Reconsidering α-Synuclein inclusion pathology in neurons, mice, and humans with an antibody sensing NAC engagement during α-Synuclein amyloid conversion

Reconsidering α-Synuclein inclusion pathology in neurons, mice, and humans with an antibody sensing NAC engagement during α-Synuclein amyloid conversion | 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 Method Article Reconsidering α-Synuclein inclusion pathology in neurons, mice, and humans with an antibody sensing NAC engagement during α-Synuclein amyloid conversion Francesca De Giorgi, Ænora Letourneur, Marianna Kashyrina, Federica Zinghirino, and 11 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-3921168/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract The neuropathology of α-Synucleinopathies (αSP) is characterized by the spread of subcellular inclusions containing fibrils made of stacked-up α-Synuclein (α-Syn) monomers. The repetitive amyloid fold adopted by α-Syn has now been characterized at the atomic scale. However, the direct observation of amyloid α-Syn using routine immuno-histological procedures remains an issue. In particular, the widely used phosphorylated α-Syn (pS129) is only a surrogate marker of aggregation. We report here that pS129 is misleading in overexpression-based models in which it detects the overflow of soluble α-Syn while no fibrillization takes place. Further, frequent pS129-negative α-Syn inclusions are observed when seeding with preformed fibrils (PFFs) is used to force fibrillization in neurons overexpressing α-Syn. This prompted us to scrutinize a series of routine antibodies for their genuine ability to discriminate α-Syn monomers engaged or not into amyloid fibrils, irrespective of phosphorylation. We observed unexpected antibody properties and utilized these latter in neurons and brain sections to detect the loss of accessibility of interlocked NAC domains when the monomers engage into fibrils. In cultured neurons, we observed that α-Syn mutations associated with familial Parkinson’s disease (PD), or S129A which prevents α-Syn phosphorylation, are neither sufficient to trigger spontaneous α-Syn fibrillization nor aggravate the process seeded by PFFs. Further challenging the pathogenic role of fibrillization, our results also indicated that the pS129-positive α-Syn inclusions detected in the brains of mice inoculated with PFFs and of a sporadic PD patient are not exclusively amyloid. This not only points to the notion that pS129 positivity is not tantamount to amyloid α-Syn but also indicates that the experimental α-Syn inclusions seeded in mice as well as the Lewy bodies forming in PD are populated by non-amyloid species which might represent alternative proxies of the α-Syn mutations endowed with a pathogenic potential. Neurobiology of Disease α-Synuclein amyloid fibrils detection methodology antibody Syn-1 Clone 42 NAC Parkinson’s disease Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Background In the group of neurogenerative diseases called α-Synucleinopathies (αSP), which includes Parkinson’s Disease (PD), Dementia with Lewy Bodies (DLB), and Multiple System Atrophy (MSA), the underlying molecular pathology is characterized by the amyloid aggregation of the presynaptic protein α-Synuclein (α-Syn)( 1 – 3 ). α-Syn is an intrinsically disordered protein that normally binds to the synaptic vesicle membranes as an α-helical oligomer and is thought to play a role in the modulation of neurosecretion. Initiating a pathological cascade, α-Syn can instead form multimeric assemblies in which the protein assumes a flat and rigid conformation with folded β-strands that is transmitted to the neighboring monomers by templating and progressive stacking, reminiscent of prions ( 4 , 5 ). Although the pathophysiological causes for the initiation of this process inside neurons remain unknown, experimental seeding of this conformational conversion by preformed assemblies has been well established ( 6 – 11 ). Amyloid aggregation of α-Syn can be experimentally achieved in vitro as a protein-only process using recombinant α-Syn, in spontaneous or seeded conditions, leading to highly structured fibrils ( 12 , 13 ). These assemblies most often appear as two intertwined protofilaments forming a fibril with an axial symmetry and are easily detectable with fluorescent probes that bind the surface grooves of the amyloid structure, among which the most used is Thioflavin T ( 14 ). The organization of α-Syn fibrils extracted from post-mortem brain tissue has been resolved at the atomic scale by cryoEM substantiating the existence of distinct amyloid fold polymorphs in the different αSP ( 15 , 16 ). From a neuropathological point of view, the α-Syn fibrils accumulate inside subcellular inclusions forming defined cytological objects (for instance: Lewy bodies LBs, Lewy neurites LNs, glial cytoplasmic inclusion GCI) ( 17 – 19 ). In about 50% of PD patients, the progression of the disease has been correlated with the apparent spread of these objects in new brain regions, leading to the definition of Braak staging ( 20 – 22 ). In addition, the sarkosyl-insoluble α-Syn fibrils extracted from DLB or MSA brain tissue can experimentally seed a de novo α-Syn pathology made of identifiable inclusions in the brain of non-transgenic mice( 10 ) ( 23 ): α-Syn fibrils are thus considered to be the cause of spread and of disease progression ( 4 ). However, the mechanisms linking amyloid α-Syn spread and neurodegeneration are unclear, and it has thus been proposed that neurodegeneration could be associated with other non-fibrillar (non-amyloid) forms of assemblies referred to as oligomers ( 24 ). Yet their structure, their presence/distribution in the brain, and their ability to template their own growth and spread remain elusive. Several cell-based or animal models, either based on α-Syn overexpression or on the induction of a seeded aggregation by α-Syn fibril injections have been set up, with the aim of understanding the biological mechanisms linking α-Syn amyloid conversion and fibril formation to neuronal failure and death ( 25 ). However, addressing genuine amyloid conversion of α-Syn in a cellular context remains challenging. Moreover, in contrast to in vitro conditions using pure recombinant α-Syn, the conversion of α-Syn from a monomeric to a structured supramolecular amyloid state is certainly a much more complex process inside neurons, since it can be affected by interactions with other proteins and lipids, post-translational modifications, and transport mechanisms. In addition, inside neurons and glial cells aggregated α-Syn is processed by a variety of catabolic pathways like autophagy, proteasomal degradation, and the disaggregase chaperone complex ( 26 – 28 ). All these elements, by limiting or contributing to the pathology, could potentially represent druggable targets for the development of therapeutic strategies. Thus, the ability to specifically detect amyloid α-Syn in experimental cellular or animal models and to differentiate amyloids from the other forms of the protein is a key step required to evaluate candidate targeted therapeutics aimed at reducing αSP. In histopathology, a frequently used approach to detect aggregated α-Syn in patient tissue is the use of antibodies that can specifically detect the phosphorylated form of α-Syn at serine 129 (pS129) ( 29 ). This post-translational modification is considered a reliable surrogate marker of amyloid α-Syn since it has been shown that α-Syn assemblies extracted from patients are phosphorylated at S129 and that the distribution of phosphorylated α-Syn in patient brain sections is restricted to the pathological inclusions ( 30 ). Beyond synaptic staining, general α-Syn antibodies can also stain LB, GCI, and LN, which are identifiable by their simple morphological features due to the overwhelming concentration of the protein inside the inclusions. However, in all these cases, the amyloid state of α-Syn is only secondarily inferred. A more specific method involves the use of conformational antibodies with a higher affinity for fibrils than for other forms of the protein. However, their preference for high molecular weight α-Syn assemblies is relative ( 31 ), and the main discriminating factor remains their ability to highlight typical cytopathological inclusions. Detection and identification of such bona fide inclusions is unfortunately an issue in several preclinical models. In other words, while in the context of human disease α-Syn pathology is strictly defined as the presence of delineated subcellular inclusions, certain animal models do not reproduce them ( 32 – 36 ). To accommodate this limitation, it has been proposed that in these models pathological α-Syn could take the prodromal appearance of diffuse anatomical-scale increases of the pS129 signal analyzable by global thresholding and/or of regional resistances of the diffuse α-Syn immunoreactivity to treatment of the sections with proteases ( 35 , 36 ). However, these types of signs do not belong to the clinical neuropathology of αSP which exclusively relies on the individuation and the count of inclusions for the scoring of Lewy and MSA pathology ( 19 ). The opposite problem can be encountered in α-Syn overexpressing models in which seeded aggregation can burst without the formation of well-delineated inclusions because the ability of the neurons/oligodendrocytes to constrain the pathological assemblies in confined cytoplasmic regions is overwhelmed (see results). Without seeding, the situation is even worse: overexpressed α-Syn can remain soluble, overflow the entire cytoplasm, and get phosphorylated without forming any fibrils (see results). This results in the genesis of images that can erroneously be interpreted as the emergence of inclusions mimicking the human pathology ( 37 ). In line with our previous observations made in mice overexpressing α-Syn under the control of the PLP promoter ( 37 ), our experiments using neuronal primary cultures and mice infected with AAV-human α-Syn (hSyn) to model αSP, indicated that S129 phosphorylation most often turns out to be a false positive marker of α-Syn aggregation in overexpression conditions. To overcome this difficulty, we thus tried to find an alternative way to detect amyloid α-Syn unambiguously. To do so, we tested a set of commercially available antibodies widely employed in the literature. Unexpectedly, we stumbled upon neglected antibody features and built upon these properties to derive markers of α-Syn conversion into fibrils suitable for experimental and clinical neuropathology studies. Methods Antibodies Primary antibodies were used as follows: IF in vitro IF in PFFE sections IHC Biochemical assay EP1536Y Abcam ab51253 1:500 1:500 1:5000 1/5000 MJFR-1 Abcam ab138501 1:1000 1:1000 1:5000 1/10000 Alexa 488 MJFR-1 Abcam ab195025 1:500 1:500 n/a n/a SynF1 BioLegend 847802 1:500 n/a n/a 1/10000 D37A6 Cell Signaling, #4179 1:500 n/a n/a 1/2000 Syn1 Clone 42 BD Biosciences 610787 1:500 1:500 n/a 1/2000 EP1646Y Abcam ab51252 1:500 n/a n/a 1/5000 MJFR14 Abcam ab227047 1:500 n/a n/a 1/10000 LB509 ThermoFisher #180215 1:500 n/a 1/2000 Syn505 ThermoFisher #35-8300 1:500 n/a n/a 1/2000 EP1532Y Abcam ab137869 n/a n/a 1:5000 n/a α-Syn expression and purification E. coli strain BL21(DE3) plysS was chemically transformed with pT7-7-α-Syn vector and plated onto Lysogeny broth (LB) agar plate containing Ampicillin. A pre-culture in 5 mL LB medium was inoculated with one clone and incubated at 37°C under 200 rpm shaking for 4 hours. The expression on α-Syn was carried out in LB Medium + Glucose (1 g/L). Cells from LB pre-culture were used for inoculating 100 mL of LB medium. Cells were grown overnight at 37°C under 200 rpm shaking and then diluted in 2.5 L of culture. Protein expression was induced by adding 1 mM IPTG during exponential phase, evaluated by Optical Density at 600 nm reaching 0.6. Cells were harvested after 5 hours of culture at 30°C by 4,000*g centrifuge (JLA 8.1 Beckman Coulter) and pellet was kept at -20°C before purification. The pellets were resuspended in lysis buffer (10 mM Tris and 1 mM EDTA (pH 7.2)) and sonicated at 50% max energy, 30 sec on and 30 sec off for three rounds with a probe sonicator (Q-Sonica, Newtown, CT, USA). The sonicated pellets were centrifuged at 20,000× g for 30 min, and the supernatant was saved. The pH of the supernatant was then reduced to pH 3.5 using HCl, and the mixture stirred at room temperature (RT) for 20 min and then centrifuged at 60,000× g for 30 min. The pellets were discarded. The pH of the supernatant was then increased to pH 7.4 with NaOH and then dialyzed against 20 mM Tris-HCl (pH 7.40) and 100 mM NaCl buffer before loading onto a 75 pg HiLoad 26/600 Superdex column equilibrated with the same buffer with ÄKTA pure system. Monomeric fractions were collected and concentrated if needed by using Vivaspin 15R 2 kDa cutoff concentrator (Sartorius Stedim, Göttingen, Germany). Purification fractions were checked by using polyacrylamide gel electrophoresis (PAGE) Tris-tricine 13% dying with ProBlue Safe Stain. Protein concentration was evaluated spectrophotometrically by using absorbance at 280 nm and an extinction coefficient of 5960 M − 1 cm − 1 . α-Syn fibrillization Solutions of monomeric α-Syn at 4 to 5 mg/mL in saline (H 2 O, 100 mM NaCl, and 20 mM Tris-HCl (pH 7.40)) were sterilized by filtration through 0.22 µm Millipore single-use filters and stored in sterile 15 mL conical falcon tubes at 4°C. Sterilized stock was then distributed into safe-lock Biopur individually sterile-packaged 1.5 mL Eppendorf tubes as 500 µL aliquots. The tubes were cap-locked and additionally sealed with parafilm. All previous steps were performed aseptically in a particle-free environment under a microbiological safety laminar flow hood. The samples were loaded in a ThermoMixer (Eppendorf, Hamburg, Germany) in a 24-position 1.5 mL Eppendorf tube holder equipped with a heating lid. Temperature was set to 37°C, and continuous shaking at 2000 rpm proceeded for 4 days. Different polymorphs were obtained as described in ( 14 ). Human α-Syn AAV particles Recombinant AAV9-CMVie/SynP-syn-WPRE vectors containing the sequence of hSyn, wild-type (wt) or presenting the targeted mutation put under control of the human synapsin promoter was produced by polyethylenimine (PEI) mediated triple transfection of low passage HEK-293T /17 cells (ATCC; cat number CRL-11268). The AAV (adeno-associated virus) expression plasmids pAAV2-CMVie/hSyn-syn-WPRE-pA were co-transfected with the adeno helper pAd Delta F6 plasmid (Penn Vector Core, cat # PL-F-PVADF6) and AAV Rep Cap pAAV2/9 plasmid (Penn Vector Core, cat # PL-T-PV008). Cells are harvested 72h post transfection, resuspended in lysis buffer (150 mM NaCl, 50 mM Tris-HCl pH 8.5) and lysed by 3 freeze-thaw cycles (37°C/-80°C). The cell lysate is treated with 150 units/ml Benzonase (Sigma, St Louis, MO) for 1 hour at 37°C and the crude lysate is clarified by centrifugation. Vectors are purified by iodixanol step gradient centrifugation and concentrated and buffer-exchanged into Lactated Ringer's solution (Baxter, Deerfield, IL) using vivaspin20 100kDa cut off concentrator (Sartorius Stedim, Goettingen, Germany).The genome-containing particle (GCP) titer was determined by quantitative real-time PCR using the Light Cycler 480 SYBR green master mix (Roche, cat # 04887352001) with primers specific for the AAV2 ITRs (fwd 5′-GGAACCCCTAGTGATGGAGTT-3′; rev 5′-CGGCCTCAGTGAGCGA-3′) on a Light Cycler 480 instrument. Purity assessment of vector stocks was estimated by loading 10 µl of vector stock on 10% SDS acrylamide gels, total proteins were visualized using the Krypton Infrared Protein Stain according to the manufacturer’s instructions (Life Technologies). In vitro α-Syn Pathology Timed pregnant C57BL/6J female mice were bred at the animal facility of the IMN. Cortices were harvested from embryonic day 18 mouse embryos and dissociated enzymatically and mechanically (using neural tissue dissociation kit, C Tubes, and an OctoDissociator with heaters; Miltenyi Biotec, Bergish-Gladbach, Germany) to yield a homogenous cell suspension. The cells were then plated at 25,000 cells per well in 96-well plates (Corning BioCoat poly-Dlysine imaging plates) in neuronal medium (MACS Neuro Medium, Miltenyi Biotec, BergishGladbach, Germany) containing 0.5% penicillin-streptomycin, 0.5 mM alanyl-glutamine, and 2% NeuroBrew supplement (Miltenyi Biotec, Bergish-Gladbach, Germany). The cultures were maintained with 5% CO 2 at 37°C in humidified atmosphere. The medium was changed by one-third every 3 days, until a maximum of 30 DIV (days in vitro ). After 7 DIV, extemporaneously sonicated α-Syn fibrils were added at a final concentration of 10 nM (equivalent monomeric α-Syn concentration). When relevant, neurons were infected at DIV 10 with α-Syn AAV particles (multiplicity of infection, 1000). High Content Analysis (HCA) and Laser-Scanning Confocal Microscopy (LSCM) At 30 DIV the plates were processed for immunofluorescence. When relevant, the wells were treated 10 minutes with 25 µM digitonin in 100 mM KCl prior to fixation. All plates were fixed with a solution of 4% formaldehyde 4% sucrose in Phosphate Buffered Saline (PBS) pH 7.4. The cells were then permeabilized with a solution of 3% Bovine Serum Albumin (BSA), 0.1% Triton X in PBS and incubated overnight in a primary antibody solution in BSA 3%, and the next day with the corresponding secondary antibody in BSA 3%. High Content Analysis was performed on multichannel fluorescence images acquired 20× using the generic analysis module of the Incucyte S3 (Sartorius) and Top-Hat cellular segmentation was based on the fluorescence signal corresponding to the antibody of interest. Multichannel fluorescence optical sections of the samples were performed (thickness < 0.8 µm) using a Leica SP5 LSCM equipped spectral detector, with 488, 561 and 633 nm laser lines, with a motorized X-Y stage and with a mixed stepping motor/piezo Z controller. Urea treatments of recombinant α-Syn monomers and PFFs Recombinant α-Syn monomers and fibrils stock preparations (4–5 mg.ml-1 in Tris-Buffered Saline (TBS)) were diluted to 0.5 mg/ml final concentration in TBS. For each urea concentration, 3 µL (1.5 µg) of recombinant monomers or PFFs were mixed with urea (Sigma) in TBS to reach a final concentration of 0–8 M urea in 60 µl. Mixtures were pipet mixed prior to incubation for 2h at RT in the dark. At the end of the incubation period, treatments were stopped by quickly diluting the samples with 1260 µL TBS and directly subjecting them to filter-blot assay (science advance + companion paper). Briefly, 100 µl of treated monomers/PFFs were filtered through a nitrocellulose 0.2 µm membrane (Protran, GE) using a dot blot vacuum device (Whatman). Membranes were fixed for 30 min at RT in PBS with paraformaldehyde (PFA) (Sigma) 4% (v/v) final concentration. After three washes with PBS, membranes were saturated with 5% (w/v) skimmed powder milk in PBS-Tween20 0.05% (v/v) and probed with primary (overnight at 4°C) and secondary (1 hour at RT antibodies in PBS-T with 4% (w/v) BSA (see antibody list) with three washes in PBS-T after each step. Immunoreactivity was measured by infrared using an Odyssey Scanner and Image Studio (Li-Cor). The different α-Syn species were quantified by immunolabelling, and expressed as a percentage of related untreated samples, allowing to draw curves of relative disassembly. Sarkosyl fractionation of human brain samples homogenates Human cingulate gyrus samples were dissected from freshly frozen post-mortem brain samples from n = 3 control, sporadic PD or MSA subjects respectively. Brain tissue samples were homogenized at 10% (w/v) in solubilization buffer (SB): 10 mM Tris pH 7.5, 100 mM NaCl, 0.1 mM EDTA, 1 mM DTT, Complete EDTA-free protease inhibitors (Roche) and PhosSTOP phosphatase inhibitors (Roche) using a gentleMACS Octo Dissociator (Miltenyi Biotec) with M Tubes, and the Protein extraction program. Protein concentration was determined using Pierce 660 nm Protein Assay kit (Thermo Fisher). For the fractionation of sarkosyl-soluble and insoluble components of brain homogenates, the pelleting procedure is similar to previously published protocols termed Sarkospin [see companion paper Laferrière et al.]