Francesca De Giorgi

Aenora Letourneur

Marianna Kashyrina

Federica Zinghirino

Sandra Dovero

Nathalie Dutheil

Leslie-Ann Largitte

Marie-Laure Arotçarena

Erwan Bezard

Marie-Hélène Canron

Francesca De

Francesca Degiorgi-

Francesco De Nuccio

Dario Domenico

Florent Laferrière

François Ichas
email: ichas@inserm.fr

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

Keywords: α-Synuclein, amyloid, brils, detection, methodology, antibody, Syn-1, Clone 42, NAC, Parkinson's disease

come

Abstract

The neuropathology of α-Synucleinopathies (αSP) is characterized by the spread of subcellular inclusions containing brils 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 over ow of soluble α-Syn while no brillization takes place. Further, frequent pS129-negative α-Syn inclusions are observed when seeding with preformed brils (PFFs) is used to force brillization 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 brils, 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 brils. 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 su cient to trigger spontaneous α-Syn brillization nor aggravate the process seeded by PFFs. Further challenging the pathogenic role of brillization, 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.

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 at 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)(7)(8)(9)[START_REF] Masuda-Suzukake | Prion-like spreading of pathological α-synuclein in brain[END_REF][START_REF] Mougenot | Transmission of prion strains in a transgenic mouse model overexpressing human A53T mutated α-synuclein[END_REF]. 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 brils [START_REF] Groveman | Rapid and ultrasensitive quantitation of disease-associated α-synuclein seeds in brain and cerebrospinal uid by αSyn RT-QuIC[END_REF][START_REF] Shahnawaz | Discriminating α-synuclein strains in Parkinson's disease and multiple system atrophy[END_REF]. These assemblies most often appear as two intertwined proto laments forming a bril with an axial symmetry and are easily detectable with uorescent probes that bind the surface grooves of the amyloid structure, among which the most used is Thio avin T [START_REF] De Giorgi | Novel self-replicating αsynuclein polymorphs that escape ThT monitoring can spontaneously emerge and acutely spread in neurons[END_REF]. The organization of α-Syn brils 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 [START_REF] Yang | Structures of α-synuclein laments from human brains with Lewy pathology[END_REF]16).

From a neuropathological point of view, the α-Syn brils accumulate inside subcellular inclusions forming de ned cytological objects (for instance: Lewy bodies LBs, Lewy neurites LNs, glial cytoplasmic inclusion GCI) [START_REF] Papp | Accumulation of tubular structures in oligodendroglial and neuronal cells as the basic alteration in multiple system atrophy[END_REF](18)[START_REF] Koga | Neuropathology and molecular diagnosis of Synucleinopathies[END_REF]. 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 de nition of Braak staging [START_REF] Jellinger | Is Braak staging valid for all types of Parkinson's disease?[END_REF][START_REF] Braak | Staging of brain pathology related to sporadic Parkinson's disease[END_REF][START_REF] Beach | Uni ed staging system for Lewy body disorders: Correlation with nigrostriatal degeneration, cognitive impairment and motor dysfunction[END_REF]. In addition, the sarkosyl-insoluble α-Syn brils extracted from DLB or MSA brain tissue can experimentally seed a de novo α-Syn pathology made of identi able inclusions in the brain of non-transgenic mice(10) [START_REF] Peng | Cellular milieu imparts distinct pathological α-synuclein strains in α-synucleinopathies[END_REF]: α-Syn brils 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-brillar (non-amyloid) forms of assemblies referred to as oligomers [START_REF] Conway | Acceleration of oligomerization, not brillization, is a shared property of both alpha-synuclein mutations linked to early-onset Parkinson's disease: implications for pathogenesis and therapy[END_REF]. 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 bril injections have been set up, with the aim of understanding the biological mechanisms linking α-Syn amyloid conversion and bril formation to neuronal failure and death [START_REF] Uchihara | Propagation of alpha-synuclein pathology: hypotheses, discoveries, and yet unresolved questions from experimental and human brain studies[END_REF]. 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, posttranslational modi cations, 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 speci cally 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 speci cally detect the phosphorylated form of α-Syn at serine 129 (pS129) [START_REF] Oueslati | Implication of Alpha-Synuclein Phosphorylation at S129 in Synucleinopathies: What Have We Learned in the Last Decade?[END_REF]. This post-translational modi cation 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 [START_REF] Fujiwara | alpha-Synuclein is phosphorylated in synucleinopathy lesions[END_REF]. Beyond synaptic staining, general α-Syn antibodies can also stain LB, GCI, and LN, which are identi able 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 speci c method involves the use of conformational antibodies with a higher a nity for brils than for other forms of the protein. However, their preference for high molecular weight α-Syn assemblies is relative [START_REF] Kumar | How speci c are the conformation-speci c α-synuclein antibodies? Characterization and validation of 16 α-synuclein conformation-speci c antibodies using well-characterized preparations of α-synuclein monomers, brils and oligomers with distinct structures and morphology[END_REF], and the main discriminating factor remains their ability to highlight typical cytopathological inclusions.

