Chrystelle Aillaud

Christophe Bosc

Leticia Peris

Anouk Bosson

Pierre Heemeryck

Juliette Van Dijk

Julien Le Friec

Benoit Boulan

Frédérique Vossier

Laura E Sanman

Juliette Van Dijk

Salahuddin Syed

Neri Amara

Yohann Couté

Laurence Lafanechère

Eric Denarier

Christian Delphin

Laurent Pelletier

Sandrine Humbert

Matthew Bogyo

Annie Andrieux
email: annie.andrieux@univ-grenoble-alpes.fr

Krzysztof Rogowski
email: krzysztof.rogowski@igh.cnrs.fr.

Marie-Jo Moutin
email: moutinm@univ-grenoble-alpes.fr

Vasohibins/SVBP are tubulin carboxypeptidases (TCPs) that regulate neuron differentiation

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Main text:

Microtubules are cytoskeletal polymers of α/β tubulin dimers centrally involved in cell division, motility and morphogenesis. In the detyrosination/tyrosination cycle of tubulin, the C-terminal tyrosine of α-tubulin is removed by an elusive peptidase (TCP) and re-added by the tubulin tyrosine ligase (TTL, for review see [START_REF] Barra | Posttranslational tyrosination/detyrosination of tubulin[END_REF]). This cycle, which is unique to tubulin and mostly conserved from chordates to mammals, has a vital role in vivo [START_REF] Erck | A vital role of tubulintyrosine-ligase for neuronal organization[END_REF]. Tubulin de/tyrosination of α-tubulin is an important regulatory signal for mitosis [START_REF] Badin-Larcon | Suppression of nuclear oscillations in Saccharomyces cerevisiae expressing Glu tubulin[END_REF][START_REF] Barisic | Mitosis. Microtubule detyrosination guides chromosomes during mitosis[END_REF][START_REF] Peris | Tubulin tyrosination is a major factor affecting the recruitment of CAP-Gly proteins at microtubule plus ends[END_REF], for neuronal physiology [START_REF] Gobrecht | Promotion of Functional Nerve Regeneration by Inhibition of Microtubule Detyrosination[END_REF][START_REF] Konishi | Tubulin tyrosination navigates the kinesin-1 motor domain to axons[END_REF][START_REF] Marcos | Tubulin tyrosination is required for the proper organization and pathfinding of the growth cone[END_REF], and for muscle mechanotransduction [START_REF] Kerr | Detyrosinated Submitted Manuscript: Confidential microtubules modulate mechanotransduction in heart and skeletal muscle[END_REF][START_REF] Robison | Detyrosinated microtubules buckle and bear load in contracting cardiomyocytes[END_REF]. Consequently, abnormal tyrosination levels are associated with cell transformation and tumor aggressiveness [START_REF] Lafanechere | Suppression of tubulin tyrosine ligase during tumor growth[END_REF][START_REF] Whipple | Epithelial-to-mesenchymal transition promotes tubulin detyrosination and microtentacles that enhance endothelial engagement[END_REF], neuronal disorganization [START_REF] Erck | A vital role of tubulintyrosine-ligase for neuronal organization[END_REF], and heart failure and cardiomyopathies [START_REF] Robison | Detyrosinated microtubules buckle and bear load in contracting cardiomyocytes[END_REF][START_REF] Belmadani | Posttranslational modifications of cardiac tubulin during chronic heart failure in the rat[END_REF]. Although the detyrosination reaction was first described 40 years ago [START_REF] Hallak | Release of tyrosine from tyrosinated tubulin. Some common factors that affect this process and the assembly of tubulin[END_REF], the identity of TCP has remained unknown.

To enrich for TCP, we designed a three step purification procedure using taxol-stabilized radiolabelled tyrosinated microtubules as a substrate to follow activity. A typical procedure gave a final purification factor of nearly 400-fold (Figs. 1A andS1A). The last fraction (IV) was able to cleave the C-terminal tyrosine from tubulin incorporated in microtubules but not from EB1 (Fig. S1B). EB1 is a protein that shares a similar C-terminal sequence with α-tubulin (-QEEY instead of -GEEY) and is generally not a substrate for TCP in physiological contexts [START_REF] Bosson | Cap-Gly proteins at microtubule plus ends: is EB1 detyrosination involved?[END_REF].

To isolate the protein(s) responsible for TCP activity from fraction IV, we reasoned that an irreversible inhibitor could be used as in other chemical proteomic studies [START_REF] Child | Smallmolecule inhibition of a depalmitoylase enhances Toxoplasma host-cell invasion[END_REF]. We tested the sensitivity of brain TCP to various commercial protease inhibitors. The activity was inhibited by several serine/cysteine protease inhibitors (AEBSF, TLCK, TPCK, E-64, parthenolide) and by the thiol-reactive compound N-Ethylmaleimide (Figs. S2A and1B). These results, in agreement with prior studies [START_REF] Fonrose | Parthenolide inhibits tubulin carboxypeptidase activity[END_REF], strongly suggested that the catalytic activity of the putative TCP depends on a catalytic cysteine.