. Samples were mixed 1:1 with SB 4% (w/v) N-lauroyl-sarcosine (sarkosyl, Sigma), 2 U.µl-1 Benzonase (Novagen) and 4 mM MgCl 2 , reaching a final volume of 500 µl. Solubilization was then performed by incubating the samples at 37°C under constant shaking at 600 rpm (Thermomixer, Eppendorf ) for 45 min. Samples were then mixed 1:1 with SB 40% (w/v) sucrose, without sarkosyl, MgCl 2 or Benzonase, in 1 ml polycarbonate ultracentrifuge tubes (Beckman Coulter) and centrifuged at 250,000 g for 1 hour at RT with a TLA 120.2 rotor using an Optima XP benchtop ultracentrifuge (Beckman Coulter). Supernatant were collected by pipetting. Pellets were resuspended directly in the tube with 100 µL of the buffer corresponding to the supernatant (SB 1% sarkosyl 20% sucrose), and mixed with the same buffer in a fresh tube for reaching 1 ml (equal volumes to supernatant). For filter-blot assays, 50 µl of native supernatant/pellet fractions were loaded on the filter-blot mounting and immunolabelled and quantified as described above. The signal for each primary antibody is expressed and plotted as percentage fraction/total (supernatant + pellet). In vivo human α-Syn overexpression and in vivo α-syn Pathology Wt mice (2 months old) received unilaterally 1 µl of -hSyn AAV (concentration: 4.05 × 1013 gcp/ml) mixed either with 1 µl of sonicated α-Syn fibrils (5 mg/ml) or with 1µl of saline by stereotactic delivery to the region immediately above the right Substantia nigra (SN) (coordinates from bregma: AP, − 2.9, L, − 1.3, DV, − 4.5) at a flow rate of 0.4 µl/min, and the pipette was left in place for 5 minutes after injection to avoid leakage. Animals were euthanized after 4 months. Ten mice were used in each group — male and female mixed. The brains were perfused with saline, postfixed for 3 days in 10 ml of 4% PFA at 4°C, cryoprotected in gradient 20% sucrose in PBS before being frozen by immersion in a cold isopentane bath (–60°C) for at least 5 minutes, and stored immediately at − 80°C until sectioning for immunohistochemistry (ICH). Immunohistochemistry in mouse brains sections IHC staining of phospho-S129 α-Synuclein (pS129 α-Syn) and tyrosine hydroxylase (TH) neurons on coronal serial sections was performed as previously described ( 38 ). The monoclonal rabbit anti-pS129 α-Syn antibody EP1536Y (ab51253, Abcam, Cambridge, UK) was used, followed by incubation with labelled polymer-HRP anti-rabbit (Dako EnVision + TM Kit, K4011, Agilent, Santa Clara, CA, USA). Visualization of pS129 α-Syn staining was performed with Dako DAB (K3468), and sections were counterstained with the Nissl stain. The actual number of pS129 α-Syn aggregates per structure ( Striatum and SN) and the total number of pS129 α-Syn aggregates were assessed using whole-section acquisition by Panoramic Scan II (3DHISTECH, Hungary) and further processed with the ad-hoc developed QuPath algorithm. For TH neurons quantification, ICH was performed for each animal on every fourth midbrain sections spanning the entire rostro-caudal Substantia Nigra pars compacta (SNpc). TH-positive cells in ipsilateral and controlateral SN were segmented and counted on each section using Qpath and the loss between the injected and the non-injected side was calculated for each animal. Immunofluorescence in FFPE mouse and human brain sections. Adult male 129SV (6–8 weeks old) unilaterally received 2 µL of sonicated α-Syn fibrils (4 mg/mL) by stereotactic delivery at a flow rate of 0.4 µL/min, and the pipette was left in place for 5 min after injection to avoid leakage. Delivery was performed within the right Striatum (AP, − 0.1; L, + 2.5; DV, + 3.8). Animals were euthanized after 6 months and were transcardially perfused with TBS (pH = 7.4) followed by 4% PFA in PBS pH = 7.4 at 4°C. Brains were subsequently postfixed in the same fixative, paraffin embedded, and 10 µm sections were obtained with a rotative microtome (Leica, Milan, Italy). The sections of interest were deparaffinized and processed for epitope retrieval: the slides were immersed in citrate buffer pH 6 (Dako Agilent Technologies, Les Ulis, France) and placed in a TintoRetriever Pressure Cooker (Bio SB, Santa Barbara, CA, USA) at high pressure, 114–120°C for 10 min. After a cooling period of 20 min, the slides were washed twice for 5 min in PBS at RT. They were then processed for immunofluorescence. Draq7 Thermo Fisher Scientific was used to image nuclei. The Alexa Fluor-coupled secondary antibodies were from Thermo Fisher (Alexa 488, 568, and 674). The sections were acquired using a Pannoramic slide scanner (3D HISTECH, MM France) in epifluorescence mode, and multichannel fluorescence optical sections of the samples were performed (thickness < 0.8 µm) using a Leica SP5 Laser Scanning Confocal Microscope. Human PD brain sections were deparaffinized and processed as described above and sequentially stained with (i) Syn1 (Clone 42, BD), and EP1536Y (Abcam) and revealed with the respective secondary antibodies, and only then with (ii) Alexa Fluor-coupled MJFR1. Draq7 is used according to the manufacturer’s instructions. Multichannel fluorescence optical sections of the samples were performed (thickness < 0.8 µm) using a Leica SP5 Laser Scanning Confocal Microscope equipped with a spectral detector, 488, 561, and 633 nm laser lines, a motorized X-Y stage, and a mixed stepping motor/piezo Z controller. Results α-Syn overexpression using AAVs: filling of the neuronal cytoplasm and pS129 positivity without assembly of fibrils and without inclusions. The overexpression of hSyn is commonly used to model αSP in mouse neurons, both in vitro and in vivo . It is supposed to facilitate the emergence of α-Syn aggregation by bringing its concentration closer to the nucleation threshold. It also allows studying the human protein in a rodent context, tentatively increasing the translational relevance of the model. Several studies reported the induction of α-Syn aggregation by simple overexpression, others reported that aggregation must be induced by an additional seeding event such as PFFs treatment ( 37 ). In both cases, the discrimination between the adverse effects due to the increased α-Syn expression levels and those due to the proper amyloid aggregation are difficult to determine and relies on the capability of specifically identifying amyloid α-Syn fibrils in situ . pS129 is a very specific marker of α-Syn inclusions in human neuropathology( 30 )( 39 ) ( 40 ). Similarly, when α-Syn PFFs are used as seeds and directly injected into the brain of wt mice, pS129 antibodies specifically allow the visualization of the α-Syn inclusions that develop and spread into the mouse brain( 9 )( 10 ) ( 41 ). Four months after an injection of PFFs at the level of the SN, several types of pS129-positive (pSyn) inclusions formed that were reminiscent of those observed in patients, both locally in the SN and at distance in the Striatum of the same hemisphere (Fig. 1 A-E). This experimental αSP phenotype can also be observed after the intracerebral injection in mice of α-Syn fibrils extracted from DLB patient brain samples using detergent, with the formation of typical pS129-positive α-Syn inclusions ( 10 ). Note, however, that in agreement with the latter publication, we found that 4 months after injection, the αSP seeded by PFFs was not associated with any specific loss of TH-positive neurons in the SNpc. Indeed, the loss observed, which was around 28% compared to the non-injected side, was not different in sham-injected controls which received an injection of saline (Fig. S1). For the sake of comparison, we injected AAV particles carrying the cDNA of hSyn under the control of a synapsin promoter (AAV-syn) to induce an overexpression of hSyn in the SN neurons (Fig. S1). In these AAV-injected animals, hSyn is readily detectable at 4 months post-injection in the cell bodies of the dopaminergic neurons populating the SN as well as in their striatal projections/terminals (Fig. S1). In these animals, bona fide intracellular inclusions cannot be identified inside the somata of the dopaminergic neurons (the cell bodies become globally/diffusely pS129-positive), and most strikingly, distant striatal pS129-positive inclusions are absent (Fig. 1 A-E). Such lack of long-distance spread as well as the diffuse perikaryal morphology of the pS129 signal is reminiscent of previous in vivo experiments in which α-Syn overexpression resulted in S129 phosphorylation without fibrillar aggregation ( 37 ). Note that 4 months after the intracerebral injection of AAV-syn, on top of the absence of αSP inclusions, no specific loss of TH-positive neurons was detected in the SNpc. Compared to the non-injected side, the loss observed was identical to sham-injected controls (Fig. S1). In the last experiment, we mixed AAV-syn particles with PFFs and injected them at the level of the SN. In these “seeded plus AAV-infected” animals, hSyn was readily detectable in the cell bodies of the dopaminergic neurons of the SN as well as in their striatal projections/terminals (Fig. S1). While bona fide intracellular inclusions were still not identified inside the somata of the dopaminergic neurons (here also the cell bodies were globally/diffusely pS129-positive), distant striatal pS129 positive inclusions were present like in the PFF-only condition (Fig. 1 A-E). Induction of an inclusion pathology with long-distance spread by adding PFFs to AAV-syn particles clearly indicates that the processes triggered by AAV-syn and PFFs are completely different (compare “AAV-Syn” and “AAV-syn + PFFs” in Fig. 1 C), yet they are both associated with the emergence of a pS129-positive signal at the injection site (compare “AAV-Syn” and “PFFs” in Fig. 1 B). This indicates that S129 phosphorylation is a misleading marker in conditions of neuronal α-Syn overexpression in vivo . Note that here also, 4 months after the intracerebral injection of the AAV-syn plus PFFs mix, no specific loss of TH-positive neurons was detected in the SNpc. The loss observed compared to the non-injected side was identical to sham-injected controls (Fig. S1). In parallel to these in vivo experiments, we evaluated the relationships between α-Syn overexpression, phosphorylation, and amyloid aggregation in primary cultures of cortical neurons infected or not with AAV-syn and seeded or not with PFFs. In line with the in vivo experiment, pS129 was not detected in non-infected control neurons and only appeared upon seeding with PFFs. Like in αSP pathology, the seeded structures which are positive for pS129 are bona fide cytological inclusions (LNs, perinuclear and intranuclear inclusions (Fig. 2 A,B). In addition, pS129 staining was fully resistant to permeabilization of the plasma membrane with digitonin prior to fixation, indicating that it corresponds to large insoluble α-Syn assemblies – i.e., fibrils – unable to diffuse out of the neurons through the digitonin pores (Fig. 2 C,D). In contrast, in non-seeded conditions, AAV-syn-infected neurons already showed a diffuse pS129 signal with no identifiable inclusion (Fig. 2 A,B). At major variance from the previous experiment, this diffuse staining was completely lost upon membrane permeabilization, indicating that S129 phosphorylation concerned in this case soluble α-Syn species that can freely diffuse out of the neurons and not insoluble fibrils (Fig. 2 C,D). However, we observe that if the AAV-syn-infected neurons were also seeded with PFFs, pS129 staining became fully resistant to permeabilization revealing that fibril assembly could still be seeded at the expense of the soluble pS129 α-Syn species that were formed in reaction to the overexpression overflow (Fig. 2 C,D). These data show that in conditions of α-Syn overexpression, pS129 cannot be considered a reliable surrogate marker of neuronal α-Syn fibrillization and aggregation. In addition, even if the pS129 signal integral is increased by seeding (Fig. 2 A-D), phosphorylation is not discriminant per se : phosphorylated fibrils cannot be distinguished from the phosphorylated soluble species “artifactually” emerging in these neurons. To identify aggregated α-Syn more specifically in these experiments we tested the routine conformational antibody LB509. LB509 has a double selectivity, detecting hSyn mostly under its aggregated form. We found in our conditions that this conformational preference was very relative - that is, in hSyn overexpressing conditions (AAV-syn) LB509 detects non-aggregated α-Syn in neurons, particularly inside the synapses. This staining is completely lost upon plasma membrane permeabilization prior to fixation confirming its non-fibrillar nature. When such AAV-syn infected neurons were seeded with PFFs, aggregation could only be inferred from morphological changes and not be quantified by measuring an increase of the LB509 signal integral in the intact cells (Fig. 2 E). The fibrillar status of the assemblies seeded by PFFs and recognized by LB509 could, however, be revealed by their resistance to plasma membrane permeabilization (Fig. 2 E). Interestingly, by comparing the signal distribution of pS129 and LB509 by double immunofluorescence in the latter permeabilized neurons, we observe that the signals are not completely overlapping (Fig. 2 F). This indicates that pS129-positive amyloid α-Syn fibrils coexist with unphosphorylated fibrils, and that some pS129-positive fibrils are not discovered by LB509. This suggests a conformation and phosphorylation heterogeneity of the C-terminus (the target of both antibodies) belonging to the monomers engaged into the amyloid fibril core. In conclusion, in experimental models introducing and manipulating the concentration of hSyn monomers in neurons, pS129 cannot be considered a reliable marker of aggregation because overexpressed α-Syn is phosphorylated under its soluble form. Appropriate tools are needed for identifying amyloid assemblies, which are less affected by the detection of the basal overexpression and targeting the proper amyloid conversion. Analysis of a set of commercial antibodies reveals overlooked yet interesting features. Using recombinant hSyn we produced and selected 3 different strains of PFFs which we characterized in previous studies: iso1, iso3, and 1B ( 14 ). For this study, we tested a panel of “routine” commercial antibodies on these different PFFs to investigate their possible ability to differentially detect the fibrils strains and the monomeric protein (Fig. 3 A). As expected, MJFR1 ( 42 ) and EP1646Y, respectively targeting the C- and the N-terminus of the protein, did not show any preference for the monomeric or the fibril α-Syn assemblies. MJFR14 which has been described as preferentially binding to fibrils ( 43 ) did not show any conformation specificity in our experimental settings. In contrast, LB509 ( 44 ) which targets the C-terminus ( 45 ) recognized both α-Syn forms, but with a clear preference for fibrils. A similar conformation-dependent preference was observed for Syn505 which binds to the 1–12 end of the N-terminus ( 46 ), and SynF1 which is targeted to the C-terminus ( 47 ) appeared most selective of amyloid fibrils vs. monomers. While these results are globally in line with the expectations (excepted for MJFR14), we identified two particularly striking – yet overlooked – properties for 2 antibodies widely used in the literature: (i) Syn1 (Clone 42) which targets the NAC region of the protein( 48 ) is often referred to as a “pan-α-Syn” antibody held capable of recognizing all forms of the protein. In fact, we found that it selectively recognized the monomeric protein and that it did not bind to the fibrils. This confirms repeated previous observations made by us and others ( 14 , 49 , 50 ); (ii) D37A6 which is held a rodent specific antibody targeting an upstream region of the C-terminus (surrounding E105), did indeed not recognize the human monomer. However, and most strikingly, it did show an affinity for hSyn engaged into amyloid fibrils. To further explore these conformation specificities, we used increasing concentrations of urea to provoke the gradual disassembly of the 3 fibrils strains back into monomers and to measure the impact of disassembly on immunoreactivity (Fig. 3 B). In agreement with the previous experiment (Fig. 3 A), disassembly did not modify the signal of the conformation-independent antibodies MJFR1, EP1646Y and MJFR14, while it collapsed the signal of all the fibril-specific antibodies which we identified, i.e., LB509, Syn505, SynF1 and D37A6. As a mirror image, progressive fibril disassembly led to the progressive appearance of Syn1 immunoreactivity as more monomers were released from the amyloid assemblies, confirming the monomer-selectivity of Syn1. Finally, we tested the above antibodies on PD and MSA brain homogenates comparing their sarkosyl insoluble and soluble fractions (Fig. 3 C and Fig. S3). The results confirm that in a biochemical assay, LB509, SynF1 and Syn505 recognize pathological α-Syn and showed that Syn1 and D37A6 behave as amyloid state-dependent antibodies. Note that to our knowledge, changes in immunoreactivity towards Syn1 is the first amyloid conversion-dependent change of α-Syn taking place at the proper fibril core level and amenable to detection by an immunological method. Amyloid conversion assays in neurons Considering these results on synthetic and extracted fibrils, we explored the use of the antibodies which we retained conformation-dependent (see above) in primary cultures of mouse cortical neurons overexpressing hSyn. We first performed double-immunofluorescence imaging using SynF1 or Syn505 in combination with EP1536Y which detects pS129 α-Syn. To discriminate aggregated α-Syn and its soluble forms, we performed the same staining on neurons permeabilized prior to fixation (Figs. 4 A,B). Both antibodies confirmed their preferential affinity for aggregated α-Syn, the quantification of the signal integral showing a significant increase for each of the antibodies in the PFF-treated neurons compared to the control conditions (Fig. 4 A). SynF1 was also sensitive to overexpression, but the differential intensity between PFF-treated and untreated conditions was larger than with LB509 (Compare Fig. 2 E and Fig. 4 A,B). The signal shown by Syn505 in these experimental conditions was generally weak but provided a more clearcut readout of α-Syn aggregation in the PFF-treated neurons. For both antibodies, permeabilization of the neurons prior to fixation, which allowed the retention of aggregated α-Syn and the release of the non-aggregated forms, improved the difference between the PFF-treated and non-treated conditions, providing a “filtered/contrasted” image of the α-Syn pathology (Figs. 4 A). However, when we considered pS129 co-staining with EP1536Y, we observed that similarly to what we noted with LB509, the neurons positive to the conformational antibodies SynF1 or Syn505, and those positive to EP1536Y only partially overlapped, both in non-permeabilized and permeabilized conditions (Fig. 4 B). This suggested that pS129 is not a comprehensive readout of aggregated α-Syn, since in some neurons or in some areas of the same neuron, aggregated α-Syn is not phosphorylated. This could be due to a difference in neuronal types, or, in the second case, it could be due to the “snapshot” of an evolving process inside a single neuron (progressive phosphorylation or dephosphorylation of the aggregates). On the other hand, we also observed neurons bearing pS129-positive aggregates that were insoluble, yet were negative to the conformational antibodies SynF1 or Syn505 (Fig. 4 B). Aiming at detecting/quantifying amyloid conversion in intact neurons irrespective of pS129 we decided to exploit the conformational properties of the Syn1 antibody. Again, the epitope targeted by this antibody (aa91-99) is part of the NAC domain of the protein which gets engaged and is instrumental in the protein-protein interactions established between stacked α-Syn monomers during assembly of the amyloid core. As a result, once the monomers are trapped inside the fibrillar structure, the epitope is no longer accessible to the antibody. Indeed, when we treated AAV-syn-infected neurons with PFFs we observed the appearance of inclusions