Detection and identi cation of such bona de inclusions is unfortunately an issue in several preclinical models. In other words, while in the context of human disease α-Syn pathology is strictly de ned as the presence of delineated subcellular inclusions, certain animal models do not reproduce them [START_REF] Recasens | Lewy body extracts from Parkinson disease brains trigger α-synuclein pathology and neurodegeneration in mice and monkeys[END_REF][START_REF] Arotcarena | Bidirectional gut-to-brain and brain-to-gut propagation of synucleinopathy in non-human primates[END_REF][START_REF] Bourdenx | Identi cation of distinct pathological signatures induced by patient-derived α-synuclein structures in nonhuman primates[END_REF][START_REF] Teil | Brain injections of glial cytoplasmic inclusions induce a multiple system atrophy-like pathology[END_REF](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 [START_REF] Teil | Brain injections of glial cytoplasmic inclusions induce a multiple system atrophy-like pathology[END_REF]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 [START_REF] Koga | Neuropathology and molecular diagnosis of Synucleinopathies[END_REF].

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 con ned cytoplasmic regions is overwhelmed (see results). Without seeding, the situation is even worse: overexpressed α-Syn can remain soluble, over ow the entire cytoplasm, and get phosphorylated without forming any brils (see results). This results in the genesis of images that can erroneously be interpreted as the emergence of inclusions mimicking the human pathology [START_REF] Laferrière | Overexpression of α-Synuclein by Oligodendrocytes in Transgenic Mice Does Not Recapitulate the Fibrillar Aggregation Seen in Multiple System Atrophy[END_REF].

In line with our previous observations made in mice overexpressing α-Syn under the control of the PLP promoter [START_REF] Laferrière | Overexpression of α-Synuclein by Oligodendrocytes in Transgenic Mice Does Not Recapitulate the Fibrillar Aggregation Seen in Multiple System Atrophy[END_REF], 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 di culty, we thus tried to nd 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 brils suitable for experimental and clinical neuropathology studies. 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. 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 humidi ed 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 brils were added at a nal 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).

Methods

Antibodies

High Content Analysis (HCA) and Laser-Scanning Confocal Microscopy (LSCM)

At 30 DIV the plates were processed for immuno uorescence. When relevant, the wells were treated 10 minutes with 25 µM digitonin in 100 mM KCl prior to xation. All plates were xed 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 uorescence images acquired 20× using the generic analysis module of the Incucyte S3 (Sartorius) and Top-Hat cellular segmentation was based on the uorescence signal corresponding to the antibody of interest. Multichannel uorescence 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 brils stock preparations (4-5 mg.ml-1 in Tris-Buffered Saline (TBS)) were diluted to 0.5 mg/ml nal 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 nal 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 lter-blot assay (science advance + companion paper). Brie y, 100 µl of treated monomers/PFFs were ltered through a nitrocellulose 0.2 µm membrane (Protran, GE) using a dot blot vacuum device (Whatman). Membranes were xed for 30 min at RT in PBS with paraformaldehyde (PFA) (Sigma) 4% (v/v) nal 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 quanti ed 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 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 lter-blot assays, 50 µl of native supernatant/pellet fractions were loaded on the lter-blot mounting and immunolabelled and quanti ed 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 brils (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 ow 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, post xed 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 quanti cation, 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.

Immuno uorescence in FFPE mouse and human brain sections.

Adult male 129SV (6-8 weeks old) unilaterally received 2 µL of sonicated α-Syn brils (4 mg/mL) by stereotactic delivery at a ow 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 post xed in the same xative, para n embedded, and 10 µm sections were obtained with a rotative microtome (Leica, Milan, Italy). The sections of interest were depara nized 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 immuno uorescence. Draq7

Thermo Fisher Scienti c 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 epi uorescence mode, and multichannel uorescence optical sections of the samples were performed (thickness < 0.8 µm) using a Leica SP5 Laser Scanning Confocal Microscope.

Human PD brain sections were depara nized 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 uorescence 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: lling of the neuronal cytoplasm and pS129 positivity without assembly of brils 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 [START_REF] Laferrière | Overexpression of α-Synuclein by Oligodendrocytes in Transgenic Mice Does Not Recapitulate the Fibrillar Aggregation Seen in Multiple System Atrophy[END_REF]. 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 di cult to determine and relies on the capability of speci cally identifying amyloid α-Syn brils in situ.

pS129 is a very speci c marker of α-Syn inclusions in human neuropathology(30)(39) [START_REF] Nishe | Accumulation of phosphorylated alpha-synuclein in the brain and peripheral ganglia of patients with multiple system atrophy[END_REF]. Similarly, when α-Syn PFFs are used as seeds and directly injected into the brain of wt mice, pS129 antibodies speci cally allow the visualization of the α-Syn inclusions that develop and spread into the mouse brain(9)(10) [START_REF] De Nuccio | Oligodendrocytes Prune Axons Containing α-Synuclein Aggregates In Vivo: Lewy Neurites as Precursors of Glial Cytoplasmic Inclusions in Multiple System Atrophy? Biomolecules[END_REF]. Four months after an injection of PFFs at the level of the SN, several types of pS129positive (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. 1A-E). This experimental αSP phenotype can also be observed after the intracerebral injection in mice of α-Syn brils extracted from DLB patient brain samples using detergent, with the formation of typical pS129-positive α-Syn inclusions [START_REF] Masuda-Suzukake | Prion-like spreading of pathological α-synuclein in brain[END_REF]. 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 speci c 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 shaminjected 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 de intracellular inclusions cannot be identi ed 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. 1A-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 brillar aggregation [START_REF] Laferrière | Overexpression of α-Synuclein by Oligodendrocytes in Transgenic Mice Does Not Recapitulate the Fibrillar Aggregation Seen in Multiple System Atrophy[END_REF].

