cem\u003eet al.\u003c/em\u003e Detection of elevated levels of soluble alpha-synuclein oligomers in post-mortem brain extracts from patients with dementia with Lewy bodies. \u003cem\u003eBrain\u003c/em\u003e 132, 1093\u0026ndash;1101 (2009). \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org:10.1093/brain/awn349\u003c/span\u003e\u003cspan address=\"https://doi.org:10.1093/brain/awn349\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eRoberts, R. F., Wade-Martins, R. \u0026amp; Alegre-Abarrategui, J. Direct visualization of alpha-synuclein oligomers reveals previously undetected pathology in Parkinson's disease brain. \u003cem\u003eBrain\u003c/em\u003e 138, 1642\u0026ndash;1657 (2015). \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org:10.1093/brain/awv040\u003c/span\u003e\u003cspan address=\"https://doi.org:10.1093/brain/awv040\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eJensen, N. M. \u003cem\u003eet al.\u003c/em\u003e MJF-14 proximity ligation assay detects early non-inclusion alpha-synuclein pathology with enhanced specificity and sensitivity. \u003cem\u003eNPJ Parkinsons Dis\u003c/em\u003e 10, 227 (2024). \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org:10.1038/s41531-024-00841-9\u003c/span\u003e\u003cspan address=\"https://doi.org:10.1038/s41531-024-00841-9\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eJensen, N. M. \u003cem\u003eet al.\u003c/em\u003e Abundant non-inclusion α-synuclein pathology in Lewy body-negative LRRK2-mutant cases. \u003cem\u003eActa Neuropathol\u003c/em\u003e 149, 41 (2025). \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org:10.1007/s00401-025-02871-w\u003c/span\u003e\u003cspan address=\"https://doi.org:10.1007/s00401-025-02871-w\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eSekiya, H. \u003cem\u003eet al.\u003c/em\u003e Wide distribution of alpha-synuclein oligomers in multiple system atrophy brain detected by proximity ligation. \u003cem\u003eActa Neuropathol\u003c/em\u003e 137, 455\u0026ndash;466 (2019). \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org:10.1007/s00401-019-01961-w\u003c/span\u003e\u003cspan address=\"https://doi.org:10.1007/s00401-019-01961-w\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eSekiya, H. \u003cem\u003eet al.\u003c/em\u003e Widespread distribution of α-synuclein oligomers in LRRK2-related Parkinson's disease. \u003cem\u003eActa Neuropathol\u003c/em\u003e 149, 42 (2025). \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org:10.1007/s00401-025-02872-9\u003c/span\u003e\u003cspan address=\"https://doi.org:10.1007/s00401-025-02872-9\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eSanderson, J. B. \u003cem\u003eet al.\u003c/em\u003e Analysis of α-synuclein species enriched from cerebral cortex of humans with sporadic dementia with Lewy bodies. \u003cem\u003eBrain Commun\u003c/em\u003e 2, fcaa010 (2020). \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org:10.1093/braincomms/fcaa010\u003c/span\u003e\u003cspan address=\"https://doi.org:10.1093/braincomms/fcaa010\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eLassen, L. B. \u003cem\u003eet al.\u003c/em\u003e ELISA method to detect α-synuclein oligomers in cell and animal models. \u003cem\u003ePLoS One\u003c/em\u003e 13, e0196056 (2018). \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org:10.1371/journal.pone.0196056\u003c/span\u003e\u003cspan address=\"https://doi.org:10.1371/journal.pone.0196056\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eJensen, P. H. \u003cem\u003eet al.\u003c/em\u003e Microtubule-associated protein 1B is a component of cortical Lewy bodies and binds alpha-synuclein filaments. \u003cem\u003eJ Biol Chem\u003c/em\u003e 275, 21500\u0026ndash;21507 (2000). \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org:10.1074/jbc.M000099200\u003c/span\u003e\u003cspan address=\"https://doi.org:10.1074/jbc.M000099200\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eJensen, P. H., Li, J. Y., Dahlstr\u0026ouml;m, A. \u0026amp; Dotti, C. G. Axonal transport of synucleins is mediated by all rate components. \u003cem\u003eEur J Neurosci\u003c/em\u003e 11, 3369\u0026ndash;3376 (1999). \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org:10.1046/j.1460-9568.1999.00754.x\u003c/span\u003e\u003cspan address=\"https://doi.org:10.1046/j.1460-9568.1999.00754.x\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eColla, E. \u003cem\u003eet al.