with a collapse of Syn1 immunoreactivity in inclusions yet positive to MJFR1 (Fig. 4 C). Thus, using a double staining with a conformation-independent antibody like MJFR1 (or EP1646Y, not shown) in combination with Syn1, we can easily differentiate soluble α-Syn from amyloid α-Syn assemblies and identify directly the neurons containing fibrils (Fig. 4 C). Indeed, in neurons not seeded with PFFs, MJFR1 and Syn1 staining completely overlapped and both signals disappeared if the neurons were permeabilized prior to fixation. This indicates that normal neurons only contain soluble, non-amyloid α-Syn. When neurons were seeded with PFFs instead, a Syn1 negative MJFR1-positive population appeared which was unsensitive to permeabilization, corresponding to the neurons in which α-Syn underwent amyloid conversion (Fig. 4 C). Note that amyloid conversion can similarly be tracked using EP1536Y to reveal pS129 α-Syn together with Syn1 (Fig. 4 D). In this case, amyloid α-Syn is EP1536Y-positive and Syn1-negative. In conclusion, pS129 is a surrogate marker of α-Syn aggregation that can generate both false positives and false negatives, especially in condition of α-Syn overexpression. The commercial conformation-dependent antibodies we tested and that do bind preferentially to aggregated α-Syn present only a relative specificity and can miss a significant fraction of the amyloid α-Syn fibrils present in neurons (false negatives). In other words, they are not able to properly discriminate aggregation in situ . Moreover, it should be highlighted that these conformation-dependent antibodies target the arrangements the N or the C terminals which derive only secondarily from the aggregation process and are not involved in the assembly of the amyloid core. In contrast, the loss of accessibility of the NAC epitope recognized by Syn1 is a direct indication of the enrollment of the protein in an amyloid structure and can thus be used in combination with conformation-independent antibodies to specifically highlight amyloid α-Syn assemblies in situ. High Content Analysis amyloid α-Syn conversion in 96 well primary neuronal cultures: revisiting the impact of α-Syn mutations. We thus decided to take advantage of this new method for investigating a series of α-Syn mutations – either disease-relevant or preventing S129 phosphorylation – to determine their propensities to act as a stand-alone trigger of α-Syn amyloid conversion inside neurons and/or to accelerate the process once seeded by PFFs (Fig. 5 ). We infected neurons with 7 distinct AAVs carrying the cDNA of hSyn bearing point mutations that have been associated with the emergence of autosomal-dominant inherited PD: A29E, A30P, E46K, G51D, H50Q, A53E, A53T ( 51 ). The mechanisms that link these mutations to PD are generally thought to depend on a facilitation of the amyloid assembly of α-Syn, but although appealing, this assumption is mostly based on historical protein-only observations made in vitro . We also included in our exploration the experimental S129A mutant, coding for a non-phosphorylatable form of the protein at S129 because here also, there is a debate on the functional impact of this phosphorylation on the process of spontaneous or of seeded α-Syn assembly ( 52 – 54 ). Using MJFR1 we quantified the levels of neuronal overexpression achieved for variants and found that the different AAV infections yielded comparable expression levels, except for A30P which was slightly less expressed than its counterparts, and for E46K which was barely expressed and detectable (this observation was repeated with different AVV-E46K α-Syn production batches, not shown) (Fig. S5). In addition, expression of E46K appeared to be neurotoxic in our primary cultures of cortical neurons, confirming previous observations ( 55 ). We thus did not consider or discuss the possible impact of the E46K mutation on the amyloid conversion of α-Syn in situ . Figure 5 A shows that as it was the case for wt hSyn, all disease-associated mutants were significantly phosphorylated in basal conditions under the simple effect of overexpression (compare with S129A which cannot be phosphorylated on this residue). The phosphorylation level in the unseeded conditions was comparable for all disease-associated mutants. Upon seeding with PFFs, a modest 2–3 fold increase of pS129A was observed for all variants, excepted for S129A which is non phosphorylatable (note that with this scale adapted for overexpression conditions, the phosphorylation of endogenous α-Syn upon seeding exists but is invisible), E46K which showed virtually no response because it was barely expressed, and A30P which exhibited the strongest basal phosphorylation in spite of its lower expression level, with little room left to detect a further impact of seeding on pS129 . Figure 5 A underlines that under overexpression conditions, little can be said on the impact of specific mutations on either basal or seeded α-Syn aggregation compared to wt α-Syn, apart perhaps the basal pS129 levels of A30P which could be interpreted as the indication of a more pronounced spontaneous aggregation of this variant. The next experiments indicate that this is not the case. In order to track true α-Syn amyloid conversion in these conditions, we used the MJFR1-Syn1 staining combination and derived the % of neurons bearing amyloid inclusions (i.e., bearing MJFR1-positive Syn1-negative inclusions) (Figs. 5 B,C). As for wt α-Syn, in unseeded conditions virtually all the neurons were perfectly double stained for all mutants (including S129A), indicative that none of the variants induced spontaneous aggregation with amyloid conversion. This indicates that the pS129 signal observed in unseeded conditions (Fig. 5 A) is not due to spontaneous aggregation but simply to overexpression and phosphorylation of non-amyloid forms of the protein. These results reveal that in cortical neurons, the α-Syn mutations that cause autosomal dominant PD fail to trigger the spontaneous amyloid conversion of α-Syn into fibrils. The same holds true for the S129A variant showing that preventing phosphorylation at S129 is not sufficient not trigger spontaneous fibrillization. We thus reasoned that in a neuronal context, these mutations might instead favor the seeded assembly of α-Syn into amyloid fibrils. Upon seeding with PFFs, we observed a massive burst of the population of neurons bearing amyloid inclusions (MJFR1-positive Syn1-negative) (Figs. 5 B,C). Unexpectedly enough, the extent of amyloid conversion here also appeared comparable for all the variants: none of the mutations appeared to favor the seeded assembly process. At the opposite, the A53E variant even inhibited the amyloid conversion of α-Syn in neurons confirming several previous observations made in vitro regarding this mutant ( 56 , 57 ). Collectively, these data suggest that the mechanisms by which familial α-Syn mutations might cause autosomal dominant PD are neither related to triggering nor to facilitation of α-Syn fibrillization in intact neurons. In addition, phosphorylation of S129 which can be misleading as a marker of fibrillization, does not seem to inhibit spontaneous or seeded α-Syn fibrillization either. Probing amyloid conversion in histological brain sections: revisiting the status of α-Syn in pathological inclusions These results prompted us to put amyloid α-Syn conversion under scrutiny in brain sections presenting clear signs of α-Syn inclusion pathology. We used in parallel sections from wt mice sacrificed 6 months after an intra-striatal injection of human PFFs and post-mortem sections from a patient with sporadic PD (Fig. 6 ). In mice (Fig. 6 A-C), the inclusion pathology was revealed using the antibody pair pS129 and Syn1 like in the primary cultures of Fig. 4 D. The figure shows a region of the right basolateral amygdala (BLA) filled with pS129-positive α-Syn neuronal inclusions of 3 types: LNs, neuronal perikaryal inclusions with a more or less compacted “Lewy Body-like” appearance, depending on the maturation level of the inclusion, and a few neuronal intranuclear inclusions (Fig. 6 A, in green). Co-staining with Syn1 (Fig. 6 B, in purple) revealed the physiological pool of non-amyloid α-Syn present in the synapses that were scattered all over the field of view. However, while as expected many pS129 inclusions appeared Syn1-negative (green inclusions in the overlay of Fig. 6 C, see a few examples pointed by empty arrowheads), indicating inclusions exclusively made of amyloid α-Syn, a significant number of inclusions alternatively appeared partially or totally Syn1-positive (white inclusions in the overlay of Fig. 6 C, see a few examples pointed by empty arrows), indicative of the presence of non-amyloid α-Syn inside the inclusions. It can be noted that the presence of non-amyloid α-Syn in experimental inclusions seeded in mice by PFFs does not seem to depend on the inclusion type since all of them can be concerned by the possible presence of non-amyloid α-Syn detectable by Syn1. Panels of Fig. 6 D-J and K-Q show the focused exploration of the amyloid status of α-Syn respectively in a LN and in a Lewy body both present in a post-mortem SN brain section of a sporadic PD patient. The section was first double-labeled and revealed using EP1536Y and Syn1 as before. The section was then hybridized with a third fluorophore-coupled MJFR1 antibody which detects hSyn irrespective of its conformation and phosphorylation (see Figs. 3 , 5 and S5 ). The LN of Fig. 6 is pS129-positive on most of its length (green) with a superposable staining pattern shown by MJFR1 (red) (see in particular the pS129 and MJFR1 traces in the line scan of the overlay Fig. 6 J). Instead, staining with Syn1 (purple) is variable along the length of the LN: some regions are Syn1-negative indicating a purely amyloid composition (empty arrowhead), while others are Syn1-positive showing the presence of non-amyloid α-Syn (empty arrows). The line scan of Fig. 6 J makes it clear that the MJFR1 and the Syn1 signals were completely decoupled in the left end of the LN highlighting the exclusive presence of fibrils in this portion of the inclusion. At major variance, it appeared difficult to identify amyloid-only subregions in the LB. The 3 antibodies showed comparable staining patterns within the inclusion, with spatially correlated MJFR1 and Syn-1 signals (see the MJFR1 and Syn1 traces in the line scan of the overlay Fig. 6 Q and compare with Fig. 6 J). In contrast with the somatic inclusions seeded in mice (Fig. 6 A-C), and with the LN shown before, Syn1 positivity was observed for all the LBs we investigated. This indicated that LBs in PD are pathological inclusions containing non-amyloid α-Syn. This is in line with the poor amyloid status of PD brain extracts compared to MSA ones [see companion paper Lafrerrière et al.] and confirms the pioneering observations of Shahmoradian and colleagues who reported that α-Syn fibrils were identifiable in many but not all α-Syn inclusions in PD ( 58 ). Discussion Our results indicate that pS129 which is widely used as a surrogate marker of α-Syn aggregation in experimental and clinical settings leads to the positive scoring of brain cells in which α-Syn aggregation does not take place. This is particularly evident for models presenting no identifiable inclusion pathology but only diffuse anatomical-scale brain staining ( 32 – 36 ). This however also applies to models in which overexpressing α-Syn produces images mistaken for intracellular inclusions and corresponding to high concentrations of soluble phosphorylated α-Syn species ( 37 ) unrelated to an amyloid aggregation process (Fig. 1 ). It is worth highlighting that beyond the case of the PLP-α-Syn mouse, and of the AAV-Syn infections shown here, the brain sections of several transgenic mouse models yielding a neuronal overexpression of α-Syn present widespread pS129-positive images with a distribution and intensities which are uncorrelated with the patterns revealed by the amyloid probe h-FTAA ( 59 ). This questions the amyloid nature of the images revealed using pS129. This issue with pS129 represents a problem for the quantitative neuropathology of α-Syn in cellular and animal models, but also for the correct interpretation of post-mortem brain sections from patients in which pS129-positive images do not grant the presence of amyloid α-Syn fibrils. Unfortunately, relying on conformation-dependent antibodies which are commercially available is not an alternative because their preference for amyloid α-Syn fibrils with regards to other soluble species is partial ( 31 ). In brain sections, their signal-to-noise ratio for α-Syn inclusion detection is low because α-Syn physiologically addressed to pre-synapses produces an overwhelming background (for instance with SynF1, and to a lesser degree with Syn 303 and Syn505 (not shown)). Our observation that the NAC epitope recognized by Syn1 is rendered inaccessible during fibril assembly offers the possibility to characterize inclusions revealed by standard antibodies and to establish their amyloid status. Syn1 is a mouse monoclonal antibody which can be used in combination with any other rabbit antibody recognizing the C-terminal region of α-Syn. In double immunofluorescence settings, any cytological structure/region positively stained with the latter types of anti-α-Syn antibodies and concomitantly appearing Syn1-negative corresponds to a purely amyloid inclusion made of α-Syn fibrils. Using this approach to score α-Syn fibrillization in primary neurons, we made the unexpected observation that under overexpression conditions, wt hSyn as well as α-Syn mutants associated with familial PD do not trigger spontaneous fibrillization. The massive pS129 signal which is observed corresponds to non-amyloid forms of the protein. Further, these α-Syn mutants do not facilitate the process of seeded fibrillization, and even inhibit it in the case of A53E. Altogether, this goes against the notion that the α-Syn mutants found in familial PD would provoke the onset of PD because they would tend to facilitate α-Syn fibrillization. Though intellectually appealing, this generic hypothesis is mainly based on the results of historical protein-only experiments( 60 – 64 ) or obtained using reconstituted systems with α-Syn and lipids( 65 ) or deduced from manipulations in yeast and other non-neuronal cells( 66 ) ( 67 ). It is worth noting however that the inhibiting effect of A53E on fibrillization was reported several times in such systems( 56 , 57 , 67 , 68 ) but the bearings of this observation for the possible causal role of fibrillization in PD did not catch a general attention. It is worth noting that a similar problem was also repeatedly encountered in vitro for the A30P mutant ( 24 , 63 , 69 ), which led Lansbury and his colleagues to conclude 23 years ago that fibrillization was not playing a prime pathogenic role in PD. Our present observation that α-Syn mutations causing autosomal dominant transmission of PD with a 100% penetrance neither trigger nor facilitate the seeded fibrillization of α-Syn in primary neuronal cultures is in line with the conclusion of Lansbury and colleagues. This indeed suggests that besides fibrils, non-amyloid α-Syn form(s) might play a prominent role in the pathophysiology of PD ( 24 ). Though counterintuitive, it should be noted that structural analyses have also shown that familial PD α-Syn point mutations are all strategically located in the protein to either prevent the proper stabilization of the canonical type I amyloid fold or to interfere with the “orthodox” intertwining of the protofilaments during α-Syn fibril assembly ( 70 ). Besides the mutations of familial PD, the prominent phosphorylation of S129 in pathological α-Syn inclusions has prompted investigations aimed at determining the functional role of this modification ( 29 ). This led to the conclusion that this modification has indeed an impact on α-Syn fibrillization. However, for some, pS129 facilitates seeded fibrillization( 53 ) while for others pS129 inhibits the process( 54 ) ( 52 ). Using the overexpression of the non-phosphorylatable α-Syn mutant S129A and our amyloid α-Syn detection method, we find that preventing S129 phosphorylation in primary neurons has no impact on either spontaneous or seeded fibrillization, pointing to a different and yet unknown role of the modification. One possibility to explain that pS129 appears on fibrils or on overexpressed monomers is that during α-Syn fibrillization or α-Syn mis-sorting, the protein no longer interacts with VAMP2 as it normally does when α-Syn is addressed to the presynaptic vesicles. Indeed, this α-Syn/VAMP2 interaction concerns the C-terminus of α-Syn in a region encompassing S129 ( 71 ). It is thus tempting to speculate that in normal conditions this interaction shields S129 from casein kinase, the constitutively active protein kinase responsible for the phosphorylation of S129 ( 29 ). In protein mis-sorting conditions (due to overexpression or to fibril formation), shielding is likely to be lost thus exposing S129 to phosphorylation by casein kinase. We next applied our methodology to address the amyloid status of the α-Syn inclusions observed in wt mice injected with PFFs and in the brain of a deceased sporadic PD patient. In line with the observations regarding mutations, we found that the α-Syn inclusions observed either in the disease or experimentally produced in the mouse are far from being exclusively constituted of fibrils and are populated by non-amyloid species. Interestingly in PD Lewy neurites present a “purer” amyloid constitution than the LBs which are rich in non-amyloid species. This observation is reminiscent of the ones of Shahmoradian et al. who reported the existence of fibril-less LBs ( 58 ). It suggests that the prominence of non-amyloid α-Syn in the LBs compared to LNs or to experimental inclusions in mice could result from the maturation level of the inclusions. Maturation could involve delayed fibril disassembly and breakdown in the latest phases of LB constitution, associated with the emergence of non-amyloid, yet pathological species. Note that the possibility that the familial mutation A30P could play its role in PD by exacerbating the release of pathogenic fragments from the fibrils was proposed by Hasegawa and colleagues ( 63 ). From a purely methodological point of view, it is worth noting that in our conditions of overexpression achieved in 96 well primary cultures of cortical neurons, spontaneous fibrillization of α-Syn is virtually absent. This could represent a model well suited to explore at a reasonable throughput the mechanisms that could trigger amyloid aggregation in the absence of internalization of a preformed seed. Indeed, while this latter step is the main candidate process invoked to explain the intercellular spread of the α-Syn inclusions, the spontaneous emergence of intraneuronal aggregates by dysregulation of cellular processes involved in α-Syn catabolism or by the disruption of heterologous protein-protein interactions could be key to the emergence of an inclusion pathology. Abbreviations AAV adeno-associated virus BLA basolateral amygdala BSA Bovine Serum Albumin DIV days in vitro DLB Dementia with Lewy bodies FFPE formalin-fixed paraffin-embedded GCI glial cytoplasmic inclusion GCP genome-containing particle HCA High Content Analysis hSyn human α-Syn IHC immmunohistochemistry LB Lewy body LN Lewy neurite LSCM Laser-Scanning Confocal Microscopy MSA multiple system atrophy PAGE polyacrylamide gel electrophoresis PBS Phosphate Buffered Saline PD Parkinson’s disease PEI polyethylenimine PFA paraformaldehyde PFFs Preformed fibrils pS129 S129-phosphorylated pSyn pS129 α-Synuclein RT room temperature SB solubilization buffer SN Substantia Nigra SNpc Substantia Nigra pars compacta TBS Tris-buffered saline TH tyrosine hydroxylase wt wild-type αSP α-Synucleinopathies α-Syn α-Synuclein Declarations Ethics approval The brain samples from MSA and PD patients were obtained from the Brain Bank GIE NeuroCEB (BRIF number 0033-00011) The animal study protocol was approved by the French Ministry of Research (protocol APAFIS #33147-2021091711598830 v6, 2021–2026). Consent for publication Not applicable Availability of data and materials The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request. Competing interests The Authors declare no competing interests. Funding This project received funding from the French Agence Nationale de la Recherche (ANR), under grant ANR-22-CE16-0002 (project ASAPS), the Department of Excellence Initiative of the Italian Ministry of Research, the Center of Excellence in Neurodegeneration (CoEN) Bordeaux Initiative for Neurodegenerative Disorders (BIND) reference CHUBX 2022/07, the MSA Coalition Global Seed Grant (project FibrilloScreen) and the Institut Roche, Paris, France. AL is supported by the Institut Roche, France (CIFRE doctoral fellowship). Authors' contributions FI, FDG and FL conceived and designed the work, performed the analysis and the interpretation of data; FDG, AL & FZ performed in vitro experiments on primary neuronal cultures; FZ, ND, LAD produced plasmids and AAVs; FL designed and performed all biochemical assays; SD, EB performed and managed AAV induced overexpression in mice and IHC experiments; MLA performed IHC experiments; MK, FDN & DDL performed and managed in vivo synucleinopathy experiments by intrastriatal injection of PFFs and IF experiments in FFPE sections; MHC helped with immunofluorescence experiments in human FFPE sections; FI and FDG wrote the original version of the manuscript; FL, AL, WM, DDL made substantial contributions to the drafting and/or the substantive revision of the manuscript. Acknowledgements The Authors thank Benjamin Dehay for making available the AAV vectors regarding the familial PD α-Syn mutations. References Tu PH, Galvin JE, Baba M, Giasson B, Tomita T, Leight S, et al. 