Although E-64 only showed modest inhibitory activity (IC 50 around 300 µM, Fig. 1B), it is an ideal starting point for inhibitor design because its reactive epoxide electrophile can display a peptide or amino acid mimicking native protein C-terminus. Furthermore, parthenolide, which is widely used to down-regulate detyrosination levels in cells [START_REF] Barisic | Mitosis. Microtubule detyrosination guides chromosomes during mitosis[END_REF][START_REF] Gobrecht | Promotion of Functional Nerve Regeneration by Inhibition of Microtubule Detyrosination[END_REF][START_REF] Kerr | Detyrosinated Submitted Manuscript: Confidential microtubules modulate mechanotransduction in heart and skeletal muscle[END_REF][START_REF] Robison | Detyrosinated microtubules buckle and bear load in contracting cardiomyocytes[END_REF], contains an epoxide function which is essential to its cellular effect [START_REF] Fonrose | Parthenolide inhibits tubulin carboxypeptidase activity[END_REF]. We thus synthesized three inhibitors, epoY, epoEY and epoEEY, which contain the epoxide coupled to one, two or three amino-acids from the α-tubulin C-terminus (Fig. S2B). EpoY was the most potent inhibitor of the TCP activity (IC 50 around 500 nM, Fig. 1B). Alkyne-epoY, which retained a strong inhibitory potency (Fig. 1B,C) and irreversibly inhibited the activity (Fig. S2C,D), was used to perform TAMRA labeling of fraction IV by click chemistry (Fig. 1D). This labeling showed specific modification of a small number of proteins (Fig. 1E). We then purified the inhibitor targets using agarose beads and, after on-beads trypsin digestion and quantitative mass spectrometry analyses of the resulting peptides from three independent experiments, we identified the protein vasohibin-1 (VASH1) as the most likely TCP candidate (Fig 1D, Tables S1-S2). Peptides covering the almost full VASH1 sequence were detected (Fig. S2E). Furthermore, recent bioinformatics data show that Vasohibin-1 and its homologue vasohibin-2 (VASH2) possess a non-canonical Cys-His-Ser catalytic triad and are members of the transglutaminase-like cysteine proteases family (18) (Fig. 1F).

Vasohibin proteins (41-42 kDa) have been extensively characterized as angiogenesis regulators but are poorly understood on a molecular level [START_REF] Sato | The vasohibin family: a novel family for angiogenesis regulation[END_REF]. SVBP (Ccdc23) is a high affinity binding partner of vasohibins that has a chaperone-like function [START_REF] Suzuki | Isolation of a small vasohibin-binding protein (SVBP) and its role in vasohibin secretion[END_REF]. We examined the ability of VASH proteins to detyrosinate α-tubulin in cells in the absence or presence of SVBP. Expression of vasohibins alone in HEK293T cells resulted in a slight increase of detyrosinated tubulin, whereas expression of either protein with SVBP resulted in a substantial increase in detyrosinated tubulin corresponding to a nearly complete loss of endogenous tyrosinated tubulin (Fig. 2A). Mutation of the putative catalytic cysteine on vasohibins (C179A for VASH1, and C158A for VASH2 [START_REF] Sanchez-Pulido | Vasohibins: new transglutaminase-like cysteine proteases possessing a non-canonical Cys-His-Ser catalytic triad[END_REF]) abolished their capacity to produce detyrosinated tubulin. Similarly, in murine embryonic fibroblasts (MEF), expression of the vasohibins with SVBP resulted in complete detyrosination of endogenous α-tubulin (Figs. 2B andS3A).

Alpha-tubulins are generally encoded with a C-terminal tyrosine preceded by two glutamates.

Alpha4-tubulin lacks the C-terminal tyrosine and α8-tubulin contains a C-terminal phenylalanine residue. Phenylalanine can be incorporated in place of tyrosine in tubulin and be a possible cause of neuronal dysfunction [START_REF] Ditamo | Post-Translational Incorporation of L-Phenylalanine into the C-Terminus of alpha-Tubulin as a Possible Cause of Neuronal Dysfunction[END_REF]. We tested the substrate specificity of the vasohibins by overexpressing α1B-and α8-tubulin together with VASH1 or VASH2 and SVBP in HEK293T cells. Both tubulin isotypes were cleaved by active vasohibins when expressed with SVBP (Figs. 2C andS3B).

Vasohibins were unable to cleave the C-terminal residue when tyrosine was mutated to alanine (Figs. 2C andS3B), confirming the specificity of VASH proteins for C-terminal tyrosine and phenylalanine residues.