Note that 4 months after the intracerebral injection of AAV-syn, on top of the absence of αSP inclusions, no speci c 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 de intracellular inclusions were still not identi ed 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. 1A-E). Induction of an inclusion pathology with longdistance 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. 1C), yet they are both associated with the emergence of a pS129-positive signal at the injection site (compare "AAV-Syn" and "PFFs" in Fig. 1B). 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 speci c 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 noninfected control neurons and only appeared upon seeding with PFFs. Like in αSP pathology, the seeded structures which are positive for pS129 are bona de cytological inclusions (LNs, perinuclear and intranuclear inclusions (Fig. 2A,B). In addition, pS129 staining was fully resistant to permeabilization of the plasma membrane with digitonin prior to xation, indicating that it corresponds to large insoluble α-Syn assemblies -i.e., brils -unable to diffuse out of the neurons through the digitonin pores (Fig. 2C,D).

In contrast, in non-seeded conditions, AAV-syn-infected neurons already showed a diffuse pS129 signal with no identi able inclusion (Fig. 2A,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 brils (Fig. 2C,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 bril assembly could still be seeded at the expense of the soluble pS129 α-Syn species that were formed in reaction to the overexpression over ow (Fig. 2C,D). These data show that in conditions of α-Syn overexpression, pS129 cannot be considered a reliable surrogate marker of neuronal α-Syn brillization and aggregation. In addition, even if the pS129 signal integral is increased by seeding (Fig. 2A-D), phosphorylation is not discriminant per se: phosphorylated brils cannot be distinguished from the phosphorylated soluble species "artifactually" emerging in these neurons.

To identify aggregated α-Syn more speci cally 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 xation con rming its non-brillar nature. When such AAV-syn infected neurons were seeded with PFFs, aggregation could only be inferred from morphological changes and not be quanti ed by measuring an increase of the LB509 signal integral in the intact cells (Fig. 2E). The brillar status of the assemblies seeded by PFFs and recognized by LB509 could, however, be revealed by their resistance to plasma membrane permeabilization (Fig. 2E).

Interestingly, by comparing the signal distribution of pS129 and LB509 by double immuno uorescence in the latter permeabilized neurons, we observe that the signals are not completely overlapping (Fig. 2F). This indicates that pS129-positive amyloid α-Syn brils coexist with unphosphorylated brils, and that some pS129-positive brils 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 bril 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 [START_REF] De Giorgi | Novel self-replicating αsynuclein polymorphs that escape ThT monitoring can spontaneously emerge and acutely spread in neurons[END_REF]. For this study, we tested a panel of "routine" commercial antibodies on these different PFFs to investigate their possible ability to differentially detect the brils strains and the monomeric protein (Fig. 3A).

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 bril α-Syn assemblies. MJFR14 which has been described as preferentially binding to brils [START_REF] Paleologou | Detection of elevated levels of soluble alpha-synuclein oligomers in post-mortem brain extracts from patients with dementia with Lewy bodies[END_REF] did not show any conformation speci city 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 brils. 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 Cterminus (47) appeared most selective of amyloid brils vs. monomers. While these results are globally in line with the expectations (excepted for MJFR14), we identi ed 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 brils. This con rms repeated previous observations made by us and others (14, 49, 50); (ii) D37A6 which is held a rodent speci c 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 a nity for hSyn engaged into amyloid brils.

To further explore these conformation speci cities, we used increasing concentrations of urea to provoke the gradual disassembly of the 3 brils strains back into monomers and to measure the impact of disassembly on immunoreactivity (Fig. 3B). In agreement with the previous experiment (Fig. 3A), disassembly did not modify the signal of the conformation-independent antibodies MJFR1, EP1646Y and MJFR14, while it collapsed the signal of all the bril-speci c antibodies which we identi ed, i.e., LB509, Syn505, SynF1 and D37A6. As a mirror image, progressive bril disassembly led to the progressive appearance of Syn1 immunoreactivity as more monomers were released from the amyloid assemblies, con rming 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. 3C and Fig. S3). The results con rm 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 rst amyloid conversion-dependent change of α-Syn taking place at the proper bril core level and amenable to detection by an immunological method.

Amyloid conversion assays in neurons

Considering these results on synthetic and extracted brils, we explored the use of the antibodies which we retained conformation-dependent (see above) in primary cultures of mouse cortical neurons overexpressing hSyn.

We rst performed double-immuno uorescence 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 xation (Figs. 4A,B).

Both antibodies con rmed their preferential a nity for aggregated α-Syn, the quanti cation of the signal integral showing a signi cant increase for each of the antibodies in the PFF-treated neurons compared to the control conditions (Fig. 4A). SynF1 was also sensitive to overexpression, but the differential intensity between PFF-treated and untreated conditions was larger than with LB509 (Compare Fig. 2E and Fig. 4A,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 xation, 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 " ltered/contrasted" image of the α-Syn pathology (Figs. 4A).

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. 4B). 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. 4B).

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 proteinprotein interactions established between stacked α-Syn monomers during assembly of the amyloid core.

As a result, once the monomers are trapped inside the brillar 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. 4C). 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 brils (Fig. 4C). Indeed, in neurons not seeded with PFFs, MJFR1 and Syn1 staining completely overlapped and both signals disappeared if the neurons were permeabilized prior to xation. 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. 4C). Note that amyloid conversion can similarly be tracked using EP1536Y to reveal pS129 α-Syn together with Syn1 (Fig. 4D). In this case, amyloid α-Syn is EP1536Ypositive 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 conformationdependent antibodies we tested and that do bind preferentially to aggregated α-Syn present only a relative speci city and can miss a signi cant fraction of the amyloid α-Syn brils 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 speci cally 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 mutationseither 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 [START_REF] Oliveira | Alpha-synuclein research: de ning strategic moves in the battle against Parkinson's disease[END_REF]. 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 [START_REF] Tenreiro | Phosphorylation modulates clearance of alpha-synuclein inclusions in a yeast model of Parkinson's disease[END_REF][START_REF] Karampetsou | Phosphorylated exogenous alpha-synuclein brils exacerbate pathology and induce neuronal dysfunction in mice[END_REF][START_REF] Ghanem | α-Synuclein phosphorylation at serine 129 occurs after initial protein deposition and inhibits seeded bril formation and toxicity[END_REF].