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New insights on the structure of alpha-synuclein fibrils using cryo-electron microscopy. Curr Opin Neurobiol [Internet]. 2020 Apr 1 [cited 2023 Oct 19];61:89–95. Available from: https://pubmed.ncbi.nlm.nih.gov/32112991/ Sun J, Wang L, Bao H, Premi S, Das U, Chapman ER, et al. Functional cooperation of α-synuclein and VAMP2 in synaptic vesicle recycling. Proc Natl Acad Sci U S A. 2019;166(23):11113–5. Additional Declarations The authors declare no competing interests. Supplementary Files Slide2.tif S1 Slide5.tif S3 Slide8.tif S5 SupplementaryLegends.docx Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. 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Bordeaux, CNRS, IMN, UMR 5293, F-33000 Bordeaux, France and CHU Bordeaux, Service de Neurologie des Maladies Neurodégénératives, IMNc, NS-Park/FCRIN Network, Bordeaux, France and Department Medicine, University of Otago, Christchurch, and New Zealand Brain Research Institute, Christchurch, New Zealand","correspondingAuthor":false,"prefix":"","firstName":"Wassilios","middleName":"","lastName":"Meissner","suffix":""},{"id":270704928,"identity":"c635edf6-e238-4b7e-84b2-1d4675d4c297","order_by":11,"name":"Francesco De Nuccio","email":"","orcid":"","institution":"Univ. Salento, DiSTeBA, Anatomia Umana, I-73100 Lecce, Italy","correspondingAuthor":false,"prefix":"","firstName":"Francesco","middleName":"","lastName":"De Nuccio","suffix":""},{"id":270704929,"identity":"2115bea9-062a-442e-a9ad-a51ac1d77c05","order_by":12,"name":"Dario Domenico Lofrumento","email":"","orcid":"","institution":"Univ. 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Bordeaux, CNRS, IMN, UMR 5293, F-33000 Bordeaux, France","correspondingAuthor":false,"prefix":"","firstName":"Florent","middleName":"","lastName":"Laferrière","suffix":""},{"id":270704931,"identity":"f6479cdc-08e1-474c-8855-2bc8417ede77","order_by":14,"name":"François Ichas","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA9klEQVRIiWNgGAWjYBAC9gYwJcHAwMzABmTY8EAl5HBq4TmAqiUNpsWYkBYwAGk5DOPg0SJ9+NmDHxUW9gzszEDGn/My5vyHj326wWCQj1MLX5q5Yc8ZicQGZjZzw9622zyWM9KSZ+cwGFg24NBiz8NgJs3YJpHAwMzDJsHbcJvH4AaPMXMOwx8DnLbwsH8DabEHaZH88+ccj8H585+BWgzwaOEB28LYANQizcN2gMfgQA4zIS1lkiC/tDGzmUnLtiUDHZYGdJgBPi3s2yR+VNTZ8/Mffib55o+dvcH5w4+Zcypwa4EDNlQuYQ2jYBSMglEwCvAAAFxmQTlMIJDzAAAAAElFTkSuQmCC","orcid":"https://orcid.org/0000-0001-8184-5248","institution":"Univ. Bordeaux, CNRS, IMN, UMR 5293, F-33000 Bordeaux, France and Univ. Salento, DiSTeBA, Anatomia Umana, I-73100 Lecce, Italy","correspondingAuthor":true,"prefix":"","firstName":"François","middleName":"","lastName":"Ichas","suffix":""}],"badges":[],"createdAt":"2024-02-02 14:18:33","currentVersionCode":1,"declarations":{"humanSubjects":true,"vertebrateSubjects":true,"conflictsOfInterestStatement":false,"humanSubjectEthicalGuidelines":true,"humanSubjectConsent":true,"humanSubjectClinicalTrial":false,"humanSubjectCaseReport":false,"vertebrateSubjectEthicalGuidelines":true},"doi":"10.21203/rs.3.rs-3921168/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-3921168/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":50819121,"identity":"4ab5e79e-c9fc-4c0b-a0fc-a09bfd5402c0","added_by":"auto","created_at":"2024-02-07 20:25:58","extension":"tif","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":276070,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003epS129 α-Syn is not a specific surrogate marker of aggregation when α-Syn is overexpressed.\u003c/strong\u003e A) Schematical representation of the experimental conditions. A saline solution, AAV-syn, PFFs or a combination of these latter were injected in the SNpc of mice and animals were sacrified 4 months later. B-C) The antibody EP1536Y is used in IHC experiments for detecting pS129 α-Syn in the SNpc (injection site) and in the \u003cem\u003eStriatum\u003c/em\u003e. (B) The injection of PFFs in the SN of adult mice generates pS129 α-Syn-positive aggregates locally (SNpc) and a distance (\u003cem\u003eStriatum\u003c/em\u003e) in the brain. In overexpression conditions a strong signal is detected in the SNpc both in AAV-syn alone and in the presence of PFFs. (C) In the \u003cem\u003eStriatum\u003c/em\u003e the aggregates were detected exclusively in the PFF-treated mice. D-E) Quantification of the number of pSyn-positive objects in the different treatment conditions. Each circle corresponds to one animal, with n = 10.\u003c/p\u003e","description":"","filename":"Slide1.tif","url":"https://assets-eu.researchsquare.com/files/rs-3921168/v1/6607b6fa4668e3716200086e.tif"},{"id":50818446,"identity":"23a6bc43-9c7c-452a-8102-3f3854920e6e","added_by":"auto","created_at":"2024-02-07 20:17:58","extension":"tif","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":245310,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eThe overexpression of hSyn in a primary culture of mouse cortical neurons leads to the apparition of a pSyn-positive signal in the absence of aggregation\u003c/strong\u003e. A-B) pSyn detected by the EP1536Y antibody at DIV30 in mouse cortical neurons overexpressing hSyn (+AAV-syn) or not (not infected). In (A) representative images of non-infected and infected neurons in the basal condition (not seeded) and in (B) in neurons exposed to PFFs from DIV7 (B, “+ PFFs”). pSyn is shown in green with EP1536Y and is superimposed on the phase contrast (merge). C) Quantification of the integrated intensity of the pSyn signal, of wt and hSyn-overexpressing neurons in unseeded conditions, with or without permeabilization of the cell membrane prior to fixation (digitonin), showing that phosphorylation in overexpression conditions is associated with soluble α-Syn. D) Quantification of the integrated intensity of the pSyn signal, of wt and hSyn-overexpressing neurons in PFFs exposed conditions, with or without permeabilization of the cell membrane prior to fixation (digitonin). In these conditions the pSyn signal is resistant to plasma membrane permeabilization. Measurements correspond to n = 18 imaged fields. The p-value was obtained by a Two-Way ANOVA test. E) LB509 staining does not specifically detect aggregated overexpressed hSyn. Quantification of the overall signal of LB509 discriminated seeded vs non-seeded conditions only in permeabilized neurons. Each circle corresponds to one culture well, with n = 2 or n = 3. The p-value was obtained by a Two-Way ANOVA test. F) Epifluoresence microscopy image of mouse cortical neurons at DIV30, treated at DIV7 with PFFs, infected at DIV10 with AAV-syn and permeabilized with digitonin prior to fixation and co-stained with LB509 and EP1536Y, showing only a partial overlap of the signals (overlay).\u003c/p\u003e","description":"","filename":"Slide3.tif","url":"https://assets-eu.researchsquare.com/files/rs-3921168/v1/79152f20c4ef5ae104f57465.tif"},{"id":50818452,"identity":"158a4a74-c929-4956-ad06-965a7477b37d","added_by":"auto","created_at":"2024-02-07 20:17:58","extension":"tif","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":222955,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSpecificity of a panel of antibodies towards different α-Syn assembly states.\u003c/strong\u003e A) Dot Blot. Samples of monomeric (m) or fibrillar (polymorphs 1B, i1 and i3) hSyn filtered through a nitrocellulose membrane which is then immunoblotted against different α-Syn antibodies. B) Dot Blot. Samples denatured in increasing concentrations of urea, filtered through a nitrocellulose membrane which is then immunoblotted against the same panel of antibodies. The signals obtained are normalized as percentage of the signal without urea and plotted as a function of the concentration of urea. C) Dot Blot. Samples of sarkospin fractions extracted from human brain samples filtered through a nitrocellulose membrane which is then immunoblotted against the same panel of antibodies. CTL are samples from a healthy brain, PD are samples from the brain of a PD patient, MSA are samples from the brain of a MSA patient. The S fractions correspond to the supernatant after ultracentrifugation, the P fractions correspond to the pellet. The ratio of signal between the supernatant and the pellet is expressed as a % of the total signal for each antibody. The statistical difference between the samples was calculated with a Two-Way ANOVA test.\u003c/p\u003e","description":"","filename":"Slide4.tif","url":"https://assets-eu.researchsquare.com/files/rs-3921168/v1/3c7e629d6f404729c4dc7c89.tif"},{"id":50818450,"identity":"74c44312-a0b4-4967-95ef-fb6aeae4141a","added_by":"auto","created_at":"2024-02-07 20:17:58","extension":"tif","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":382073,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003e\u003cstrong\u003eIn situ\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e detection of aggregated α-Syn in a primary culture of mouse cortical neurons overexpressing hSyn.\u003c/strong\u003e A) Quantification of the integrated intensity of the Syn505 or SynF1 signal in different conditions, specified by the table underneath the graphs: PFFs corresponds to seeded cultures, AAV-syn to hSyn overexpression, and digitonin to the permeabilization of the plasma membrane prior to fixation. Each circle corresponds to one culture well, made up of the sum of 9 fields of acquisition, with n = 2 or n = 3. The p-value was obtained by a One-Way ANOVA test. B) Representative images of mouse cortical neurons at DIV30, treated at DIV7 with PFFs and infected at DIV10 with AAV-syn. pSyn (EP1536Y) is shown in green. Syn505 (left) or SynF1 (right) are shown in red. Signals only partially overlap. C) Representative images of a double immunostaining with Syn1 (red) and MJFR1 (green) showing that aggregated permeabilization-resistant α-Syn is Syn1 negative. AAV-syn cortical neurons at DIV30, untreated (left) or treated at DIV7 with PFFs are immunostained directly (intact) or after permeabilization prior to fixation (digitonin). Syn1 is shown in red, MJFR1 in green. D) Representative image of a double immunostaining with Syn1 (red) and EP1536Y (green) in cortical neurons, treated at DIV7 with PFFs , showing that in intact cells, pSyn-positive neurons could be either Syn1 negative (amyloid) or Syn1-positive (non-amyloid).\u003c/p\u003e","description":"","filename":"Slide6.tif","url":"https://assets-eu.researchsquare.com/files/rs-3921168/v1/9db6f385021c91be3c081670.tif"},{"id":50818451,"identity":"2d88f532-6fde-44a7-8e28-e1a738955192","added_by":"auto","created_at":"2024-02-07 20:17:58","extension":"tif","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":280854,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eα-Syn amyloid conversion in primary cultures of mouse cortical neurons expressing different α-Syn mutants.\u003c/strong\u003eA) Quantification of the integrated intensity of the pSyn (EP1536Y) signal in DIV30 cultures untreated (-) or treated with PFFs at DIV7 (+), infected with AAV particles of different α-Syn mutants at DIV10. Each circle corresponds to one field of acquisition of epifluorescence microscopy, with n = 18. The p-value was obtained by a One-Way ANOVA test. B) Quantification of the % of neurons with amyloid α-Syn. The % of neurons with amyloid α-Syn corresponds to the % of neurons expressing non-monomeric α-Syn (detected by MJFR1 but not by Syn1). Each circle corresponds to one field of acquisition of epifluorescence microscopy, with n = 18. The p-value was obtained by a Two-Way ANOVA test. C) Representative field of mouse cortical neurons untreated (not seeded) or treated at DIV7 with PFFs, expressing wt hSyn or mutant hSyn (S129A, A53T, A53E, H50Q, G51D). Syn-1 is shown in red, MJFR1 in green. The overlap in yellow (merge) is complete in the non-seeded condition.\u003c/p\u003e","description":"","filename":"Slide7.tif","url":"https://assets-eu.researchsquare.com/files/rs-3921168/v1/95f86f2274e0e766cc23b39b.tif"},{"id":50818453,"identity":"cb031c1d-5b9e-4e62-8ef6-3652be246a69","added_by":"auto","created_at":"2024-02-07 20:17:59","extension":"tif","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":684097,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eUsing a combination of 2 antibodies allows the characterization of pathological α-Syn.\u003c/strong\u003eA-C) Confocal microscopy on horizontal brain slices from wt mice sacrificed 6 months after an intra-striatal injection of human PFFs. The region observed is the BLA. pSyn is shown in green with EP1536Y, non-amyloid α-Syn is shown in purple with Syn1, and the nuclei are shown in cyan with Draq7 (which is a chromatin marker). pSyn-posjtive, Syn1-negative objects are shown by the empty arrowheads, while pSyn-positive, Syn1-negative objects are shown by the empty arrows. D-Q) Confocal microscopy on \u003cem\u003epost-mortem\u003c/em\u003esections from a patient with sporadic PD. The region observed is the SNpc. pSyn is shown in green with EP1536Y, non-amyloid α-Syn is shown in purple with Syn1, and total hSyn is shown in red with MJFR1. D-I) Characterization of aLN: the empty arrowhead shows a pSyn-positive, Syn1-negative portion of the aggregate, while the empty arrows show pSyn-positive, Syn1-positive portions of the aggregate. I) in white dots, the region of the line scan shown in J). In J), line scan of the fluorescence intensities of each α-Syn antibody (MJFR1 in red, Syn1 in purple, EP1536Y in green). K-P) Characterization of a LB: the empty arrow shows a pSyn-positive, Syn1-positive aggregate. P) in white dots, the region of the line scan shown in Q). In Q), line scan of the fluorescence intensities of each α-Syn antibody (MJFR1 in red, Syn1 in purple, EP1536Y in green).\u003c/p\u003e","description":"","filename":"Slide9.tif","url":"https://assets-eu.researchsquare.com/files/rs-3921168/v1/fcf8b4ebf5d113e1ab31e403.tif"},{"id":50819696,"identity":"cf782343-890f-4b59-af10-f09e6a60142a","added_by":"auto","created_at":"2024-02-07 20:34:00","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2974778,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-3921168/v1/c3b7629a-cafa-4c8d-9104-8e996b76b194.pdf"},{"id":50819120,"identity":"284c4636-458e-43c7-9dab-059d4a12724f","added_by":"auto","created_at":"2024-02-07 20:25:58","extension":"tif","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":194238,"visible":true,"origin":"","legend":"\u003cp\u003eS1\u003c/p\u003e","description":"","filename":"Slide2.tif","url":"https://assets-eu.researchsquare.com/files/rs-3921168/v1/b7f624a733b9ac70a28a4783.tif"},{"id":50818448,"identity":"f9c62ea6-575d-4728-b2e5-916e237de983","added_by":"auto","created_at":"2024-02-07 20:17:58","extension":"tif","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":324560,"visible":true,"origin":"","legend":"\u003cp\u003eS3\u003c/p\u003e","description":"","filename":"Slide5.tif","url":"https://assets-eu.researchsquare.com/files/rs-3921168/v1/1616c42f7d31ce6dd6ff175e.tif"},{"id":50818445,"identity":"cb3adc15-01c2-4fba-9911-a545bba702d8","added_by":"auto","created_at":"2024-02-07 20:17:58","extension":"tif","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":52480,"visible":true,"origin":"","legend":"\u003cp\u003eS5\u003c/p\u003e","description":"","filename":"Slide8.tif","url":"https://assets-eu.researchsquare.com/files/rs-3921168/v1/833a79e8a8bf6ef0674c2e3a.tif"},{"id":50818449,"identity":"880869d5-1bdd-4a39-b307-800ccba85aec","added_by":"auto","created_at":"2024-02-07 20:17:58","extension":"docx","order_by":4,"title":"","display":"","copyAsset":false,"role":"supplement","size":13221,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryLegends.docx","url":"https://assets-eu.researchsquare.com/files/rs-3921168/v1/12231f565dcccdebc6144393.docx"}],"financialInterests":"The authors declare no competing interests.","formattedTitle":"\u003cp\u003eReconsidering α-Synuclein inclusion pathology in neurons, mice, and humans with an antibody sensing NAC engagement during α-Synuclein amyloid conversion\u003c/p\u003e","fulltext":[{"header":"Background","content":"\u003cp\u003eIn the group of neurogenerative diseases called α-Synucleinopathies (αSP), which includes Parkinson\u0026rsquo;s Disease (PD), Dementia with Lewy Bodies (DLB), and Multiple System Atrophy (MSA), the underlying molecular pathology is characterized by the amyloid aggregation of the presynaptic protein α-Synuclein (α-Syn)(\u003cspan additionalcitationids=\"CR2\" citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eα-Syn is an intrinsically disordered protein that normally binds to the synaptic vesicle membranes as an α-helical oligomer and is thought to play a role in the modulation of neurosecretion. Initiating a pathological cascade, α-Syn can instead form multimeric assemblies in which the protein assumes a flat and rigid conformation with folded β-strands that is transmitted to the neighboring monomers by templating and progressive stacking, reminiscent of prions (\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e). Although the pathophysiological causes for the initiation of this process inside neurons remain unknown, experimental seeding of this conformational conversion by preformed assemblies has been well established (\u003cspan additionalcitationids=\"CR7 CR8 CR9 CR10\" citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e). Amyloid aggregation of α-Syn can be experimentally achieved \u003cem\u003ein vitro\u003c/em\u003e as a protein-only process using recombinant α-Syn, in spontaneous or seeded conditions, leading to highly structured fibrils (\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e, \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e). These assemblies most often appear as two intertwined protofilaments forming a fibril with an axial symmetry and are easily detectable with fluorescent probes that bind the surface grooves of the amyloid structure, among which the most used is Thioflavin T (\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e). The organization of α-Syn fibrils extracted from \u003cem\u003epost-mortem\u003c/em\u003e brain tissue has been resolved at the atomic scale by cryoEM substantiating the existence of distinct amyloid fold polymorphs in the different αSP (\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e, \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eFrom a neuropathological point of view, the α-Syn fibrils accumulate inside subcellular inclusions forming defined cytological objects (for instance: Lewy bodies LBs, Lewy neurites LNs, glial cytoplasmic inclusion GCI) (\u003cspan additionalcitationids=\"CR18\" citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e). In about 50% of PD patients, the progression of the disease has been correlated with the apparent spread of these objects in new brain regions, leading to the definition of Braak staging (\u003cspan additionalcitationids=\"CR21\" citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e). In addition, the sarkosyl-insoluble α-Syn fibrils extracted from DLB or MSA brain tissue can experimentally seed a \u003cem\u003ede novo\u003c/em\u003e α-Syn pathology made of identifiable inclusions in the brain of non-transgenic mice(\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e) (\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e): α-Syn fibrils are thus considered to be the cause of spread and of disease progression (\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e). However, the mechanisms linking amyloid α-Syn spread and neurodegeneration are unclear, and it has thus been proposed that neurodegeneration could be associated with other non-fibrillar (non-amyloid) forms of assemblies referred to as oligomers (\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e). Yet their structure, their presence/distribution in the brain, and their ability to template their own growth and spread remain elusive.