We overexpressed vasohibins in HEK293T cells in the absence or presence of SVBP and purified the resulting complexes using a cobalt resin. SVBP co-purified with both vasohibins as expected from previous affinity measurements, (K D 30-90 nM (20)), and complex formation was not dependent on catalytic activity (Fig. S4A). The two protein complexes efficiently catalyzed tubulin detyrosination while complexes containing the catalytic dead versions of the vasohibins were unable to detyrosinate tubulin (Fig. 2D). Both complexes cleaved microtubules more rapidly compared to tubulin dimers (Fig. 2E,S4B), consistent with the reported preference of brain TCP in in vitro experiments [START_REF] Barra | Posttranslational tyrosination/detyrosination of tubulin[END_REF][START_REF] Deanin | On the mechanism of turnover of the carboxy-terminal tyrosine of the alpha chain of tubulin[END_REF][START_REF] Kumar | Preferential action of a brain detyrosinolating carboxypeptidase on polymerized tubulin[END_REF]. Purified VASH1/SVBP complex was not able to cleave the C-terminal tyrosine from EB1 indicating its clear tubulin preference. Intringuingly VASH2/SVBP complex was able to partially detyrosinate EB1 in the same conditions (Fig. 2F). In most cell types, including neuronal-derived, C-terminal tyrosine cleavage is restricted to tubulin [START_REF] Barra | Posttranslational tyrosination/detyrosination of tubulin[END_REF][START_REF] Bosson | Cap-Gly proteins at microtubule plus ends: is EB1 detyrosination involved?[END_REF]. EB1 can however be detyrosinated in specific endothelial and tumor cells [START_REF] Rovini | Antimigratory effect of vinflunine in endothelial and glioblastoma cells is associated with changes in EB1 C-terminal detyrosinated/tyrosinated status[END_REF] and this may be related to their VASH2 contents or defect in a regulatory mechanism.

To confirm the functional significance of vasohibins and their role in α-tubulin detyrosination, we assessed the phenotypic effects of knocking down expression of these proteins in differentiating neurons where the de/tyrosination cycle is highly important for growth cone pathfinding and axon differentiation, i. e. for neuron polarization [START_REF] Erck | A vital role of tubulintyrosine-ligase for neuronal organization[END_REF][START_REF] Konishi | Tubulin tyrosination navigates the kinesin-1 motor domain to axons[END_REF][START_REF] Marcos | Tubulin tyrosination is required for the proper organization and pathfinding of the growth cone[END_REF]. Although we were unable to detect vasohibins and SVBP by immuno-blot in mouse neurons using commercial antibodies, we amplified their transcripts from RNA preparations of cultured hippocampal neurons as well as of adult and embryonic mouse brain tissues (Fig. 3A, S5A, S6A). We transfected hippocampal neurons with plasmids expressing short hairpin RNAs (shRNAs) targeting either the vasohibins or SVBP (validated in Fig. S5B andS6B) as well as control shRNAs. The levels of detyrosinated α-tubulin were decreased by almost 50 % when the two vasohibins, or the SVPB, were down-regulated.

Addition of inhibitor (alkyne-epoY) also largely reduced detyrosinated α-tubulin (Fig. 3B,C,S5C).

When added together with the shRNAs, levels of detyrosinated tubulin were decreased up to 75 % (Fig. 3B,C). The remaining pool of detyrosinated α-tubulin could be due to uncomplete depletion of vasohibins activities or to the presence of α4-tubulin which genetically lacks the C-terminal tyrosine and remains in detyrosinated form in the brain [START_REF] Redeker | Mass spectrometry analysis of C-terminal posttranslational modifications of tubulins[END_REF]. The occurrence of other detyrosinases can however not be excluded. Confocal images of neurons cultured 2 days in vitro (2DIV) confirmed a decrease in the levels of the native detyrosinated α-tubulin upon reduction or inhibition of vasohibins expression (Fig. 3D). The remaining detyrosinated pools were specifically concentrated in the axon, while α-tubulin in the other neurites appeared highly tyrosinated.

Vasohibins down-regulation led to a clear delay in axonal differentiation (Fig. 3E). Tau and ankyrin G staining of neurons bearing an axon confirmed normal distribution, with Tau highly expressed in axon shaft at 3DIV and ankyrin G in the axon initial segment at 10DIV (Fig. S7). 2DIV neurons knocked down for vasohibins developed an increased number of neurites and branches with overall reduced axon length (Fig. 3F). A delay of axon differentiation and similar morphological anomalies were observed when down-regulating SVBP (Fig. S6D-F). Transfection of a plasmid allowing coexpression of shRNAs targeting SVBP and a shRNA-resistant form of SVBP rescued α-tubulin detyrosination levels (Fig. S6A,C). Thus, contrary to the premature axonal differentiation observed in the absence of the reverse enzyme TTL (2), here we observe a clear delay of axon differentiation when down-regulating vasohibins or SVBP.