Using MJFR1 we quanti ed 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, con rming previous observations [START_REF] Winner | In vivo demonstration that αsynuclein oligomers are toxic[END_REF]. We thus did not consider or discuss the possible impact of the E46K mutation on the amyloid conversion of α-Syn in situ.

Figure 5A shows that as it was the case for wt hSyn, all disease-associated mutants were signi cantly 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 5A underlines that under overexpression conditions, little can be said on the impact of speci c 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 Syn1negative inclusions) (Figs. 5B,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. 5A) 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 brils. The same holds true for the S129A variant showing that preventing phosphorylation at S129 is not su cient not trigger spontaneous brillization.

We thus reasoned that in a neuronal context, these mutations might instead favor the seeded assembly of α-Syn into amyloid brils. Upon seeding with PFFs, we observed a massive burst of the population of neurons bearing amyloid inclusions (MJFR1-positive Syn1-negative) (Figs. 5B,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 con rming 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 brillization in intact neurons. In addition, phosphorylation of S129 which can be misleading as a marker of brillization, does not seem to inhibit spontaneous or seeded α-Syn brillization 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 sacri ced 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. 6A-C), the inclusion pathology was revealed using the antibody pair pS129 and Syn1 like in the primary cultures of Fig. 4D. The gure shows a region of the right basolateral amygdala (BLA) lled 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. 6A, in green). Co-staining with Syn1 (Fig. 6B, in purple) revealed the physiological pool of non-amyloid α-Syn present in the synapses that were scattered all over the eld of view. However, while as expected many pS129 inclusions appeared Syn1-negative (green inclusions in the overlay of Fig. 6C Panels of Fig. 6D-J andK-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 rst double-labeled and revealed using EP1536Y and Syn1 as before. The section was then hybridized with a third uorophore-coupled MJFR1 antibody which detects hSyn irrespective of its conformation and phosphorylation (see Figs. 3, 5 andS5). 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. 6J). 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. 6J 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 brils in this portion of the inclusion. At major variance, it appeared di cult 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. 6Q and compare with Fig. 6J). In contrast with the somatic inclusions seeded in mice (Fig. 6A-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 con rms the pioneering observations of Shahmoradian and colleagues who reported that α-Syn brils were identi able 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 identi able inclusion pathology but only diffuse anatomical-scale brain staining [START_REF] Recasens | Lewy body extracts from Parkinson disease brains trigger α-synuclein pathology and neurodegeneration in mice and monkeys[END_REF][START_REF] Arotcarena | Bidirectional gut-to-brain and brain-to-gut propagation of synucleinopathy in non-human primates[END_REF][START_REF] Bourdenx | Identi cation of distinct pathological signatures induced by patient-derived α-synuclein structures in nonhuman primates[END_REF][START_REF] Teil | Brain injections of glial cytoplasmic inclusions induce a multiple system atrophy-like pathology[END_REF](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 [START_REF] Tanriöver | Prominent microglial inclusions in transgenic mouse models of α-synucleinopathy that are distinct from neuronal lesions[END_REF]. 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 brils.

Unfortunately, relying on conformation-dependent antibodies which are commercially available is not an alternative because their preference for amyloid α-Syn brils with regards to other soluble species is partial [START_REF] Kumar | How speci c are the conformation-speci c α-synuclein antibodies? Characterization and validation of 16 α-synuclein conformation-speci c antibodies using well-characterized preparations of α-synuclein monomers, brils and oligomers with distinct structures and morphology[END_REF]. 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 bril 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 immuno uorescence 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 brils.

Using this approach to score α-Syn brillization 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 brillization. The massive pS129 signal which is observed corresponds to nonamyloid forms of the protein. Further, these α-Syn mutants do not facilitate the process of seeded brillization, 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 brillization. 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 brillization was reported several times in such systems(56, 57, 67, 68) but the bearings of this observation for the possible causal role of brillization 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 brillization 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 brillization of α-Syn in primary neuronal cultures is in line with the conclusion of Lansbury and colleagues. This indeed suggests that besides brils, non-amyloid α-Syn form(s) might play a prominent role in the pathophysiology of PD [START_REF] Conway | Acceleration of oligomerization, not brillization, is a shared property of both alpha-synuclein mutations linked to early-onset Parkinson's disease: implications for pathogenesis and therapy[END_REF]. 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 proto laments during α-Syn bril assembly [START_REF] Guerrero-Ferreira | New insights on the structure of alpha-synuclein brils using cryo-electron microscopy[END_REF].

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 modi cation [START_REF] Oueslati | Implication of Alpha-Synuclein Phosphorylation at S129 in Synucleinopathies: What Have We Learned in the Last Decade?[END_REF]. This led to the conclusion that this modi cation has indeed an impact on α-Syn brillization. However, for some, pS129 facilitates seeded brillization(53) while for others pS129 inhibits the process(54) [START_REF] Tenreiro | Phosphorylation modulates clearance of alpha-synuclein inclusions in a yeast model of Parkinson's disease[END_REF].