\u003c/p\u003e \u003cp\u003eSeveral cell-based or animal models, either based on α-Syn overexpression or on the induction of a seeded aggregation by α-Syn fibril injections have been set up, with the aim of understanding the biological mechanisms linking α-Syn amyloid conversion and fibril formation to neuronal failure and death (\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e). However, addressing genuine amyloid conversion of α-Syn in a cellular context remains challenging. Moreover, in contrast to \u003cem\u003ein vitro\u003c/em\u003e conditions using pure recombinant α-Syn, the conversion of α-Syn from a monomeric to a structured supramolecular amyloid state is certainly a much more complex process inside neurons, since it can be affected by interactions with other proteins and lipids, post-translational modifications, and transport mechanisms. In addition, inside neurons and glial cells aggregated α-Syn is processed by a variety of catabolic pathways like autophagy, proteasomal degradation, and the disaggregase chaperone complex (\u003cspan additionalcitationids=\"CR27\" citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e). All these elements, by limiting or contributing to the pathology, could potentially represent druggable targets for the development of therapeutic strategies. Thus, the ability to specifically detect amyloid α-Syn in experimental cellular or animal models and to differentiate amyloids from the other forms of the protein is a key step required to evaluate candidate targeted therapeutics aimed at reducing αSP.\u003c/p\u003e \u003cp\u003eIn histopathology, a frequently used approach to detect aggregated α-Syn in patient tissue is the use of antibodies that can specifically detect the phosphorylated form of α-Syn at serine 129 (pS129) (\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e). This post-translational modification is considered a reliable surrogate marker of amyloid α-Syn since it has been shown that α-Syn assemblies extracted from patients are phosphorylated at S129 and that the distribution of phosphorylated α-Syn in patient brain sections is restricted to the pathological inclusions (\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e). Beyond synaptic staining, general α-Syn antibodies can also stain LB, GCI, and LN, which are identifiable by their simple morphological features due to the overwhelming concentration of the protein inside the inclusions. However, in all these cases, the amyloid state of α-Syn is only secondarily inferred. A more specific method involves the use of conformational antibodies with a higher affinity for fibrils than for other forms of the protein. However, their preference for high molecular weight α-Syn assemblies is relative (\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e), and the main discriminating factor remains their ability to highlight typical cytopathological inclusions.\u003c/p\u003e \u003cp\u003eDetection and identification of such \u003cem\u003ebona fide\u003c/em\u003e inclusions is unfortunately an issue in several preclinical models. In other words, while in the context of human disease α-Syn pathology is strictly defined as the presence of delineated subcellular inclusions, certain animal models do not reproduce them (\u003cspan additionalcitationids=\"CR33 CR34 CR35\" citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e). To accommodate this limitation, it has been proposed that in these models pathological α-Syn could take the prodromal appearance of diffuse anatomical-scale increases of the pS129 signal analyzable by global thresholding and/or of regional resistances of the diffuse α-Syn immunoreactivity to treatment of the sections with proteases (\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e, \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e). However, these types of signs do not belong to the clinical neuropathology of αSP which exclusively relies on the individuation and the count of inclusions for the scoring of Lewy and MSA pathology (\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThe opposite problem can be encountered in α-Syn overexpressing models in which seeded aggregation can burst without the formation of well-delineated inclusions because the ability of the neurons/oligodendrocytes to constrain the pathological assemblies in confined cytoplasmic regions is overwhelmed (see results). Without seeding, the situation is even worse: overexpressed α-Syn can remain soluble, overflow the entire cytoplasm, and get phosphorylated without forming any fibrils (see results). This results in the genesis of images that can erroneously be interpreted as the emergence of inclusions mimicking the human pathology (\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eIn line with our previous observations made in mice overexpressing α-Syn under the control of the PLP promoter (\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e), our experiments using neuronal primary cultures and mice infected with AAV-human α-Syn (hSyn) to model αSP, indicated that S129 phosphorylation most often turns out to be a false positive marker of α-Syn aggregation in overexpression conditions. To overcome this difficulty, we thus tried to find an alternative way to detect amyloid α-Syn unambiguously. To do so, we tested a set of commercially available antibodies widely employed in the literature. Unexpectedly, we stumbled upon neglected antibody features and built upon these properties to derive markers of α-Syn conversion into fibrils suitable for experimental and clinical neuropathology studies.\u003c/p\u003e"},{"header":"Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eAntibodies\u003c/h2\u003e \u003cp\u003ePrimary antibodies were used as follows:\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"No\" id=\"Taba\" border=\"1\"\u003e \u003ccolgroup cols=\"6\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eIF \u003cem\u003ein vitro\u003c/em\u003e\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eIF in PFFE sections\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eIHC\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003eBiochemical assay\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eEP1536Y\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eAbcam ab51253\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1:500\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1:500\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e1:5000\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e1/5000\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMJFR-1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eAbcam ab138501\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1:1000\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1:1000\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e1:5000\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e1/10000\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAlexa 488 MJFR-1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eAbcam ab195025\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1:500\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1:500\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003en/a\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003en/a\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSynF1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eBioLegend 847802\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1:500\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003en/a\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003en/a\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e1/10000\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eD37A6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eCell Signaling, #4179\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1:500\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003en/a\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003en/a\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e1/2000\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSyn1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eClone 42 BD Biosciences 610787\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1:500\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1:500\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003en/a\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e1/2000\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eEP1646Y\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eAbcam ab51252\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1:500\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003en/a\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003en/a\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e1/5000\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMJFR14\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eAbcam ab227047\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1:500\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003en/a\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003en/a\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e1/10000\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eLB509\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eThermoFisher #180215\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1:500\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003en/a\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e1/2000\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSyn505\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eThermoFisher #35-8300\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1:500\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003en/a\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003en/a\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e1/2000\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eEP1532Y\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eAbcam ab137869\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003en/a\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003en/a\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e1:5000\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003en/a\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003eα-Syn expression and purification\u003c/h2\u003e \u003cp\u003e \u003cem\u003eE. coli\u003c/em\u003e strain BL21(DE3) plysS was chemically transformed with pT7-7-α-Syn vector and plated onto Lysogeny broth (LB) agar plate containing Ampicillin. A pre-culture in 5 mL LB medium was inoculated with one clone and incubated at 37\u0026deg;C under 200 rpm shaking for 4 hours. The expression on α-Syn was carried out in LB Medium\u0026thinsp;+\u0026thinsp;Glucose (1 g/L). Cells from LB pre-culture were used for inoculating 100 mL of LB medium. Cells were grown overnight at 37\u0026deg;C under 200 rpm shaking and then diluted in 2.5 L of culture. Protein expression was induced by adding 1 mM IPTG during exponential phase, evaluated by Optical Density at 600 nm reaching 0.6. Cells were harvested after 5 hours of culture at 30\u0026deg;C by 4,000*g centrifuge (JLA 8.1 Beckman Coulter) and pellet was kept at -20\u0026deg;C before purification.\u003c/p\u003e \u003cp\u003eThe pellets were resuspended in lysis buffer (10 mM Tris and 1 mM EDTA (pH 7.2)) and sonicated at 50% max energy, 30 sec on and 30 sec off for three rounds with a probe sonicator (Q-Sonica, Newtown, CT, USA). The sonicated pellets were centrifuged at 20,000\u0026times; g for 30 min, and the supernatant was saved. The pH of the supernatant was then reduced to pH 3.5 using HCl, and the mixture stirred at room temperature (RT) for 20 min and then centrifuged at 60,000\u0026times; g for 30 min. The pellets were discarded. The pH of the supernatant was then increased to pH 7.4 with NaOH and then dialyzed against 20 mM Tris-HCl (pH 7.40) and 100 mM NaCl buffer before loading onto a 75 pg HiLoad 26/600 Superdex column equilibrated with the same buffer with \u0026Auml;KTA pure system. Monomeric fractions were collected and concentrated if needed by using Vivaspin 15R 2 kDa cutoff concentrator (Sartorius Stedim, G\u0026ouml;ttingen, Germany). Purification fractions were checked by using polyacrylamide gel electrophoresis (PAGE) Tris-tricine 13% dying with ProBlue Safe Stain. Protein concentration was evaluated spectrophotometrically by using absorbance at 280 nm and an extinction coefficient of 5960 M\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003eα-Syn fibrillization\u003c/h2\u003e \u003cp\u003eSolutions of monomeric α-Syn at 4 to 5 mg/mL in saline (H\u003csub\u003e2\u003c/sub\u003eO, 100 mM NaCl, and 20 mM Tris-HCl (pH 7.40)) were sterilized by filtration through 0.22 \u0026micro;m Millipore single-use filters and stored in sterile 15 mL conical falcon tubes at 4\u0026deg;C. Sterilized stock was then distributed into safe-lock Biopur individually sterile-packaged 1.5 mL Eppendorf tubes as 500 \u0026micro;L aliquots. The tubes were cap-locked and additionally sealed with parafilm. All previous steps were performed aseptically in a particle-free environment under a microbiological safety laminar flow hood. The samples were loaded in a ThermoMixer (Eppendorf, Hamburg, Germany) in a 24-position 1.5 mL Eppendorf tube holder equipped with a heating lid. Temperature was set to 37\u0026deg;C, and continuous shaking at 2000 rpm proceeded for 4 days. Different polymorphs were obtained as described in (\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003eHuman α-Syn AAV particles\u003c/h2\u003e \u003cp\u003eRecombinant AAV9-CMVie/SynP-syn-WPRE vectors containing the sequence of hSyn, wild-type (wt) or presenting the targeted mutation put under control of the human synapsin promoter was produced by polyethylenimine (PEI) mediated triple transfection of low passage HEK-293T /17 cells (ATCC; cat number CRL-11268). The AAV (adeno-associated virus) expression plasmids pAAV2-CMVie/hSyn-syn-WPRE-pA were co-transfected with the adeno helper pAd Delta F6 plasmid (Penn Vector Core, cat # PL-F-PVADF6) and AAV Rep Cap pAAV2/9 plasmid (Penn Vector Core, cat # PL-T-PV008). Cells are harvested 72h post transfection, resuspended in lysis buffer (150 mM NaCl, 50 mM Tris-HCl pH 8.5) and lysed by 3 freeze-thaw cycles (37\u0026deg;C/-80\u0026deg;C). The cell lysate is treated with 150 units/ml Benzonase (Sigma, St Louis, MO) for 1 hour at 37\u0026deg;C and the crude lysate is clarified by centrifugation. Vectors are purified by iodixanol step gradient centrifugation and concentrated and buffer-exchanged into Lactated Ringer's solution (Baxter, Deerfield, IL) using vivaspin20 100kDa cut off concentrator (Sartorius Stedim, Goettingen, Germany).The genome-containing particle (GCP) titer was determined by quantitative real-time PCR using the Light Cycler 480 SYBR green master mix (Roche, cat # 04887352001) with primers specific for the AAV2 ITRs (fwd 5\u0026prime;-GGAACCCCTAGTGATGGAGTT-3\u0026prime;; rev 5\u0026prime;-CGGCCTCAGTGAGCGA-3\u0026prime;) on a Light Cycler 480 instrument. Purity assessment of vector stocks was estimated by loading 10 \u0026micro;l of vector stock on 10% SDS acrylamide gels, total proteins were visualized using the Krypton Infrared Protein Stain according to the manufacturer\u0026rsquo;s instructions (Life Technologies).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003eIn vitro α-Syn Pathology\u003c/h2\u003e \u003cp\u003eTimed pregnant C57BL/6J female mice were bred at the animal facility of the IMN. Cortices were harvested from embryonic day 18 mouse embryos and dissociated enzymatically and mechanically (using neural tissue dissociation kit, C Tubes, and an OctoDissociator with heaters; Miltenyi Biotec, Bergish-Gladbach, Germany) to yield a homogenous cell suspension. The cells were then plated at 25,000 cells per well in 96-well plates (Corning BioCoat poly-Dlysine imaging plates) in neuronal medium (MACS Neuro Medium, Miltenyi Biotec, BergishGladbach, Germany) containing 0.5% penicillin-streptomycin, 0.5 mM alanyl-glutamine, and 2% NeuroBrew supplement (Miltenyi Biotec, Bergish-Gladbach, Germany). The cultures were maintained with 5% CO\u003csub\u003e2\u003c/sub\u003e at 37\u0026deg;C in humidified atmosphere. The medium was changed by one-third every 3 days, until a maximum of 30 DIV (days \u003cem\u003ein vitro\u003c/em\u003e). After 7 DIV, extemporaneously sonicated α-Syn fibrils were added at a final concentration of 10 nM (equivalent monomeric α-Syn concentration). When relevant, neurons were infected at DIV 10 with α-Syn AAV particles (multiplicity of infection, 1000).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eHigh Content Analysis (HCA) and Laser-Scanning Confocal Microscopy (LSCM)\u003c/h2\u003e \u003cp\u003eAt 30 DIV the plates were processed for immunofluorescence. When relevant, the wells were treated 10 minutes with 25 \u0026micro;M digitonin in 100 mM KCl prior to fixation. All plates were fixed with a solution of 4% formaldehyde 4% sucrose in Phosphate Buffered Saline (PBS) pH 7.4. The cells were then permeabilized with a solution of 3% Bovine Serum Albumin (BSA), 0.1% Triton X in PBS and incubated overnight in a primary antibody solution in BSA 3%, and the next day with the corresponding secondary antibody in BSA 3%. High Content Analysis was performed on multichannel fluorescence images acquired 20\u0026times; using the generic analysis module of the Incucyte S3 (Sartorius) and Top-Hat cellular segmentation was based on the fluorescence signal corresponding to the antibody of interest. Multichannel fluorescence optical sections of the samples were performed (thickness\u0026thinsp;\u0026lt;\u0026thinsp;0.8 \u0026micro;m) using a Leica SP5 LSCM equipped spectral detector, with 488, 561 and 633 nm laser lines, with a motorized X-Y stage and with a mixed stepping motor/piezo Z controller.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003eUrea treatments of recombinant α-Syn monomers and PFFs\u003c/h2\u003e \u003cp\u003eRecombinant α-Syn monomers and fibrils stock preparations (4\u0026ndash;5 mg.ml-1 in Tris-Buffered Saline (TBS)) were diluted to 0.5 mg/ml final concentration in TBS. For each urea concentration, 3 \u0026micro;L (1.5 \u0026micro;g) of recombinant monomers or PFFs were mixed with urea (Sigma) in TBS to reach a final concentration of 0\u0026ndash;8 M urea in 60 \u0026micro;l. Mixtures were pipet mixed prior to incubation for 2h at RT in the dark. At the end of the incubation period, treatments were stopped by quickly diluting the samples with 1260 \u0026micro;L TBS and directly subjecting them to filter-blot assay (science advance\u0026thinsp;+\u0026thinsp;companion paper). Briefly, 100 \u0026micro;l of treated monomers/PFFs were filtered through a nitrocellulose 0.2 \u0026micro;m membrane (Protran, GE) using a dot blot vacuum device (Whatman). Membranes were fixed for 30 min at RT in PBS with paraformaldehyde (PFA) (Sigma) 4% (v/v) final concentration. After three washes with PBS, membranes were saturated with 5% (w/v) skimmed powder milk in PBS-Tween20 0.05% (v/v) and probed with primary (overnight at 4\u0026deg;C) and secondary (1 hour at RT antibodies in PBS-T with 4% (w/v) BSA (see antibody list) with three washes in PBS-T after each step. Immunoreactivity was measured by infrared using an Odyssey Scanner and Image Studio (Li-Cor). The different α-Syn species were quantified by immunolabelling, and expressed as a percentage of related untreated samples, allowing to draw curves of relative disassembly.