\u003c/em\u003e Accumulation of toxic α-synuclein oligomer within endoplasmic reticulum occurs in α-synucleinopathy in vivo. \u003cem\u003eJ Neurosci\u003c/em\u003e 32, 3301\u0026ndash;3305 (2012). \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org:10.1523/jneurosci.5368-11.2012\u003c/span\u003e\u003cspan address=\"https://doi.org:10.1523/jneurosci.5368-11.2012\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eLiekniņa, I. \u003cem\u003eet al.\u003c/em\u003e Structural basis of epitope recognition by anti-alpha-synuclein antibodies MJFR14-6-4-2. \u003cem\u003eNPJ Parkinsons Dis\u003c/em\u003e 10, 206 (2024). \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org:10.1038/s41531-024-00822-y\u003c/span\u003e\u003cspan address=\"https://doi.org:10.1038/s41531-024-00822-y\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\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":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"npj-parkinsons-disease","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"npjparkd","sideBox":"Learn more about [npj Parkinson's Disease](http://www.nature.com/npjparkd/)","snPcode":"41531","submissionUrl":"https://submission.springernature.com/new-submission/41531/3","title":"npj Parkinson's Disease","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"NPJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"","lastPublishedDoi":"10.21203/rs.3.rs-7957852/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7957852/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eThe size-distribution of α-synuclein oligomers in brain cytosol from dementia with Lewy bodies (DLB) and control cases was investigated by combining size-exclusion chromatography and an aggregate specific MJFR14-6-4-2 ELISA. The oligomers in DLB peaked at 340 kDa and extended up to 2000 kDa. In controls, oligomers were smaller. Immunoblotting revealed the existence of pools of oligomers with different sensitivity to denaturation.\u003c/p\u003e","manuscriptTitle":"Higher molecular weight alpha-synuclein oligomers are increased in brain cytosol from dementia with Lewy bodies","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-11-13 14:50:35","doi":"10.21203/rs.3.rs-7957852/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-12-19T14:40:36+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-12-09T04:39:45+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-12-05T04:18:46+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"165839917240727739685914085323746121334","date":"2025-11-28T03:39:33+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"136681501206548950503367522486977560544","date":"2025-11-27T03:10:49+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-11-17T17:43:27+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"123804737915021906156965099361795170025","date":"2025-11-10T13:13:04+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-11-04T03:13:10+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-11-02T23:56:25+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-11-02T16:49:39+00:00","index":"","fulltext":""},{"type":"submitted","content":"npj Parkinson's Disease","date":"2025-10-26T16:29:29+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"npj-parkinsons-disease","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"npjparkd","sideBox":"Learn more about [npj Parkinson's Disease](http://www.nature.com/npjparkd/)","snPcode":"41531","submissionUrl":"https://submission.springernature.com/new-submission/41531/3","title":"npj Parkinson's Disease","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"NPJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"7d0269b4-8656-4236-b2e4-1cf9c2c43629","owner":[],"postedDate":"November 13th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[{"id":57732804,"name":"Biological sciences/Biochemistry"},{"id":57732805,"name":"Health sciences/Neurology"},{"id":57732806,"name":"Biological sciences/Neuroscience"}],"tags":[],"updatedAt":"2026-03-02T16:07:22+00:00","versionOfRecord":{"articleIdentity":"rs-7957852","link":"https://doi.org/10.1038/s41531-026-01301-2","journal":{"identity":"npj-parkinsons-disease","isVorOnly":false,"title":"npj Parkinson's Disease"},"publishedOn":"2026-02-28 15:58:51","publishedOnDateReadable":"February 28th, 2026"},"versionCreatedAt":"2025-11-13 14:50:35","video":"","vorDoi":"10.1038/s41531-026-01301-2","vorDoiUrl":"https://doi.org/10.1038/s41531-026-01301-2","workflowStages":[]},"version":"v1","identity":"rs-7957852","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7957852","identity":"rs-7957852","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}