Using the overexpression of the non-phosphorylatable α-Syn mutant S129A and our amyloid α-Syn detection method, we nd that preventing S129 phosphorylation in primary neurons has no impact on either spontaneous or seeded brillization, pointing to a different and yet unknown role of the modi cation. One possibility to explain that pS129 appears on brils or on overexpressed monomers is that during α-Syn brillization 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 [START_REF] Sun | Functional cooperation of α-synuclein and VAMP2 in synaptic vesicle recycling[END_REF]. 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 [START_REF] Oueslati | Implication of Alpha-Synuclein Phosphorylation at S129 in Synucleinopathies: What Have We Learned in the Last Decade?[END_REF]. In protein mis-sorting conditions (due to overexpression or to bril 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 brils and are populated by nonamyloid 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 bril-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 bril 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 brils 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 brillization 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.

  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 nal 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

  , see a few examples pointed by empty arrowheads), indicating inclusions exclusively made of amyloid α-Syn, a signi cant number of inclusions alternatively appeared partially or totally Syn1-positive (white inclusions in the overlay of Fig. 6C, 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.

  AbbreviationsAAV adeno-associated virus

Figure 3 Speci

 3 Figure 3

Figure 4 In

 4 Figure 4

Figure 5 α

 5 Figure 5

Acknowledgements

The Authors thank Benjamin Dehay for making available the AAV vectors regarding the familial PD α-Syn mutations.

Funding

This 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).

Availability of data and materials

The datasets and/or analyzed during the current study are available from the corresponding author on reasonable request.

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

Competing interests

The Authors declare no competing interests. The overexpression of hSyn in primary culture of mouse cortical leads to the apparition of a pSyn-positive signal the absence of aggregation. A-B) pSyn detected by the EP1536Y antibody 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) Quanti cation of the integrated intensity of the pSyn signal, of wt and hSyn-overexpressing neurons in unseeded conditions, with or without permeabilization 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 ltered 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.

Figure 6 Using a combination of 2 antibodies allows the characterization of pathological α-Syn.A-C) Confocal on horizontal slices from wt mice sacri ced 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).

Supplementary Files

This is a list of supplementary les associated with this preprint. Click to download.


Sylvain Chataigner
email: sylvain.chataigner@univ-eiffel.fr

Laurent Michel
email: laurent.michel@univ-lyon1.fr

Arnaud Rolland
email: arnaud.rolland@cerema.fr

USE OF FRP REBARS IN REINFORCED CONCRETE STRUCTURES: AN OVERVIEW OF THE 2021 FRENCH GUIDELINE FROM AFGC

Keywords: FRP rebar, Reinforced concrete structures, Recommendations, Design rules, Mechanical and durability characterization, Quality control

In 2018, the French Association for Civil Engineering (AFGC) formed a dedicated working group to focus on the application of Fiber-Reinforced Polymer (FRP) bars for the internal reinforcement of concrete structures. The primary objective was to establish French recommendations by conducting a comprehensive review of existing standards and knowledge in the field. A technical report gathering those recommendations (in French) was released in 2021 and is currently undergoing translation into English. This report comprises six chapters covering various aspects, including the characterization of FRP reinforcing bars (rebars), their durability and temperature behaviors, as well as design recommendations for ultimate and serviceability limit states, encompassing flexure, shear, punching shear, fatigue, and pile reinforcement. Additionally, several case studies are presented in the Appendix.

INTRODUCTION

Fiber-Reinforced Polymer (FRP) reinforcing bars (rebars), which involve the combination of continuous high performance fibers and organic matrices, present distinct benefits over conventional options such as carbon or stainless steel rebars: they are lightweight and corosion resistant, exhibit outstanding mechanical properties, and are most often non-conductive and non-magnetic. Numerous research studies and preliminary recommendations exist at both the European level (CNR-DT 203, 2006;Fib, 2007) and the international level (CSA S806-12, 2012;ACI 440.1R-15, 2014;AASHTO, 2018;ACI 440-11, 2022) regarding the utilization of these FRP rebars in reinforced concrete (RC) structures. European normalization commissions are actively involved in integrating these reinforcements into the ongoing revision of Eurocode 2. However, it is worth noting that in France, there is currently no established reference framework to guide engineering firms and project owners in incorporating FRP reinforcement into their projects. Several years ago, the French Association of Civil Engineering (AFGC) established a dedicated working group to address this matter and develop nationallevel recommendations. This group comprises various stakeholders, including academics, research centers, companies, project owners, engineering and inspection firms, certification bodies, and composite reinforcement manufacturers. In December 2021, the initial version of these recommendations was completed and made available. Currently, efforts are underway to translate the recommendations into English. This guide covers a range of important topics including the characterization of composite reinforcements, their durability and response to temperature variations. It also provides guidance on the design of reinforced concrete (RC) structures utilizing internal composite reinforcements, as well as information on quality control and on-site implementation, along with valuable feedback. Additionally, the guide incorporates case studies from various producers and includes several design examples that showcase the maturity of the technique. The present article aims to provide an overview of the key aspects covered in the guide. Readers are encouraged to refer to the final document (AFGC, 2021) for further information.