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003eSarkosyl fractionation of human brain samples homogenates\u003c/h2\u003e \u003cp\u003eHuman cingulate gyrus samples were dissected from freshly frozen \u003cem\u003epost-mortem\u003c/em\u003e brain samples from n\u0026thinsp;=\u0026thinsp;3 control, sporadic PD or MSA subjects respectively. Brain tissue samples were homogenized at 10% (w/v) in solubilization buffer (SB): 10 mM Tris pH 7.5, 100 mM NaCl, 0.1 mM EDTA, 1 mM DTT, Complete EDTA-free protease inhibitors (Roche) and PhosSTOP phosphatase inhibitors (Roche) using a gentleMACS Octo Dissociator (Miltenyi Biotec) with M Tubes, and the Protein extraction program. Protein concentration was determined using Pierce 660 nm Protein Assay kit (Thermo Fisher).\u003c/p\u003e \u003cp\u003eFor the fractionation of sarkosyl-soluble and insoluble components of brain homogenates, the pelleting procedure is similar to previously published protocols termed Sarkospin [see companion paper Laferri\u0026egrave;re et al.]. Samples were mixed 1:1 with SB 4% (w/v) N-lauroyl-sarcosine (sarkosyl, Sigma), 2 U.\u0026micro;l-1 Benzonase (Novagen) and 4 mM MgCl\u003csub\u003e2\u003c/sub\u003e, reaching a final volume of 500 \u0026micro;l. Solubilization was then performed by incubating the samples at 37\u0026deg;C under constant shaking at 600 rpm (Thermomixer, Eppendorf ) for 45 min. Samples were then mixed 1:1 with SB 40% (w/v) sucrose, without sarkosyl, MgCl\u003csub\u003e2\u003c/sub\u003e or Benzonase, in 1 ml polycarbonate ultracentrifuge tubes (Beckman Coulter) and centrifuged at 250,000 g for 1 hour at RT with a TLA 120.2 rotor using an Optima XP benchtop ultracentrifuge (Beckman Coulter). Supernatant were collected by pipetting. Pellets were resuspended directly in the tube with 100 \u0026micro;L of the buffer corresponding to the supernatant (SB 1% sarkosyl 20% sucrose), and mixed with the same buffer in a fresh tube for reaching 1 ml (equal volumes to supernatant). For filter-blot assays, 50 \u0026micro;l of native supernatant/pellet fractions were loaded on the filter-blot mounting and immunolabelled and quantified as described above. The signal for each primary antibody is expressed and plotted as percentage fraction/total (supernatant\u0026thinsp;+\u0026thinsp;pellet).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eIn vivo human α-Syn overexpression and in vivo α-syn Pathology\u003c/h2\u003e \u003cp\u003eWt mice (2 months old) received unilaterally 1 \u0026micro;l of -hSyn AAV (concentration: 4.05 \u0026times; 1013 gcp/ml) mixed either with 1 \u0026micro;l of sonicated α-Syn fibrils (5 mg/ml) or with 1\u0026micro;l of saline by stereotactic delivery to the region immediately above the right \u003cem\u003eSubstantia nigra\u003c/em\u003e (SN) (coordinates from bregma: AP, \u0026minus;\u0026thinsp;2.9, L, \u0026minus;\u0026thinsp;1.3, DV, \u0026minus;\u0026thinsp;4.5) at a flow rate of 0.4 \u0026micro;l/min, and the pipette was left in place for 5 minutes after injection to avoid leakage. Animals were euthanized after 4 months. Ten mice were used in each group \u0026mdash; male and female mixed. The brains were perfused with saline, postfixed for 3 days in 10 ml of 4% PFA at 4\u0026deg;C, cryoprotected in gradient 20% sucrose in PBS before being frozen by immersion in a cold isopentane bath (\u0026ndash;60\u0026deg;C) for at least 5 minutes, and stored immediately at \u0026minus;\u0026thinsp;80\u0026deg;C until sectioning for immunohistochemistry (ICH).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eImmunohistochemistry in mouse brains sections\u003c/h2\u003e \u003cp\u003eIHC staining of phospho-S129 α-Synuclein (pS129 α-Syn) and tyrosine hydroxylase (TH) neurons on coronal serial sections was performed as previously described (\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e). The monoclonal rabbit anti-pS129 α-Syn antibody EP1536Y (ab51253, Abcam, Cambridge, UK) was used, followed by incubation with labelled polymer-HRP anti-rabbit (Dako EnVision\u0026thinsp;+\u0026thinsp;TM Kit, K4011, Agilent, Santa Clara, CA, USA). Visualization of pS129 α-Syn staining was performed with Dako DAB (K3468), and sections were counterstained with the Nissl stain. The actual number of pS129 α-Syn aggregates per structure (\u003cem\u003eStriatum\u003c/em\u003e and SN) and the total number of pS129 α-Syn aggregates were assessed using whole-section acquisition by Panoramic Scan II (3DHISTECH, Hungary) and further processed with the ad-hoc developed QuPath algorithm.\u003c/p\u003e \u003cp\u003eFor TH neurons quantification, ICH was performed for each animal on every fourth midbrain sections spanning the entire rostro-caudal \u003cem\u003eSubstantia Nigra pars compacta\u003c/em\u003e (SNpc). TH-positive cells in ipsilateral and controlateral SN were segmented and counted on each section using Qpath and the loss between the injected and the non-injected side was calculated for each animal.\u003c/p\u003e \u003cp\u003e \u003cem\u003eImmunofluorescence in FFPE mouse and human brain sections.\u003c/em\u003e \u003c/p\u003e \u003cp\u003eAdult male 129SV (6\u0026ndash;8 weeks old) unilaterally received 2 \u0026micro;L of sonicated α-Syn fibrils (4 mg/mL) by stereotactic delivery at a flow rate of 0.4 \u0026micro;L/min, and the pipette was left in place for 5 min after injection to avoid leakage. Delivery was performed within the right \u003cem\u003eStriatum\u003c/em\u003e (AP, \u0026minus;\u0026thinsp;0.1; L, +\u0026thinsp;2.5; DV, +\u0026thinsp;3.8). Animals were euthanized after 6 months and were transcardially perfused with TBS (pH\u0026thinsp;=\u0026thinsp;7.4) followed by 4% PFA in PBS pH\u0026thinsp;=\u0026thinsp;7.4 at 4\u0026deg;C. Brains were subsequently postfixed in the same fixative, paraffin embedded, and 10 \u0026micro;m sections were obtained with a rotative microtome (Leica, Milan, Italy). The sections of interest were deparaffinized and processed for epitope retrieval: the slides were immersed in citrate buffer pH 6 (Dako Agilent Technologies, Les Ulis, France) and placed in a TintoRetriever Pressure Cooker (Bio SB, Santa Barbara, CA, USA) at high pressure, 114\u0026ndash;120\u0026deg;C for 10 min. After a cooling period of 20 min, the slides were washed twice for 5 min in PBS at RT. They were then processed for immunofluorescence. Draq7 Thermo Fisher Scientific was used to image nuclei. The Alexa Fluor-coupled secondary antibodies were from Thermo Fisher (Alexa 488, 568, and 674). The sections were acquired using a Pannoramic slide scanner (3D HISTECH, MM France) in epifluorescence mode, and multichannel fluorescence optical sections of the samples were performed (thickness\u0026thinsp;\u0026lt;\u0026thinsp;0.8 \u0026micro;m) using a Leica SP5 Laser Scanning Confocal Microscope.\u003c/p\u003e \u003cp\u003eHuman PD brain sections were deparaffinized and processed as described above and sequentially stained with (i) Syn1 (Clone 42, BD), and EP1536Y (Abcam) and revealed with the respective secondary antibodies, and only then with (ii) Alexa Fluor-coupled MJFR1. Draq7 is used according to the manufacturer\u0026rsquo;s instructions. Multichannel fluorescence optical sections of the samples were performed (thickness\u0026thinsp;\u0026lt;\u0026thinsp;0.8 \u0026micro;m) using a Leica SP5 Laser Scanning Confocal Microscope equipped with a spectral detector, 488, 561, and 633 nm laser lines, a motorized X-Y stage, and a mixed stepping motor/piezo Z controller.\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cp\u003e \u003cem\u003eα-Syn overexpression using AAVs: filling of the neuronal cytoplasm and pS129 positivity without assembly of fibrils and without inclusions.\u003c/em\u003e \u003c/p\u003e \u003cp\u003eThe overexpression of hSyn is commonly used to model αSP in mouse neurons, both \u003cem\u003ein vitro\u003c/em\u003e and \u003cem\u003ein vivo\u003c/em\u003e. It is supposed to facilitate the emergence of α-Syn aggregation by bringing its concentration closer to the nucleation threshold. It also allows studying the human protein in a rodent context, tentatively increasing the translational relevance of the model. Several studies reported the induction of α-Syn aggregation by simple overexpression, others reported that aggregation must be induced by an additional seeding event such as PFFs treatment (\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e). In both cases, the discrimination between the adverse effects due to the increased α-Syn expression levels and those due to the proper amyloid aggregation are difficult to determine and relies on the capability of specifically identifying amyloid α-Syn fibrils \u003cem\u003ein situ\u003c/em\u003e.\u003c/p\u003e \u003cp\u003epS129 is a very specific marker of α-Syn inclusions in human neuropathology(\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e)(\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e) (\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e). Similarly, when α-Syn PFFs are used as seeds and directly injected into the brain of wt mice, pS129 antibodies specifically allow the visualization of the α-Syn inclusions that develop and spread into the mouse brain(\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e)(\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e) (\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e). Four months after an injection of PFFs at the level of the SN, several types of pS129-positive (pSyn) inclusions formed that were reminiscent of those observed in patients, both locally in the SN and at distance in the \u003cem\u003eStriatum\u003c/em\u003e of the same hemisphere (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA-E). This experimental αSP phenotype can also be observed after the intracerebral injection in mice of α-Syn fibrils extracted from DLB patient brain samples using detergent, with the formation of typical pS129-positive α-Syn inclusions (\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e). Note, however, that in agreement with the latter publication, we found that 4 months after injection, the αSP seeded by PFFs was not associated with any specific loss of TH-positive neurons in the SNpc. Indeed, the loss observed, which was around 28% compared to the non-injected side, was not different in sham-injected controls which received an injection of saline (Fig. S1).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eFor the sake of comparison, we injected AAV particles carrying the cDNA of hSyn under the control of a synapsin promoter (AAV-syn) to induce an overexpression of hSyn in the SN neurons (Fig. S1). In these AAV-injected animals, hSyn is readily detectable at 4 months post-injection in the cell bodies of the dopaminergic neurons populating the SN as well as in their striatal projections/terminals (Fig. S1). In these animals, \u003cem\u003ebona fide\u003c/em\u003e intracellular inclusions cannot be identified inside the somata of the dopaminergic neurons (the cell bodies become globally/diffusely pS129-positive), and most strikingly, distant striatal pS129-positive inclusions are absent (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA-E). Such lack of long-distance spread as well as the diffuse perikaryal morphology of the pS129 signal is reminiscent of previous \u003cem\u003ein vivo\u003c/em\u003e experiments in which α-Syn overexpression resulted in S129 phosphorylation without fibrillar aggregation (\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eNote that 4 months after the intracerebral injection of AAV-syn, on top of the absence of αSP inclusions, no specific loss of TH-positive neurons was detected in the SNpc. Compared to the non-injected side, the loss observed was identical to sham-injected controls (Fig. S1).\u003c/p\u003e \u003cp\u003eIn the last experiment, we mixed AAV-syn particles with PFFs and injected them at the level of the SN. In these \u0026ldquo;seeded plus AAV-infected\u0026rdquo; animals, hSyn was readily detectable in the cell bodies of the dopaminergic neurons of the SN as well as in their striatal projections/terminals (Fig. S1). While \u003cem\u003ebona fide\u003c/em\u003e intracellular inclusions were still not identified inside the somata of the dopaminergic neurons (here also the cell bodies were globally/diffusely pS129-positive), distant striatal pS129 positive inclusions were present like in the PFF-only condition (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA-E). Induction of an inclusion pathology with long-distance spread by adding PFFs to AAV-syn particles clearly indicates that the processes triggered by AAV-syn and PFFs are completely different (compare \u0026ldquo;AAV-Syn\u0026rdquo; and \u0026ldquo;AAV-syn\u0026thinsp;+\u0026thinsp;PFFs\u0026rdquo; in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC), yet they are both associated with the emergence of a pS129-positive signal at the injection site (compare \u0026ldquo;AAV-Syn\u0026rdquo; and \u0026ldquo;PFFs\u0026rdquo; in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB). This indicates that S129 phosphorylation is a misleading marker in conditions of neuronal α-Syn overexpression \u003cem\u003ein vivo\u003c/em\u003e.\u003c/p\u003e \u003cp\u003eNote that here also, 4 months after the intracerebral injection of the AAV-syn plus PFFs mix, no specific loss of TH-positive neurons was detected in the SNpc. The loss observed compared to the non-injected side was identical to sham-injected controls (Fig. S1).\u003c/p\u003e \u003cp\u003eIn parallel to these \u003cem\u003ein vivo\u003c/em\u003e experiments, we evaluated the relationships between α-Syn overexpression, phosphorylation, and amyloid aggregation in primary cultures of cortical neurons infected or not with AAV-syn and seeded or not with PFFs. In line with the \u003cem\u003ein vivo\u003c/em\u003e experiment, pS129 was not detected in non-infected control neurons and only appeared upon seeding with PFFs. Like in αSP pathology, the seeded structures which are positive for pS129 are \u003cem\u003ebona fide\u003c/em\u003e cytological inclusions (LNs, perinuclear and intranuclear inclusions (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e2\u003c/span\u003eA,B). In addition, pS129 staining was fully resistant to permeabilization of the plasma membrane with digitonin prior to fixation, indicating that it corresponds to large insoluble α-Syn assemblies \u0026ndash; i.e., fibrils \u0026ndash; unable to diffuse out of the neurons through the digitonin pores (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e2\u003c/span\u003eC,D).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eIn contrast, in non-seeded conditions, AAV-syn-infected neurons already showed a diffuse pS129 signal with no identifiable inclusion (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e2\u003c/span\u003eA,B). At major variance from the previous experiment, this diffuse staining was completely lost upon membrane permeabilization, indicating that S129 phosphorylation concerned in this case soluble α-Syn species that can freely diffuse out of the neurons and not insoluble fibrils (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e2\u003c/span\u003eC,D). However, we observe that if the AAV-syn-infected neurons were also seeded with PFFs, pS129 staining became fully resistant to permeabilization revealing that fibril assembly could still be seeded at the expense of the soluble pS129 α-Syn species that were formed in reaction to the overexpression overflow (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e2\u003c/span\u003eC,D).\u003c/p\u003e \u003cp\u003eThese data show that in conditions of α-Syn overexpression, pS129 cannot be considered a reliable surrogate marker of neuronal α-Syn fibrillization and aggregation. In addition, even if the pS129 signal integral is increased by seeding (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e2\u003c/span\u003eA-D), phosphorylation is not discriminant \u003cem\u003eper se\u003c/em\u003e: phosphorylated fibrils cannot be distinguished from the phosphorylated soluble species \u0026ldquo;artifactually\u0026rdquo; emerging in these neurons.\u003c/p\u003e \u003cp\u003eTo identify aggregated α-Syn more specifically in these experiments we tested the routine conformational antibody LB509. LB509 has a double selectivity, detecting hSyn mostly under its aggregated form. We found in our conditions that this conformational preference was very relative - that is, in hSyn overexpressing conditions (AAV-syn) LB509 detects non-aggregated α-Syn in neurons, particularly inside the synapses. This staining is completely lost upon plasma membrane permeabilization prior to fixation confirming its non-fibrillar nature. When such AAV-syn infected neurons were seeded with PFFs, aggregation could only be inferred from morphological changes and not be quantified by measuring an increase of the LB509 signal integral in the intact cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e2\u003c/span\u003eE). The fibrillar status of the assemblies seeded by PFFs and recognized by LB509 could, however, be revealed by their resistance to plasma membrane permeabilization (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e2\u003c/span\u003eE).\u003c/p\u003e \u003cp\u003eInterestingly, by comparing the signal distribution of pS129 and LB509 by double immunofluorescence in the latter permeabilized neurons, we observe that the signals are not completely overlapping (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e2\u003c/span\u003eF). This indicates that pS129-positive amyloid α-Syn fibrils coexist with unphosphorylated fibrils, and that some pS129-positive fibrils are not discovered by LB509. This suggests a conformation and phosphorylation heterogeneity of the C-terminus (the target of both antibodies) belonging to the monomers engaged into the amyloid fibril core.\u003c/p\u003e \u003cp\u003eIn conclusion, in experimental models introducing and manipulating the concentration of hSyn monomers in neurons, pS129 cannot be considered a reliable marker of aggregation because overexpressed α-Syn is phosphorylated under its soluble form. Appropriate tools are needed for identifying amyloid assemblies, which are less affected by the detection of the basal overexpression and targeting the proper amyloid conversion.\u003c/p\u003e \u003cp\u003e \u003cem\u003eAnalysis of a set of commercial antibodies reveals overlooked yet interesting features.\u003c/em\u003e \u003c/p\u003e \u003cp\u003eUsing recombinant hSyn we produced and selected 3 different strains of PFFs which we characterized in previous studies: iso1, iso3, and 1B (\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e). For this study, we tested a panel of \u0026ldquo;routine\u0026rdquo; commercial antibodies on these different PFFs to investigate their possible ability to differentially detect the fibrils strains and the monomeric protein (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e3\u003c/span\u003eA).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eAs expected, MJFR1 (\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e) and EP1646Y, respectively targeting the C- and the N-terminus of the protein, did not show any preference for the monomeric or the fibril α-Syn assemblies. MJFR14 which has been described as preferentially binding to fibrils (\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e) did not show any conformation specificity in our experimental settings. In contrast, LB509 (\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e) which targets the C-terminus (\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e) recognized both α-Syn forms, but with a clear preference for fibrils. A similar conformation-dependent preference was observed for Syn505 which binds to the 1\u0026ndash;12 end of the N-terminus (\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e), and SynF1 which is targeted to the C-terminus (\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e) appeared most selective of amyloid fibrils vs. monomers.