We next tested the functional significance of vasohibins in vivo in mouse brain and focused on the cerebral cortex where the de/tyrosination cycle is critical for the neocortex layer organization [START_REF] Erck | A vital role of tubulintyrosine-ligase for neuronal organization[END_REF]. During corticogenesis neuronal migration relies in part on neuron polarization which was highly dependent on vasohibins (Fig. 3D,E) and SVBP (Fig. S6D,E). We electroporated E14.5 embryos with the plasmids expressing shRNAs targeting the vasohibins as well as control shRNA, and analyzed radial neuronal migration four days later. At E18.5, most neurons from control brains had reached the upper layer (bin 1), whereas when vasohibins were downregulated a significant fraction of neurons failed to do so (Fig 4A ,B). Thus, these enzymes have a crucial role in neuronal migration during brain cortex development.

TCP remained the crucial missing element of the α-tubulin de/tyrosination cycle for 40 years.

Here, we identify vasohibins as enzymatic proteins that perform the TCP function (Fig. S8). The failure of prior attempts to identify TCPs most probably resulted from their association with SVBP for stability and activity which could likely be lost during standard purification assays. In agreement with TCP functions, vasohibins are widely distributed in eucaryotes, have broad tissue expression, and vasohibin-1 (which is generally more expressed than vasohibin-2) is abundant in brain, heart and kidney [START_REF] Sanchez-Pulido | Vasohibins: new transglutaminase-like cysteine proteases possessing a non-canonical Cys-His-Ser catalytic triad[END_REF][START_REF] Nimmagadda | Expression pattern of Vasohibin during chick development[END_REF][START_REF] Shibuya | Isolation and characterization of vasohibin-2 as a homologue of VEGF-inducible endothelium-derived angiogenesis inhibitor vasohibin[END_REF] (see also GTEx Portal on https://www.gtexportal.org/home/). Additionally, we demonstrated their critical role in neuron and brain physiology. Preparation of tyrosinated tubulin and microtubule, and EB1. Bovine brain tubulin (12 mg/mL, prepared as in ( 28)) was tyrosinated by incubation for 45 min at 30°C with purified chicken TTL (1 mg/mL, gift from Steinmetz group) and 0.1 mM L-tyrosine or [ 14 C]-L-tyrosine (0.125 µCi/nmole) in 40 mM Pipes at pH 6.7, 60 mM KCl, 2.5 mM ATP, 1 mM DTT, 12.5 mM MgCl 2 . Microtubules polymerization was allowed by one-third dilution in 100 mM PIPES at pH 6.7, 1 mM EGTA, 1 mM MgCl 2 , 30% glycerol, 1 mM GTP and incubation at 32°C for 45 min, followed by another 45 min incubation with 50 µM Paclitaxel (Calbiochem). Microtubules were sedimented by 25 min centrifugation at 100,000 g and 30°C on 60% glycerol cushions. TTL-containing supernatant was discarded and tyrosinated-microtubules were resuspended in 100 mM PIPES at pH 6.7, 1 mM EGTA, 1 mM MgCl 2 , 10% glycerol, 80 µM paclitaxel and stored at -20°C. To obtain unpolymerized tyrosinated-tubulin, paclitaxel was omitted during the whole procedure. EB1 was prepared as in [START_REF] Ramirez-Rios | Tau antagonizes end-binding protein tracking at microtubule ends through a phosphorylation-dependent mechanism[END_REF].

TCP activity assays. In radioactivity tests using [ 14 C]-tyrosinated taxol-stabilized microtubule, enzyme activity was measured in 100 mM MES at pH 6.7, 1 mM EGTA and 1 mM MgCl 2 using 2 µM radiolabeled tubulin, except in Figs. 2D and S2D (quantity indicated in legend). Reactions were performed for 45 min at 37°C (without or with previous incubation with inhibitors, see legends) and were stopped on ice by addition of 20 µg/ml bovine serum albumin and 10% perchloric acid and centrifugation. Tyrosine cleaved from microtubules was estimated by measuring radioactivity in the supernatant (a zero time control was subtracted). To compose the table of Fig. S1A, detyrosination activity from equal volumes of the various fractions was measured. In tests with inhibitors, detyrosination activity was tested after a 20 min preincubation with inhibitor (except in Figs. S2C andS2D where varying time of incubation were used as indicated in legend), and results with inhibitors were expressed as percentage of the activity in the control with corresponding solvent. In tests using immunoblots, enzyme activity was assayed with 5 µM substrates (taxol-stabilized microtubule, tubulin or EB1) in 100 mM MES at pH 6.7, 1 mM EGTA and 1 mM MgCl 2 for times indicated in figures. Positive controls were done with carboxypeptidase A (2 ng/mL). Reactions were stopped by addition of Laemmli buffer. A reaction with carboxypeptidase A (CPA), which efficiently cleaves aromatic and aliphatic C-terminal residues, was used as positive control. In tests using VASH/SVBP complexes purified on cobalt resin from HEK293T cells, enzymes concentration were approximated from in-gel Coomassie-blue staining of purified VASH1 and VASH2 together with standards of bovine serum albumin.