CHARACTERIZATION OF PHYSICAL AND MECHANICAL PROPERTIES OF FRP REBARS

The AFGC guide focuses on the use of pultruded FRP reinforcements, consist of long fibers embedded in an organic matrix. Ensuring proper identification and traceability of the component materials is crucial. Four main types of fibers are commonly used, namely carbon (CFRP), aramid (AFRP), glass (GFRP), and basalt (BFRP), each with different grades and corresponding properties that significantly impact the final mechanical properties of the reinforcements (see Fig. 1). The choice of fiber, along with its sizing, also influences the reinforcement's durability. The matrix plays a vital role in facilitating force transfer between the fibers, ensuring homogeneous stress distribution throughout the reinforcement section, and providing protection against external environments. The matrices used are predominantly organic and can be categorized into two main polymer families: thermosetting (currently the most commonly used) and thermoplastic polymers. While thermoplastic matrices offer malleability at high temperatures, their application in FRP manufacturing is more challenging, and there is limited feedback on their performance. To enhance the bond between FRP reinforcement and concrete, various techniques can be employed, including sand coating, machining, helical fiber addition, and combined treatments. It is essential to use sand coating compatible with the cementitious environment. There are various manufacturing processes for composite materials, but when it comes to FRP rebars for internal reinforcement of concrete, a highly suitable and widely adopted method involves pultrusion. In this process, a bundle of fibers is pulled through a series of stages known as a "pultrusion" chain.

The fibers undergo successive steps, including passing through resin baths, through a die to shape the desired section geometry, and through ovens for for the polymerization (crosslinking) of the thermosetting resin. This continuous process enables the production of straight reinforcements.

As part of the pultrusion process, manufacturers apply surface treatments to improve the adhesion between FRP rebars and concrete. Figure 2 provides an overview of the surface preparations commonly used in this procedure. It's important to note that pultrusion is not suitable for the production of curved FRP rebars or reinforcements with specific anchorages. In such cases, different manufacturing processes are used.

Typically, the fibers are placed on steel molds with the desired shape after passing through resin baths and dies. Subsequently, these molds are placed in ovens to initiate polymerization and achieve the final geometries of the rebars.

The first chapter of the AFGC guide focuses on the characterization of FRP reinforcements and provides a comprehensive set of methods and recommendations (test protocols, performance threshold values, etc.) covering the following parameters:

• microstructural parameters: mass fraction of fibers or fiber content, porosity or void content, water absorption; • other physical parameters: density, effective section; • thermal behavior parameters: thermal expansion coefficients (longitudinal and transverse), glass transition temperature (Tg) and degree of cure of the polymer matrix; • mechanical properties of the FRP reinforcements: tensile and compressive characteristics, transverse and interlaminar shear strength, bending strength; • assembly properties: bond properties at FRP/concrete interfaces, strength of couplers and anchorage systems (button-ended reinforcement or bent bar).

Considerable effort has been made to adapt North-American recommendations on final properties (minimum and maximum cross-sections; mechanical tensile properties: elastic modulus and tensile strength; minimum bending radius) to suit the diameters commonly encountered in the French market. Additionally, the guide provides detailed guidelines on how experimental investigations should be conducted.

Table 1 presents a list of the main characteristics discussed in the AFGC guide, along with the recommended determination methods and threshold values. European standards were adopted whenever available. For further details regarding specific threshold values, such as the minimum tensile strengths for straight and bent bars that vary according to the diameter, readers are encouraged to consult the AFGC guide (AFGC, 2021).

DURABILITY, TEMPERATURE BEHAVIOR AND FIRE RESISTANCE OF FRP REBARS

Durability of FRP rebars

In general, FRP materials can experience alterations in the microstructure of the matrix, fibers, or fiber/matrix interface when exposed to external aggressive agents. These alterations can result in significant loss of mechanical performance. Additionally, medium-and long-term mechanical stresses, such as creep and fatigue, can cause damage and impact the durability of FRPs (Figure 3).

Numerous laboratory studies have focused on evaluating the durability of Glass-FRP (GFRP) bars, considered as the most economically viable material. These studies often involve accelerated aging tests, including immersion in alkaline environments that simulate the condition of early age concrete and its potential aggressiveness towards glass fibers. The findings from these studies have contributed to the development of evaluation and qualification procedures for assessing the durability of FRP reinforcements. Efforts have also been made to establish correlations between laboratory results and actual on-site aging conditions. Despite significant progress in this area (better representation of the alkaline concrete environment, consideration of creep load effects during aging), challenges still remain in proposing reliable predictive approaches for on-site degradation kinetics of FRP reinforcements. Further research is also necessary to enhance the understanding of the durability of the FRP/concrete interface. We now have approximately twenty years of experience with the in-situ aging of FRP rebars in concrete structures that are in service. The feedback obtained from these real-life conditions demonstrates that natural aging conditions are much less aggressive compared to accelerated laboratory aging. This emphasizes the conservative nature of the North American standards, which served as a basis for the development of the AFGC guide. Additionally, the quality of reinforcement has improved significantly since the first applications in North America. As a result, it is likely that some environmental partial safety coefficients can be revised after new investigations on longer aging periods are carried out.

In this context, the second chapter of the AFGC guide focuses on a range of characterization methods specifically dedicated to the durability assessment of FRP rebars. This chapter also provides recommendations, including test protocols and performance threshold values, in relation to fatigue, creep, and alkali resistance. Table 2 presents a compilation of the recommended experimental methods and key specifications.