\u003c/p\u003e \u003cp\u003eWhile these results are globally in line with the expectations (excepted for MJFR14), we identified two particularly striking \u0026ndash; yet overlooked \u0026ndash; properties for 2 antibodies widely used in the literature: (i) Syn1 (Clone 42) which targets the NAC region of the protein(\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e) is often referred to as a \u0026ldquo;pan-α-Syn\u0026rdquo; antibody held capable of recognizing all forms of the protein. In fact, we found that it selectively recognized the monomeric protein and that it did not bind to the fibrils. This confirms repeated previous observations made by us and others (\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e, \u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e, \u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e); (ii) D37A6 which is held a rodent specific antibody targeting an upstream region of the C-terminus (surrounding E105), did indeed not recognize the human monomer. However, and most strikingly, it did show an affinity for hSyn engaged into amyloid fibrils.\u003c/p\u003e \u003cp\u003eTo further explore these conformation specificities, we used increasing concentrations of urea to provoke the gradual disassembly of the 3 fibrils strains back into monomers and to measure the impact of disassembly on immunoreactivity (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e3\u003c/span\u003eB). In agreement with the previous experiment (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e3\u003c/span\u003eA), disassembly did not modify the signal of the conformation-independent antibodies MJFR1, EP1646Y and MJFR14, while it collapsed the signal of all the fibril-specific antibodies which we identified, i.e., LB509, Syn505, SynF1 and D37A6. As a mirror image, progressive fibril disassembly led to the progressive appearance of Syn1 immunoreactivity as more monomers were released from the amyloid assemblies, confirming the monomer-selectivity of Syn1.\u003c/p\u003e \u003cp\u003eFinally, we tested the above antibodies on PD and MSA brain homogenates comparing their sarkosyl insoluble and soluble fractions (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e3\u003c/span\u003eC and Fig. S3). The results confirm that in a biochemical assay, LB509, SynF1 and Syn505 recognize pathological α-Syn and showed that Syn1 and D37A6 behave as amyloid state-dependent antibodies. Note that to our knowledge, changes in immunoreactivity towards Syn1 is the first amyloid conversion-dependent change of α-Syn taking place at the proper fibril core level and amenable to detection by an immunological method.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eAmyloid conversion assays in neurons\u003c/h2\u003e \u003cp\u003eConsidering these results on synthetic and extracted fibrils, we explored the use of the antibodies which we retained conformation-dependent (see above) in primary cultures of mouse cortical neurons overexpressing hSyn.\u003c/p\u003e \u003cp\u003eWe first performed double-immunofluorescence imaging using SynF1 or Syn505 in combination with EP1536Y which detects pS129 α-Syn. To discriminate aggregated α-Syn and its soluble forms, we performed the same staining on neurons permeabilized prior to fixation (Figs.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e4\u003c/span\u003eA,B).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eBoth antibodies confirmed their preferential affinity for aggregated α-Syn, the quantification of the signal integral showing a significant increase for each of the antibodies in the PFF-treated neurons compared to the control conditions (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e4\u003c/span\u003eA). SynF1 was also sensitive to overexpression, but the differential intensity between PFF-treated and untreated conditions was larger than with LB509 (Compare Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e2\u003c/span\u003eE and Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e4\u003c/span\u003eA,B). The signal shown by Syn505 in these experimental conditions was generally weak but provided a more clearcut readout of α-Syn aggregation in the PFF-treated neurons. For both antibodies, permeabilization of the neurons prior to fixation, which allowed the retention of aggregated α-Syn and the release of the non-aggregated forms, improved the difference between the PFF-treated and non-treated conditions, providing a \u0026ldquo;filtered/contrasted\u0026rdquo; image of the α-Syn pathology (Figs.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e4\u003c/span\u003eA).\u003c/p\u003e \u003cp\u003eHowever, when we considered pS129 co-staining with EP1536Y, we observed that similarly to what we noted with LB509, the neurons positive to the conformational antibodies SynF1 or Syn505, and those positive to EP1536Y only partially overlapped, both in non-permeabilized and permeabilized conditions (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e4\u003c/span\u003eB). This suggested that pS129 is not a comprehensive readout of aggregated α-Syn, since in some neurons or in some areas of the same neuron, aggregated α-Syn is not phosphorylated. This could be due to a difference in neuronal types, or, in the second case, it could be due to the \u0026ldquo;snapshot\u0026rdquo; of an evolving process inside a single neuron (progressive phosphorylation or dephosphorylation of the aggregates). On the other hand, we also observed neurons bearing pS129-positive aggregates that were insoluble, yet were negative to the conformational antibodies SynF1 or Syn505 (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e4\u003c/span\u003eB).\u003c/p\u003e \u003cp\u003eAiming at detecting/quantifying amyloid conversion in intact neurons irrespective of pS129 we decided to exploit the conformational properties of the Syn1 antibody. Again, the epitope targeted by this antibody (aa91-99) is part of the NAC domain of the protein which gets engaged and is instrumental in the protein-protein interactions established between stacked α-Syn monomers during assembly of the amyloid core. As a result, once the monomers are trapped inside the fibrillar structure, the epitope is no longer accessible to the antibody. Indeed, when we treated AAV-syn-infected neurons with PFFs we observed the appearance of inclusions with a collapse of Syn1 immunoreactivity in inclusions yet positive to MJFR1 (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e4\u003c/span\u003eC). Thus, using a double staining with a conformation-independent antibody like MJFR1 (or EP1646Y, not shown) in combination with Syn1, we can easily differentiate soluble α-Syn from amyloid α-Syn assemblies and identify directly the neurons containing fibrils (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e4\u003c/span\u003eC). Indeed, in neurons not seeded with PFFs, MJFR1 and Syn1 staining completely overlapped and both signals disappeared if the neurons were permeabilized prior to fixation. This indicates that normal neurons only contain soluble, non-amyloid α-Syn. When neurons were seeded with PFFs instead, a Syn1 negative MJFR1-positive population appeared which was unsensitive to permeabilization, corresponding to the neurons in which α-Syn underwent amyloid conversion (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e4\u003c/span\u003eC). Note that amyloid conversion can similarly be tracked using EP1536Y to reveal pS129 α-Syn together with Syn1 (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e4\u003c/span\u003eD). In this case, amyloid α-Syn is EP1536Y-positive and Syn1-negative.\u003c/p\u003e \u003cp\u003eIn conclusion, pS129 is a surrogate marker of α-Syn aggregation that can generate both false positives and false negatives, especially in condition of α-Syn overexpression. The commercial conformation-dependent antibodies we tested and that do bind preferentially to aggregated α-Syn present only a relative specificity and can miss a significant fraction of the amyloid α-Syn fibrils present in neurons (false negatives). In other words, they are not able to properly discriminate aggregation \u003cem\u003ein situ\u003c/em\u003e. Moreover, it should be highlighted that these conformation-dependent antibodies target the arrangements the N or the C terminals which derive only secondarily from the aggregation process and are not involved in the assembly of the amyloid core. In contrast, the loss of accessibility of the NAC epitope recognized by Syn1 is a direct indication of the enrollment of the protein in an amyloid structure and can thus be used in combination with conformation-independent antibodies to specifically highlight amyloid α-Syn assemblies \u003cem\u003ein situ.\u003c/em\u003e\u003c/p\u003e \u003cp\u003e \u003cem\u003eHigh Content Analysis amyloid α-Syn conversion in 96 well primary neuronal cultures: revisiting the impact of α-Syn mutations.\u003c/em\u003e \u003c/p\u003e \u003cp\u003eWe thus decided to take advantage of this new method for investigating a series of α-Syn mutations \u0026ndash; either disease-relevant or preventing S129 phosphorylation \u0026ndash; to determine their propensities to act as a stand-alone trigger of α-Syn amyloid conversion inside neurons and/or to accelerate the process once seeded by PFFs (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e5\u003c/span\u003e). We infected neurons with 7 distinct AAVs carrying the cDNA of hSyn bearing point mutations that have been associated with the emergence of autosomal-dominant inherited PD: A29E, A30P, E46K, G51D, H50Q, A53E, A53T (\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e). The mechanisms that link these mutations to PD are generally thought to depend on a facilitation of the amyloid assembly of α-Syn, but although appealing, this assumption is mostly based on historical protein-only observations made \u003cem\u003ein vitro\u003c/em\u003e. We also included in our exploration the experimental S129A mutant, coding for a non-phosphorylatable form of the protein at S129 because here also, there is a debate on the functional impact of this phosphorylation on the process of spontaneous or of seeded α-Syn assembly (\u003cspan additionalcitationids=\"CR53\" citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eUsing MJFR1 we quantified the levels of neuronal overexpression achieved for variants and found that the different AAV infections yielded comparable expression levels, except for A30P which was slightly less expressed than its counterparts, and for E46K which was barely expressed and detectable (this observation was repeated with different AVV-E46K α-Syn production batches, not shown) (Fig. S5). In addition, expression of E46K appeared to be neurotoxic in our primary cultures of cortical neurons, confirming previous observations (\u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e). We thus did not consider or discuss the possible impact of the E46K mutation on the amyloid conversion of α-Syn \u003cem\u003ein situ\u003c/em\u003e.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eFigure\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e5\u003c/span\u003eA shows that as it was the case for wt hSyn, all disease-associated mutants were significantly phosphorylated in basal conditions under the simple effect of overexpression (compare with S129A which cannot be phosphorylated on this residue). The phosphorylation level in the unseeded conditions was comparable for all disease-associated mutants.\u003c/p\u003e \u003cp\u003eUpon seeding with PFFs, a modest 2\u0026ndash;3 fold increase of pS129A was observed for all variants, excepted for S129A which is non phosphorylatable (note that with this scale adapted for overexpression conditions, the phosphorylation of endogenous α-Syn upon seeding exists but is invisible), E46K which showed virtually no response because it was barely expressed, and A30P which exhibited the strongest basal phosphorylation in spite of its lower expression level, with little room left to detect a further impact of seeding on pS129 .\u003c/p\u003e \u003cp\u003eFigure\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e5\u003c/span\u003eA underlines that under overexpression conditions, little can be said on the impact of specific mutations on either basal or seeded α-Syn aggregation compared to wt α-Syn, apart perhaps the basal pS129 levels of A30P which could be interpreted as the indication of a more pronounced spontaneous aggregation of this variant. The next experiments indicate that this is not the case.\u003c/p\u003e \u003cp\u003eIn order to track true α-Syn amyloid conversion in these conditions, we used the MJFR1-Syn1 staining combination and derived the % of neurons bearing amyloid inclusions (i.e., bearing MJFR1-positive Syn1-negative inclusions) (Figs.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e5\u003c/span\u003eB,C). As for wt α-Syn, in unseeded conditions virtually all the neurons were perfectly double stained for all mutants (including S129A), indicative that none of the variants induced spontaneous aggregation with amyloid conversion. This indicates that the pS129 signal observed in unseeded conditions (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e5\u003c/span\u003eA) is not due to spontaneous aggregation but simply to overexpression and phosphorylation of non-amyloid forms of the protein. These results reveal that in cortical neurons, the α-Syn mutations that cause autosomal dominant PD fail to trigger the spontaneous amyloid conversion of α-Syn into fibrils. The same holds true for the S129A variant showing that preventing phosphorylation at S129 is not sufficient not trigger spontaneous fibrillization.\u003c/p\u003e \u003cp\u003eWe thus reasoned that in a neuronal context, these mutations might instead favor the seeded assembly of α-Syn into amyloid fibrils. Upon seeding with PFFs, we observed a massive burst of the population of neurons bearing amyloid inclusions (MJFR1-positive Syn1-negative) (Figs.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e5\u003c/span\u003eB,C). Unexpectedly enough, the extent of amyloid conversion here also appeared comparable for all the variants: none of the mutations appeared to favor the seeded assembly process. At the opposite, the A53E variant even inhibited the amyloid conversion of α-Syn in neurons confirming several previous observations made \u003cem\u003ein vitro\u003c/em\u003e regarding this mutant (\u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e56\u003c/span\u003e, \u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e57\u003c/span\u003e). Collectively, these data suggest that the mechanisms by which familial α-Syn mutations might cause autosomal dominant PD are neither related to triggering nor to facilitation of α-Syn fibrillization in intact neurons. In addition, phosphorylation of S129 which can be misleading as a marker of fibrillization, does not seem to inhibit spontaneous or seeded α-Syn fibrillization either.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003eProbing amyloid conversion in histological brain sections: revisiting the status of α-Syn in pathological inclusions\u003c/h2\u003e \u003cp\u003eThese results prompted us to put amyloid α-Syn conversion under scrutiny in brain sections presenting clear signs of α-Syn inclusion pathology. We used in parallel sections from wt mice sacrificed 6 months after an intra-striatal injection of human PFFs and \u003cem\u003epost-mortem\u003c/em\u003e sections from a patient with sporadic PD (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e6\u003c/span\u003e). In mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e6\u003c/span\u003eA-C), the inclusion pathology was revealed using the antibody pair pS129 and Syn1 like in the primary cultures of Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e4\u003c/span\u003eD. The figure shows a region of the right basolateral amygdala (BLA) filled with pS129-positive α-Syn neuronal inclusions of 3 types: LNs, neuronal perikaryal inclusions with a more or less compacted \u0026ldquo;Lewy Body-like\u0026rdquo; appearance, depending on the maturation level of the inclusion, and a few neuronal intranuclear inclusions (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e6\u003c/span\u003eA, in green). Co-staining with Syn1 (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e6\u003c/span\u003eB, in purple) revealed the physiological pool of non-amyloid α-Syn present in the synapses that were scattered all over the field of view. However, while as expected many pS129 inclusions appeared Syn1-negative (green inclusions in the overlay of Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e6\u003c/span\u003eC, see a few examples pointed by empty arrowheads), indicating inclusions exclusively made of amyloid α-Syn, a significant number of inclusions alternatively appeared partially or totally Syn1-positive (white inclusions in the overlay of Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e6\u003c/span\u003eC, see a few examples pointed by empty arrows), indicative of the presence of non-amyloid α-Syn inside the inclusions. It can be noted that the presence of non-amyloid α-Syn in experimental inclusions seeded in mice by PFFs does not seem to depend on the inclusion type since all of them can be concerned by the possible presence of non-amyloid α-Syn detectable by Syn1.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003ePanels of Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e6\u003c/span\u003eD-J and K-Q show the focused exploration of the amyloid status of α-Syn respectively in a LN and in a Lewy body both present in a \u003cem\u003epost-mortem\u003c/em\u003e SN brain section of a sporadic PD patient. The section was first double-labeled and revealed using EP1536Y and Syn1 as before. The section was then hybridized with a third fluorophore-coupled MJFR1 antibody which detects hSyn irrespective of its conformation and phosphorylation (see Figs.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e3\u003c/span\u003e, \u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e5\u003c/span\u003e and \u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003eS5\u003c/span\u003e). The LN of Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e6\u003c/span\u003e is pS129-positive on most of its length (green) with a superposable staining pattern shown by MJFR1 (red) (see in particular the pS129 and MJFR1 traces in the line scan of the overlay Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e6\u003c/span\u003eJ). Instead, staining with Syn1 (purple) is variable along the length of the LN: some regions are Syn1-negative indicating a purely amyloid composition (empty arrowhead), while others are Syn1-positive showing the presence of non-amyloid α-Syn (empty arrows). The line scan of Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e6\u003c/span\u003eJ makes it clear that the MJFR1 and the Syn1 signals were completely decoupled in the left end of the LN highlighting the exclusive presence of fibrils in this portion of the inclusion. At major variance, it appeared difficult to identify amyloid-only subregions in the LB. The 3 antibodies showed comparable staining patterns within the inclusion, with spatially correlated MJFR1 and Syn-1 signals (see the MJFR1 and Syn1 traces in the line scan of the overlay Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e6\u003c/span\u003eQ and compare with Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e6\u003c/span\u003eJ). In contrast with the somatic inclusions seeded in mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e6\u003c/span\u003eA-C), and with the LN shown before, Syn1 positivity was observed for all the LBs we investigated. This indicated that LBs in PD are pathological inclusions containing non-amyloid α-Syn. This is in line with the poor amyloid status of PD brain extracts compared to MSA ones [see companion paper Lafrerri\u0026egrave;re et al.] and confirms the pioneering observations of Shahmoradian and colleagues who reported that α-Syn fibrils were identifiable in many but not all α-Syn inclusions in PD (\u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e58\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eOur results indicate that pS129 which is widely used as a surrogate marker of α-Syn aggregation in experimental and clinical settings leads to the positive scoring of brain cells in which α-Syn aggregation does not take place. This is particularly evident for models presenting no identifiable inclusion pathology but only diffuse anatomical-scale brain staining (\u003cspan additionalcitationids=\"CR33 CR34 CR35\" citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e). This however also applies to models in which overexpressing α-Syn produces images mistaken for intracellular inclusions and corresponding to high concentrations of soluble phosphorylated α-Syn species (\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e) unrelated to an amyloid aggregation process (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). It is worth highlighting that beyond the case of the PLP-α-Syn mouse, and of the AAV-Syn infections shown here, the brain sections of several transgenic mouse models yielding a neuronal overexpression of α-Syn present widespread pS129-positive images with a distribution and intensities which are uncorrelated with the patterns revealed by the amyloid probe h-FTAA (\u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e59\u003c/span\u003e). This questions the amyloid nature of the images revealed using pS129.