Enzyme enrichment. We designed a three-step purification procedure from brain lysate inspired from previous efforts [START_REF] Kumar | Preferential action of a brain detyrosinolating carboxypeptidase on polymerized tubulin[END_REF][START_REF] Argarana | Tubulinyl-tyrosine carboxypeptidase from chicken brain: properties and partial purification[END_REF]. All steps were carried out at 4°C. Adult mice brains were homogenized in 50 mM phosphate buffer at pH 6.7, 1 mM EGTA, 1 mM MgCl 2 , 1 µg/ml DNAse with a protease inhibitor cocktail (cOmplete EDTA-free, Roche) and centrifuged 1 h at 100,000 g.

The supernatant was collected (Fraction I). In step 1 (ammonium sulfate fractionation), Fraction I was slowly brought to 45% ammonium sulfate, incubated for 15 min and centrifuged 20 min at 15,000 g. Resulting supernatant underwent the same procedure at 65% ammonium sulfate. The pellet was re-suspended in one-eighth of initial volume with 50 mM MES pH 6.7, 1 mM EGTA, 1 mM MgCl 2 and desalted using Bio-Gel P30 (Bio-Rad) equilibrated with the same buffer (Fraction II). In step 2 (strong anion column), Fraction II was loaded onto a 5 mL Hitrap Q XL column (GE Healthcare) connected on a BioLogic DuoFlow chromatography systems (Biorad) and unbound proteins were collected (Fraction III). In step 3 (strong cation column): Fraction III adjusted to pH 6.2 and 0.12 M NaCl, was loaded on a 5mL Hitrap SP XL column (GE Heathcare). Proteins were eluted with a 0.12 M to 1 M NaCl gradient. Protein fractions were equilibrated in 50 mM MES at pH 6.7, 1 mM EGTA, 1 mM MgCl 2 (by desalting with BioGel P-30). TCP activity assay was performed on these fractions and activity containing fractions were pooled (Fraction IV).

Purification data of a typical experiment are shown in Figs. 1A andS1A. For the quick enzyme enrichment (used in Fig. S1A,C,D), brain homogenate was directly proceeded to step 3, and then desalted.

Click chemistry (Cu-catalyzed azide-alkyne cycloaddition, CuCAAC reaction). Fraction IV

was equilibrated in phosphate buffered solution at pH 7.4 and concentrated to 2 mg/ml. Following 1 h incubation at 37°C with 10 µM of alkyne-epoY or 10 µM of epoY (respectively clickable and control irreversible inhibitors, see structures in Fig. 1C,S2B), two equivalent volumes of azideagarose beads (Jena Biosciences) were then added and click reaction was started by addition of 1 mM CuSO 4 , 100 µM TBTA and 1 mM TCEP. Reaction was completed in 3 h at room temperature.

For fluorescence labeling, 100 µM 5/6-TAMRA-PEG3-azide (Jena Bioscience) was used instead of azide-agarose beads.

Mass Spectrometry sample preparation. Removal of non-specifically bound proteins, and peptides preparation to be analyzed were achieved according to the Click Chemistry Capture Kit protocol (Jena Bioscience). Quickly, agarose-bound proteins were reduced and alkylated. Beads were washed with 1% SDS and 8 M urea. On-beads tryptic proteolysis (using Sequencing Grade Modified Trypsin, Promega) was performed and released peptides were purified on C18 cartridges (Ultra-Micro spin columns, Harvard Apparatus).

Mass spectrometry-based proteomic analyses.

Peptides were analyzed by nanoliquid chromatography coupled to tandem mass spectrometry (Ultimate 3000 coupled to LTQ-Orbitrap Velos Pro, Thermo Scientific) using a 120-min gradient as described in [START_REF] Milbradt | Proteomic analysis of the multimeric nuclear egress complex of human cytomegalovirus[END_REF]. Peptides and proteins were identified through concomitant searches against Uniprot (March 2017 version, Mus musculus taxonomy), classical contaminants (homemade) and the corresponding reversed databases using Mascot (version 2.5.1). The Proline software (http://proline.profiproteomics.fr) was used to filter the results (conservation of rank 1 peptides, peptide identification FDR < 1% as calculated on peptide scores by employing the reverse database strategy, and minimum of 1 specific peptide per identified protein group) before performing a compilation, grouping and comparison of the protein groups from the control and positive samples. Proteins from the contaminants database and additional keratins were discarded from the final list of identified proteins. Only proteins identified with a minimum of 3 specific spectral counts in the 3 replicates of positive samples and absent from the control samples were further considered.