Temperature behavior and fire resistance of FRP rebars

As temperatures increases, FRP rebars experience a more significant decrease in their tensile mechanical properties (elastic modulus and tensile strength) compared to steel rebars. Experimental curves depicting these decreasing trends are presented in (Bisby, 2003) (Figure 4) for CFRP, AFRP, and GFRP. It should be noted that these evolutions are influenced by various factors, including the component materials (fiber and matrix type), fabrication process, and other variables. Hence, it is crucial to characterize the property evolutions over a wide temperature range for each new FRP rebar introduced to the market. Typically, the tensile strength of FRP rebars is divided by a factor of two (with respect to the reference strength at 20°C) at temperatures of around 325-350°C for CFRPs and 250-280°C for GFRPs (Wang et al., 2005;Bisby et al., 2007). It is important to note that these temperatures are well above the glass transition temperature (Tg) of the polymer matrix. Furthermore, it appears that CFRP rebars have better fire resistance than GFRP rebars.

Limited studies have examined the impact of temperature on the FRP/concrete interface. However, it has been observed that the interfacial properties exhibit a more pronounced decrease with temperature compared to the tensile properties of the FRP itself. For instance, Solyom et al. ( 2020) conducted pullout tests at different temperatures and demonstrated that the bond strength decreased by 30% near the glass transition temperature (Tg) of GFRPs, and even by 90% around 300°C (Figure 4). It is important to note that these results are influenced by factors such as the type of rebar, fabrication process, surface treatment, and concrete formulation. Therefore, it is essential to specify a maximum service temperature when designing concrete reinforced with FRP rebars. AFGC ( 2021) recommends maximum service temperatures 20°C lower than the characteristic Tg of the FRP rebars, in agreement with (CSA S6-14, 2014).

Regarding fire design, similar approaches to those adopted for steel RC structures can be applied, as outlined in (ACI 440.1R-15, 2014). The Eurocode 2 approach defines the critical temperature as the temperature at which the FRP's tensile strength decreases by 50%. Additionally, it is essential to ensure that anchorage zones are located in areas with low risks of fire exposure.

DESIGN OF CONCRETE STRUCTURES REINFORCED WITH FRP REBARS

The third chapter of the guide presents a comprehensive set of design methods covering various aspects, including:

• the determination of an appropriate cover thickness for the reinforcement;

• the establishment of safety coefficients for calculating strength design values, including partial safety factors that vary depending on the type of loading and environmental factors based on exposure class and fiber type; • justifications in bending, considering both ultimate and serviceability limit states with regards to stresses, deflections, and crack openings; • justifications with respect to shear forces; • reinforcement design for columns subjected to simple compression or combined bending; • verification of slab resistance against punching shear; • verifications with respect to fatigue;

• specific constructive provisions for each type of loading, such as reinforcement ratios, rebar spacing, diameters,…; • general recommendations for anchorage length and lap length.

These design methods provide guidance for incorporating FRP reinforcement effectively and safely in RC structures.

The recommendations provided in each section of the guide are based on the general methodology outlined in Eurocode 2, with adaptations specifically tailored to the use of FRP reinforcements. These adaptations take into account guidelines from various sources, including (CSA S806-12, 2012), (CSA S6-14, 2014), (AASHTO, 2018), (CNR DT 203, 2006), and(ACI 440.1R-15, 2014). It is important to note that due to the brittle elastic behavior of FRP reinforcements, it is not recommended to consider plastic redistribution within the structure. Additionally, the contribution of compression reinforcements can be neglected in the design justifications for limit states. The guide includes three comprehensive design examples that are developed with detailed design considerations. These examples cover a double-span T-beam (Figure 5), a retaining wall, and an aboveground parking column. An important observation, supported by existing literature, is that for the first two cases, the serviceability limit states tend to be critical in concrete structures reinforced with FRP bars. These serviceability limit states commonly involve considerations such as crack opening and deflection. This differs from structures reinforced with steel, where the ultimate limit states usually govern the design considerations.

QUALITY CONTROL AND ON-SITE APPLICATION

Currently in France, there is no established regulatory framework for certifying FRP reinforcement or affixing a CE or NF marking, similar to the case of steel reinforcements that are certified under AFCAB (independant body for certification). This limitation hinders the broader adoption and development of FRP rebar technology. Nevertheless, the recommendations presented in the guide align with the quality control practices followed in Europe for construction products, as outlined in the EU Construction Products Regulation No. 305/2011 (CPR) (EU, 2011).

The approach proposed in the guide involves:

 initial evaluation of the essential characteristics of the FRP reinforcement, where performance levels associated with each essential characteristic are determined. This evaluation is typically conducted through testing, under the responsibility of an external assessment body;  ongoing evaluation and verification of performance consistency through the implementation of a control program. This control program outlines the responsibilities of the producer in terms of internal factory production control, as well as the tasks assigned to an external control body, including periodic audits and sample testing.

The guide's final chapter provides recommendations for internal or third-party evaluation programs, encompassing aspects such as defining production lots, identifying characteristics to be determined, specifying evaluation methods, determining the number of samples and testing frequency.

Additionally, the guide offers guidance on storage, handling, and on-site implementation of FRP reinforcement. These recommendations aim to ensure proper practices are followed throughout the lifecycle of FRP reinforcement, promoting its reliable and effective use in construction projects.

CONCLUSIONS

The purpose of the developed guide (AFGC, 2021), as briefly introduced here, is to provide support to engineers and design offices involved in the design and verification of concrete structures reinforced with FRP rebars. At this stage, the guide offers recommendations and does not have any normative intent. However, it should be noted that in Europe, standards are being developed regarding the design and certification aspects, including CE marking. Therefore, the AFGC guide may undergo revisions based on the progress of ongoing European standards and associated techniques.