\u003c/p\u003e \u003cp\u003eThis issue with pS129 represents a problem for the quantitative neuropathology of α-Syn in cellular and animal models, but also for the correct interpretation of \u003cem\u003epost-mortem\u003c/em\u003e brain sections from patients in which pS129-positive images do not grant the presence of amyloid α-Syn fibrils.\u003c/p\u003e \u003cp\u003eUnfortunately, relying on conformation-dependent antibodies which are commercially available is not an alternative because their preference for amyloid α-Syn fibrils with regards to other soluble species is partial (\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e). In brain sections, their signal-to-noise ratio for α-Syn inclusion detection is low because α-Syn physiologically addressed to pre-synapses produces an overwhelming background (for instance with SynF1, and to a lesser degree with Syn 303 and Syn505 (not shown)).\u003c/p\u003e \u003cp\u003eOur observation that the NAC epitope recognized by Syn1 is rendered inaccessible during fibril assembly offers the possibility to characterize inclusions revealed by standard antibodies and to establish their amyloid status. Syn1 is a mouse monoclonal antibody which can be used in combination with any other rabbit antibody recognizing the C-terminal region of α-Syn. In double immunofluorescence settings, any cytological structure/region positively stained with the latter types of anti-α-Syn antibodies and concomitantly appearing Syn1-negative corresponds to a purely amyloid inclusion made of α-Syn fibrils.\u003c/p\u003e \u003cp\u003eUsing this approach to score α-Syn fibrillization in primary neurons, we made the unexpected observation that under overexpression conditions, wt hSyn as well as α-Syn mutants associated with familial PD do not trigger spontaneous fibrillization. The massive pS129 signal which is observed corresponds to non-amyloid forms of the protein. Further, these α-Syn mutants do not facilitate the process of seeded fibrillization, and even inhibit it in the case of A53E. Altogether, this goes against the notion that the α-Syn mutants found in familial PD would provoke the onset of PD because they would tend to facilitate α-Syn fibrillization. Though intellectually appealing, this generic hypothesis is mainly based on the results of historical protein-only experiments(\u003cspan additionalcitationids=\"CR61 CR62 CR63\" citationid=\"CR60\" class=\"CitationRef\"\u003e60\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR64\" class=\"CitationRef\"\u003e64\u003c/span\u003e) or obtained using reconstituted systems with α-Syn and lipids(\u003cspan citationid=\"CR65\" class=\"CitationRef\"\u003e65\u003c/span\u003e) or deduced from manipulations in yeast and other non-neuronal cells(\u003cspan citationid=\"CR66\" class=\"CitationRef\"\u003e66\u003c/span\u003e) (\u003cspan citationid=\"CR67\" class=\"CitationRef\"\u003e67\u003c/span\u003e). It is worth noting however that the inhibiting effect of A53E on fibrillization was reported several times in such systems(\u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e56\u003c/span\u003e, \u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e57\u003c/span\u003e, \u003cspan citationid=\"CR67\" class=\"CitationRef\"\u003e67\u003c/span\u003e, \u003cspan citationid=\"CR68\" class=\"CitationRef\"\u003e68\u003c/span\u003e) but the bearings of this observation for the possible causal role of fibrillization in PD did not catch a general attention. It is worth noting that a similar problem was also repeatedly encountered \u003cem\u003ein vitro\u003c/em\u003e for the A30P mutant (\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e, \u003cspan citationid=\"CR63\" class=\"CitationRef\"\u003e63\u003c/span\u003e, \u003cspan citationid=\"CR69\" class=\"CitationRef\"\u003e69\u003c/span\u003e), which led Lansbury and his colleagues to conclude 23 years ago that fibrillization was not playing a prime pathogenic role in PD.\u003c/p\u003e \u003cp\u003eOur present observation that α-Syn mutations causing autosomal dominant transmission of PD with a 100% penetrance neither trigger nor facilitate the seeded fibrillization of α-Syn in primary neuronal cultures is in line with the conclusion of Lansbury and colleagues. This indeed suggests that besides fibrils, non-amyloid α-Syn form(s) might play a prominent role in the pathophysiology of PD (\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e). Though counterintuitive, it should be noted that structural analyses have also shown that familial PD α-Syn point mutations are all strategically located in the protein to either prevent the proper stabilization of the canonical type I amyloid fold or to interfere with the \u0026ldquo;orthodox\u0026rdquo; intertwining of the protofilaments during α-Syn fibril assembly (\u003cspan citationid=\"CR70\" class=\"CitationRef\"\u003e70\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eBesides the mutations of familial PD, the prominent phosphorylation of S129 in pathological α-Syn inclusions has prompted investigations aimed at determining the functional role of this modification (\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e). This led to the conclusion that this modification has indeed an impact on α-Syn fibrillization. However, for some, pS129 facilitates seeded fibrillization(\u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e) while for others pS129 inhibits the process(\u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e) (\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e). Using the overexpression of the non-phosphorylatable α-Syn mutant S129A and our amyloid α-Syn detection method, we find that preventing S129 phosphorylation in primary neurons has no impact on either spontaneous or seeded fibrillization, pointing to a different and yet unknown role of the modification. One possibility to explain that pS129 appears on fibrils or on overexpressed monomers is that during α-Syn fibrillization or α-Syn mis-sorting, the protein no longer interacts with VAMP2 as it normally does when α-Syn is addressed to the presynaptic vesicles. Indeed, this α-Syn/VAMP2 interaction concerns the C-terminus of α-Syn in a region encompassing S129 (\u003cspan citationid=\"CR71\" class=\"CitationRef\"\u003e71\u003c/span\u003e). It is thus tempting to speculate that in normal conditions this interaction shields S129 from casein kinase, the constitutively active protein kinase responsible for the phosphorylation of S129 (\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e). In protein mis-sorting conditions (due to overexpression or to fibril formation), shielding is likely to be lost thus exposing S129 to phosphorylation by casein kinase.\u003c/p\u003e \u003cp\u003eWe next applied our methodology to address the amyloid status of the α-Syn inclusions observed in wt mice injected with PFFs and in the brain of a deceased sporadic PD patient. In line with the observations regarding mutations, we found that the α-Syn inclusions observed either in the disease or experimentally produced in the mouse are far from being exclusively constituted of fibrils and are populated by non-amyloid species. Interestingly in PD Lewy neurites present a \u0026ldquo;purer\u0026rdquo; amyloid constitution than the LBs which are rich in non-amyloid species. This observation is reminiscent of the ones of Shahmoradian et al. who reported the existence of fibril-less LBs (\u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e58\u003c/span\u003e). It suggests that the prominence of non-amyloid α-Syn in the LBs compared to LNs or to experimental inclusions in mice could result from the maturation level of the inclusions. Maturation could involve delayed fibril disassembly and breakdown in the latest phases of LB constitution, associated with the emergence of non-amyloid, yet pathological species. Note that the possibility that the familial mutation A30P could play its role in PD by exacerbating the release of pathogenic fragments from the fibrils was proposed by Hasegawa and colleagues (\u003cspan citationid=\"CR63\" class=\"CitationRef\"\u003e63\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eFrom a purely methodological point of view, it is worth noting that in our conditions of overexpression achieved in 96 well primary cultures of cortical neurons, spontaneous fibrillization of α-Syn is virtually absent. This could represent a model well suited to explore at a reasonable throughput the mechanisms that could trigger amyloid aggregation in the absence of internalization of a preformed seed. Indeed, while this latter step is the main candidate process invoked to explain the intercellular spread of the α-Syn inclusions, the spontaneous emergence of intraneuronal aggregates by dysregulation of cellular processes involved in α-Syn catabolism or by the disruption of heterologous protein-protein interactions could be key to the emergence of an inclusion pathology.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cp\u003eAAV \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;adeno-associated virus\u003c/p\u003e\n\u003cp\u003eBLA \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; basolateral amygdala\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eBSA \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; Bovine Serum Albumin\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eDIV \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; days \u003cem\u003ein vitro\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eDLB \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;Dementia with Lewy bodies\u003c/p\u003e\n\u003cp\u003eFFPE \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; formalin-fixed \u0026nbsp;paraffin-embedded\u003c/p\u003e\n\u003cp\u003eGCI \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; glial cytoplasmic inclusion\u003c/p\u003e\n\u003cp\u003eGCP \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;genome-containing particle\u003c/p\u003e\n\u003cp\u003eHCA \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;High Content Analysis\u0026nbsp;\u003c/p\u003e\n\u003cp\u003ehSyn \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; human \u0026alpha;-Syn\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eIHC \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;immmunohistochemistry\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eLB \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; Lewy body\u003c/p\u003e\n\u003cp\u003eLN \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;Lewy neurite\u003c/p\u003e\n\u003cp\u003eLSCM \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;Laser-Scanning Confocal Microscopy\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eMSA \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; multiple system atrophy\u003c/p\u003e\n\u003cp\u003ePAGE \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;polyacrylamide gel electrophoresis\u003c/p\u003e\n\u003cp\u003ePBS \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; Phosphate Buffered Saline\u003c/p\u003e\n\u003cp\u003ePD \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;Parkinson\u0026rsquo;s disease\u003c/p\u003e\n\u003cp\u003ePEI \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;polyethylenimine\u0026nbsp;\u003c/p\u003e\n\u003cp\u003ePFA \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; paraformaldehyde\u003c/p\u003e\n\u003cp\u003ePFFs \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; Preformed fibrils\u0026nbsp;\u003c/p\u003e\n\u003cp\u003epS129 \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;S129-phosphorylated\u003c/p\u003e\n\u003cp\u003epSyn \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; pS129\u0026nbsp;\u0026alpha;-Synuclein\u003c/p\u003e\n\u003cp\u003eRT \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; room temperature\u003c/p\u003e\n\u003cp\u003eSB \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; solubilization buffer\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eSN \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u003cem\u003eSubstantia Nigra\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eSNpc \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u003cem\u003eSubstantia Nigra pars compacta\u003c/em\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eTBS \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;Tris-buffered saline\u003c/p\u003e\n\u003cp\u003eTH \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; tyrosine hydroxylase\u0026nbsp;\u003c/p\u003e\n\u003cp\u003ewt \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; wild-type\u0026nbsp;\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u0026alpha;SP \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026alpha;-Synucleinopathies\u003c/p\u003e\n\u003cp\u003e\u0026alpha;-Syn \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026alpha;-Synuclein\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eEthics approval\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe brain samples from MSA and PD patients were obtained from the Brain Bank GIE NeuroCEB (BRIF number 0033-00011)\u003c/p\u003e\n\u003cp\u003eThe animal study protocol was approved by the French Ministry of Research (protocol APAFIS #33147-2021091711598830 v6, 2021\u0026ndash;2026).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and materials\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe Authors declare no competing interests.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis project received funding from the French Agence Nationale de la Recherche (ANR), under grant ANR-22-CE16-0002 (project ASAPS), the Department of Excellence Initiative of the Italian Ministry of Research, the Center of Excellence in Neurodegeneration (CoEN) Bordeaux Initiative for Neurodegenerative Disorders (BIND) reference CHUBX 2022/07, the MSA Coalition Global Seed Grant (project FibrilloScreen) and the Institut Roche, Paris, France. AL is supported by the Institut Roche, France (CIFRE doctoral fellowship).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors\u0026apos; contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFI, FDG and FL conceived and designed the work, performed the analysis and the interpretation of data; FDG, AL \u0026amp; FZ performed\u0026nbsp;\u003cem\u003ein vitro\u003c/em\u003e experiments on primary neuronal cultures;\u0026nbsp;FZ, ND, LAD produced plasmids and AAVs; FL designed and performed all biochemical assays;\u0026nbsp;SD, EB performed and managed AAV induced overexpression in mice and IHC experiments; MLA performed IHC experiments;\u0026nbsp;MK, FDN \u0026amp; DDL performed and managed\u0026nbsp;\u003cem\u003ein vivo\u003c/em\u003e synucleinopathy experiments by intrastriatal injection of PFFs and IF experiments in FFPE sections;\u0026nbsp;MHC helped with immunofluorescence experiments in human FFPE sections; FI and FDG wrote the original version of the manuscript; FL, AL, WM, DDL made substantial contributions to the drafting and/or the substantive revision of the manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe Authors thank Benjamin Dehay for making available the AAV vectors regarding the familial PD \u0026alpha;-Syn mutations.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eTu PH, Galvin JE, Baba M, Giasson B, Tomita T, Leight S, et al. 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Functional cooperation of α-synuclein and VAMP2 in synaptic vesicle recycling. Proc Natl Acad Sci U S A. 2019;166(23):11113\u0026ndash;5.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":true,"highlight":"","institution":"University of Bordeaux","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"α-Synuclein, amyloid, fibrils, detection, methodology, antibody, Syn-1, Clone 42, NAC, Parkinson’s disease","lastPublishedDoi":"10.21203/rs.3.rs-3921168/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-3921168/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eThe neuropathology of α-Synucleinopathies (αSP) is characterized by the spread of subcellular inclusions containing fibrils made of stacked-up α-Synuclein (α-Syn) monomers. The repetitive amyloid fold adopted by α-Syn has now been characterized at the atomic scale. However, the direct observation of amyloid α-Syn using routine immuno-histological procedures remains an issue. In particular, the widely used phosphorylated α-Syn (pS129) is only a surrogate marker of aggregation. We report here that pS129 is misleading in overexpression-based models in which it detects the overflow of soluble α-Syn while no fibrillization takes place. Further, frequent pS129-negative α-Syn inclusions are observed when seeding with preformed fibrils (PFFs) is used to force fibrillization in neurons overexpressing α-Syn. This prompted us to scrutinize a series of routine antibodies for their genuine ability to discriminate α-Syn monomers engaged or not into amyloid fibrils, irrespective of phosphorylation. We observed unexpected antibody properties and utilized these latter in neurons and brain sections to detect the loss of accessibility of interlocked NAC domains when the monomers engage into fibrils. In cultured neurons, we observed that α-Syn mutations associated with familial Parkinson\u0026rsquo;s disease (PD), or S129A which prevents α-Syn phosphorylation, are neither sufficient to trigger spontaneous α-Syn fibrillization nor aggravate the process seeded by PFFs. Further challenging the pathogenic role of fibrillization, our results also indicated that the pS129-positive α-Syn inclusions detected in the brains of mice inoculated with PFFs and of a sporadic PD patient are not exclusively amyloid. This not only points to the notion that pS129 positivity is not tantamount to amyloid α-Syn but also indicates that the experimental α-Syn inclusions seeded in mice as well as the Lewy bodies forming in PD are populated by non-amyloid species which might represent alternative proxies of the α-Syn mutations endowed with a pathogenic potential.\u003c/p\u003e","manuscriptTitle":"Reconsidering α-Synuclein inclusion pathology in neurons, mice, and humans with an antibody sensing NAC engagement during α-Synuclein amyloid conversion","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-02-07 20:17:53","doi":"10.21203/rs.3.rs-3921168/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"d477b962-2240-43d0-a51f-096f2c1968a0","owner":[],"postedDate":"February 7th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":28540383,"name":"Neurobiology of Disease"}],"tags":[],"updatedAt":"2024-02-07T20:17:54+00:00","versionOfRecord":[],"versionCreatedAt":"2024-02-07 20:17:53","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-3921168","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-3921168","identity":"rs-3921168","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}