Expression constructs. Mouse vasohibin-1 (VASH1) and vasohibin-2 (VASH2) cDNAs (accession numbers NM_177354 and NM_144879, respectively) were PCR-amplified and inserted into a home-made CAG promoter-containing vector, that generates proteins with a Flag tag at the Nterminus and with superfolder-GFP (sfGFP( 31)) and a 6 His tag at the C-terminus (Flag-vasohibin-sfGFP-His). The corresponding control plasmid encodes Flag-sfGFP-His protein. Bicistronic plasmids allowing coupled expression of both vasohibins and SVBP were obtained by introducing, downstream of the Flag-vasohibin-sfGFP-His cDNA, a cassette containing the encephalomyocarditis virus IRES sequence followed by the cDNA encoding mouse SVBP (accession number NM_024462) with a C-terminal Myc-tag (Flag-vasohibin-sfGFP-His/SVBP-Myc). Point mutations were introduced by PCR to generate enzymatically dead versions of vasohibins: C179A for VASH1 and C158A for VASH2, according to the numbering of accession numbers NP_796328 and NP_659128, respectively. The plasmid encoding mouse SVBP with Cterminal Myc and Flag tags (SVBP-Myc-Flag) was obtained from OriGene. Mouse Eb1 cDNA (accession number NM_007896) was inserted into an EGFP-tagged vector in order to generate EB1-EGFP. Plasmid encoding His-EB1 for protein production in E. coli was described in [START_REF] Bosson | Cap-Gly proteins at microtubule plus ends: is EB1 detyrosination involved?[END_REF]. cDNAs coding for human tubulin α1B and mouse tubulin α8 (accession numbers NM_006082 and NM_017379, respectively) were PCR-amplified and inserted into a vector with N-terminal mCherry tag. Point mutations in α-tubulin cDNAs were introduced by PCR to replace the last aromatic residue by an alanine: Y451A for α1B-tubulin and F449A for α8-tubulin, according to the numbering of accession numbers NP_006073 and NP_059075, respectively. Plasmid expressing mouse-specific shRNAs were from OriGene: TL511800B for Vash1, TL506751C for Vash2, TL517601B for Svbp and TR30021 for control. For rescue experiment with SVBP, tGFP of the plasmid TL51701B containing the Svbp shRNA was extracted by digestion with Mlu I and Pme I and replaced by a cDNA encoding mouse Svbp with myc and FLAG epitopes at the C-terminus.

The mutations G60A, G63A, T66C, G69A and G72A in the SVBP cDNA generate shRNAresistance. All constructs were verified by DNA sequencing.

Purification of His-tagged vasohibins from HEK293T cells. HEK293T cells cotransfected with

plasmids allowing expression of GFP or active/inactive forms of vasohibins and SVBP (Flag-sfGFP-His or Flag-(dead)VASH1/2-sfGFP-His and SVBP-Myc-Flag) were lysed in Tris buffer at pH 8.0, 0.5% TritonX100, 1 mM MgCl 2 , 200 mM NaCl, 5 mM imidazole in the presence of protease inhibitor cocktail (cOmplete EDTA-free, Roche). After centrifugation (10 min at 16,000 g and 4°C), supernatants were collected and added onto 20 µL of cobalt resin (Sigma) and incubated for 3 h at 4°C. After 3 washes with lysis buffer, proteins were eluted using 200 mM imidazole, Tris buffer at pH 8.0, 1 mM MgCl 2 , 200 mM NaCl. Purified proteins were equilibrated by dialysis at 4°C in 100 mM Pipes, 1 mM EGTA and 1 mM MgCl 2 and directly used in radioactivity assays.

Cell culture and transfection. Hippocampal neurons and MEFs were prepared as previously described [START_REF] Erck | A vital role of tubulintyrosine-ligase for neuronal organization[END_REF]. HEK293T cells were maintained under standard conditions. HEK293T cells were transfected with JetPRIME transfection reagent (Polyplus-Transfection). MEFs and neurons were transfected using Amaxa Nucleofector kits (Lonza). A ratio of 1:1 was generally used for cDNA cotransfections (for VASH1/2 with SVBP, or for mCherry-tubulin with bicistronic plasmids allowing coupled expression of VASH1/2 and SVBP).

Antibodies, immuno-blotting and immune-fluorescence. Detyrosination activity was detected using antibodies specific for tyrosinated tubulin (YL 1/2 , Tyr-tub) or native EB1 (Tyr-EB1), and Cterminally detyrosinated tubulin (deTyr-tub) or EB1 (deTyr-EB1). Control of total tubulin or EB1 was estimated with antibodies that recognizes both species (total α-tub or α3A1, total EB1 from BD Transduction lab.). All these antibodies were described and characterized in [START_REF] Bosson | Cap-Gly proteins at microtubule plus ends: is EB1 detyrosination involved?[END_REF], except an anti-deTyr-tub that we recently developed (using the same peptide) in guinea pig which was used in immunofluorescence (1:1000, Fig. 3). The other primary antibodies were used as follow: mouse anti-His from Qiagen in immunofluorescence (1:5000), rabbit anti-Flag from Molecular Probes in immunofluorescence (1:10000), rabbit anti-GFP from Chromotek in immunoblot (1:5000), chicken anti-GFP from Millipore in immunofluorescence (1:500), rabbit anti-turboGFP from Invitrogen in immunofluorescence (1:1000) and in immunoblot (1:10000), mouse RFP antibody (for mCherry) from Sigma in immunoblot (1:2500), mouse anti-Tau from Millipore in immunofluorescence (1:500) and mouse anti-ankyrin G from SantaCruz in immunofluorescence (1:300).