Furthermore, efforts are underway to assess the environmental impact of FRP reinforcements. An overview of this subject is presented at the end of the guide and summarized in Table 3. It is interesting to note that GFRP and BFRP reinforcements have the lowest impact on global warming compared to CFRP and AFRP reinforcements. Additionally, the choice of matrix material significantly influences the overall environmental impact of these reinforcements. While the impact of GFRP reinforcements per unit of mass is higher than that of steel reinforcements, it is important to consider that the density of FRP reinforcements is much lower than that of steel reinforcements, as shown in Table 4. On the other hand, when considering impact per unit of volume, GFRP and BFRP exhibit lower environmental impacts than steel. Similar findings have been confirmed in (Seacon, 2015) for various environmental impacts, such as global warming, acidification, photochemical oxidation potential, and eutrophication. However, further studies are needed to validate the databases used and enable comparisons at the scale of global projects, considering that the reinforcement ratio strongly depends on the component materials of the FRP rebar.

Figure 1 :

 1 Figure 1: Tensile behavior range of the most common types of fibers used in FRP reinforcing bars (black : carbon, orange : aramid, blue : glass or basalt).

Figure 2 :

 2 Figure 2: Examples of surface finishes commonly found on commercial-grade FRP rebars.

Figure 3 :

 3 Figure 3: Synergistic effects of environmental and mechanical factors, based on (ISIS, 2007).

Figure 4 :

 4 Figure 4: Variations in tensile properties of CFRP, AFRP, and GFRP rebars, as well as changes in GFRP/concrete bond strength with temperature, according to (Bisby, 2003) (Solyom et al., 2020).

Figure 5 :

 5 Figure 5: Arrangement of longitudinal and transverse FRP reinforcements (shear force) in the T section of the beam studied at mid-span and on support, extracted from the example of (AFGC, 2021).

Table 1 :

 1 List of the main characteristics and specifications reported in the guide(AFGC, 2021)

	Characteristic/property	Symbol	Standard method	AFGC specification
	Microstructural parameters
	Mass fraction of fiber or fiber content	M f	ISO 14127: 2008 (CFRP) BFRP) NF EN ISO 1172: 1999 (GFRP and	M f > 50%
	Porosity or void content		ISO 14127: 2008
	Water absorption	NF EN ISO 62: 2008 (method 1 at 50°C) < 1% at saturation
	Other physical parameters
	Density		NF EN ISO 1183-1: 2019 (Method A)
	Effective section	A PRF, eff	ISO 10406-1: 2015 (Chap. 5)	cf (AFGC, 2021)
	Parameters related to the thermal behavior

Table 2 :

 2 Main durability characteristics and specifications discussed in the guide(AFGC, 2021)

	Characteristic/property	Standard method	AFGC specification
			> 35% fPRF,k (GFRP and BFRP) ;
	Fatigue strength	ISO 10406-1: 2015 (Chap. 10)	> 45% fPRF,k (AFRP) ;
			> 75% fPRF,k (CFRP) ;
			> 35% fPRF,k (GFRP and BFRP) ;
	Creep rupture strength (in tension)	ISO 10406-1: 2015 (Chap. 12)	> 45% fPRF,k (AFRP) ;
			> 75% fPRF,k (CFRP) ;
	Alkali resistance in high pH solution (without load)	ISO 10406-1: 2015 (Chap. 11)	Residual tensile strength > 80% fPRF,k ; Residual interlaminar shear strength > 80% τi ;
	Alkali resistance in high pH solution (with load)	CSA S806-12 Annex M	Residual tensile strength > 60% fPRF,k ;

Table 3 :

 3 Literature review on CO 2 emission and potential for soils and water acidification associated with various materials

	Material	References	Impact on global warming	Soils and water acidification potential
	Steel	(Inman et al., 2016), (Stoiber et al., 2020), (Perier et al., 2013)	0.8-2.8 (kgCO 2 eq/kg)	16.7-27.3 (MJ/kg)

DATA AVAILABILITY

Concrete

 (Stoiber et al., 2020), (Perier et al., 2013) 232-431 (kgCO 2 eq/m 3 ) 845-1567 (MJ/m 3 ) Carbon fiber (Stoiber et al., 2020), (Perier et al., 2013) In order to enhance the recommendations provided in the AFGC guide, it is crucial to conduct further research in various areas related to FRP reinforcements. The identified areas requiring additional investigation include, but are not limited to:  additional studies on the environmental and mechanical durability of these FRP reinforcements, including creep, fatigue, durability in alkaline environments, and combined effects, are necessary to refine the proposed safety coefficients and verify the adequacy of the evaluation methods;  research on the fire behavior and post-fire behavior of these reinforcements is required;  the development of couplers and anchoring systems for all geometries is needed With respect to the design of concrete structures reinforced with FRP rebars, the following additional studies are recommended:

 more research on the behavior under shear force, including straight reinforcements, bent reinforcements, and systems with anchoring heads, as well as punching;  studies on the behavior of FRP reinforcements in compression zones to improve recommendations in flexure;  investigation of the use of mixed reinforcement (FRP and steel);  work on the seismic behavior of these structures to study dissipation mechanisms and their reversibility.

ACKNOWLEDGEMENT

The authors would like to express their gratitude to all the contributors and members of the AFGC group for their valuable contributions and dedicated efforts in conducting this work. Special thanks are also extended to the AFGC for their support in organizing and facilitating the project, as well as for their assistance in the dissemination of the guide.

CONFLICT OF INTEREST

The authors declare that they have no conflicts of interest related to the work presented in this paper.