For immunoblotting, cells were collected after 24 h of transfection. After washing with phosphate-buffered saline (PBS) medium at 37°C, cells were directly lysed in Laemmli buffer.

Protein extracts were loaded on 10% acrylamide gels (Mini-PROTEAN® TGX Stain-Free™, Biorad) and transferred with Trans-Blot® Turbo (BioRad). Membranes were incubated with primary antibodies, with secondary antibodies conjugated with HRP (1:10 000, from Jackson ImmunoResearch), and finally revealed with Chemidoc camera (Biorad). For analysis and graphical representations of immunoblots (Fig. 3B,C and S6C), protein bands were quantified from triplicate blots of 3 different experiments using ImageJ software (National Institutes of Health, Bethesda, MD). Detyrosinated α-tubulin signal was normalized to the total protein content of the sample estimated from a stain-free gel image (the trihalo compounds in the Mini-PROTEAN® TGX Stain-Free™ gel react with tryptophan residues in proteins in a UV-induced 1-minute reaction to produce fluorescence).

For immunofluorescence, cells were generally fixed at 37°C in 4% paraformaldehyde, 4.2% sucrose, phosphate buffered saline medium (PBS) (except for ankyrin G staining for which methanol fixation at -20°C was used) and permeabilized using 0.1% Triton X-100, PBS. Cells were then incubated with primary antibodies, followed by incubation with secondary antibodies (see below) conjugated with either alexa-488, cyanine-3 or cyanine-5 fluorophores (1:1000). Nuclei were stained using Hoescht 33258 (1 μg/ml). SDS-PAGE, immuno-blot or immunofluorescence data presented all over the manuscript are representative results from experiments performed at least 3-times.

RT-PCR and RT-qPCR amplifications. For RT-PCR, messenger RNAs from cells and tissues

were prepared with the Dynabeads purification kit (Invitrogen). RT-PCR were performed with the Superscript One step RT-PCR System (Invitrogen) in 12.5 µL with 50 ng RNA. Products of respectively 697, 748 and 154 bp from mouse Vash1, Vash2 and Svbp were amplified at 58°C by 45 with a length inferior to 12 µm were not considered in order to avoid skeletonization artifacts. An axon was defined among primary neurites of a single neuron if its length was at least 48 µm and 1.3-times longer than any other primary neurite. Using this macro we selected a minimum of 27 neurons for each condition and compared the mean axonal length, mean primary neurite number (excluding axon) and branching frequency.

In utero electroporation, tissue processing, immunohistochemistry and analyses. A full description is available in [START_REF] Barnat | Huntingtin-Mediated Multipolar-Bipolar Transition of Newborn Cortical Neurons Is Critical for Their Postnatal Neuronal Morphology[END_REF]. Briefly, embryos from anesthetized timed-pregnant mice were electropored at E14.5 with plasmids allowing expression of shRNAs (see above). Four days later (E18.5), embryonic brains were dissected, fixed, cryosectioned and placed onto slides for analyses.

Anti-tGFP primary antibodies (1:300) was incubated overnight at 4°C. Nuclei were counterstained with DAPI (Roche). Images were acquired with a 20X N.A 0.5 objective on a DMI6000 Leica microscope with a motorized stand and analyzed with Image J. The cortical region where transfected cells were detected was divided into 6 bins of equal surface in which GFP positive (GFP + ) neurons were counted (5 embryos per condition, 3 slides per embryo). At least 260 GFP + neurons were counted per embryo. incubation with 30% TFA/DCM (v/v) for 2 hrs at room temperature. The progress of reaction was monitored by LC-MS. After completion of reaction, the solvent was evaporated under vacuum and the product was then co-evaporated with toluene to afford Alkyne-epoY (SD-186) as a colorless solid (31 mg, 93 %). 1 H NMR (400 MHz, Chloroform-d) δ 6.91 -6.86 (m, 2H), 6.72 -6.66 (m, 2H), 4.64 (ddd, J = 6.7, 5.5, 3.1 Hz, 1H), 3.94 (dd, J = 2.6, 0.8 Hz, 1H), 3.43 (d, J = 2.0 Hz, 1H), 3.17

Syntheses and analytical characterization

Synthesis of epoEY (SD-180):

Compound SD-180 was synthesized from SD-158 (50 mg, 0.08 mmol) by following the same standard procedure described for the t-Butyl deprotection in the synthesis of SD-142 to afford epoEY (SD-180) as white solid (31 mg, 86%). S2. Proteomic analyses of mouse brain clicked proteins (Auxiliary Table ). The protein contents of 3 independently prepared replicates using either the clickable inhibitor (TCP-R1 to TCP-R3) or the unclickable inhibitor (Ctrl-R1 to Ctrl-R3) were analyzed by mass spectrometrybased proteomics. Lower amounts of purified brain extract were used in R2 and R3 than in R1 (3-and 2-fold, respectively). For each identified protein are given the entry, accession, protein and gene names extracted from the Uniprot knowledgebase, and the validated number of peptides (Pep), spectral counts (SC) and specific spectral counts (SSC) observed in each sample.

Replicates with SSC = 0 despite SC > 0 were not considered (grey). To evidence proteins enriched in the replicates prepared with the clickable inhibitor, ratios of SSC (SSC TCP-R / SSC Ctrl-R) were calculated. Proteins exhibiting a SSC ≥ 3 and identified only in TCP samples or enriched at least 5 fold in TCP samples compared to corresponding Ctrl samples were considered as enriched in TCP samples (green). Among the 124 proteins enriched in TCP sample in at least one replicate, Vasohibin-1 (yellow) is the only protein enriched in all three replicates.

Fig. 1 .

 1 Fig. 1. Purification and identification of mouse brain TCP. (A) SDS-PAGE (silver staining) of

Fig. 2 .

 2 Fig. 2. Vasohibins associated to SVBP are potent tubulin tyrosine carboxypeptidases. (A)

Fig. 3 .

 3 Fig. 3. Down-regulation of VASH1 and VASH2 affects neuronal differentiation. (A) Vash1,

Fig. 4 .

 4 Fig. 4. Down-regulation of vasohibins affects radial migration of newborn cortical neurons.

  of designed TCP inhibitors. Synthesis steps, structure, molecular weight, 1 H and 13 C NMR and LC/MS analyses of epoY (A), alkyne-epoY (B), epoEY (C) and epoEEY (D).

  -2-carboxamido) propanoic acid: The t-Butyl group was removed by

  -3.08 (m, 2H), 2.94 -2.88 (m, 1H), 2.21 (t, J = 2.6 Hz, 1H).13 C NMR (101 MHz, cdcl 3 ) δ 172.80, 166.86, 166.05, 155.80, 130.20, 126.60, 115.43, 78.37, 71.73, 54.06, 53.98, 52.72, 36.20, 28.65. MS (ESI) m/z: calcd: [M+H] + 333.1, measured: [M+Na] + 355.18C. epoEYSynthesis of SD-139 (2S,3S)-ethyl 3-(S)-1-(tert-butoxy)-3-(4-hydroxyphenyl)-1-oxopropan-2yl) carbamoyl)oxirane-2-carboxylate:To the solution of L-Tyrosine tert-butyl ester (237.2 mg, 1 mmol), the Fmoc-Glu(OtBu)-OH (425 mg, 1 mmole) and and N-methylmorpholine (175 mg , 1.5 mmol) in dry THF (15 mL) was added EDC (229 mg, 1.2 mmol) and HOBt (162 mg, 1.2 mmol) in portions at 0 0 C. The reaction mixture was stirred at room temperature for 12 h. After monitoring disappearance of starting material by LC-MS, the mixture was concentrated under reduced pressure.The concentrated residue was purified by flash column chromatography over silica gel with (eluent: 1-10 % of MeOH in DCM) to afford SD-157 as a colorless solid (560 mg, 87 %).Synthesis of SD-113-Amine tert-butyl 4-amino-5-(R)-1-(tert-butoxy)-3-(4-hydroxyphenyl) -1oxopropan-2-yl)amino)-5-oxopentanoate:The Fmoc of SD-113 was removed with 20% piperidine/DMF (v/v) for 30 min. The progress of reaction was monitored by LC-MS. After completion of reaction, the solvent was evaporated under vacuum and the crude mixture was purified by flash column chromatography over silica gel with (eluent: 1-10 % of MeOH in DCM with drop of TEA) to afford SD-113_Amine as a colorless solid (171 mg, 81 %). Compound SD-158 was synthesized from SD-113-Amine (150 mg, 0.35 mmol) by following the same procedure described for the synthesis of SD-139 to afford SD-158 as white solid (132 mg, 67%).

Fig. S1 .

 S1 Fig. S1. TCP enriched fraction prefers tubulin as substrate. (A) Table of enzyme enrichment steps from mouse brain tissue. Detyrosination activity from equal volumes of the various fractions was estimated by measuring tyrosine released from [ 14 C]-tyrosine labeled microtubules (2 µM) after 45 min incubation. (B) Detyrosination of tubulin and EB1 (5 µM) by fraction IV revealed by immunoblot. A reaction with carboxypeptidase A (CPA) was used as positive control.

Fig. S3 .

 S3 Fig. S3. VASH1 and VASH2 associated to SVBP are potent tubulin Tyr/Phe carboxypeptidases. (A) Immunofluorescence analysis of de/tyrosinated α-tubulin pools from MEF cells cotransfected with plasmids encoding active or inactive VASH2 and SVBP (V2 or deadV2, respectively). Carboxypeptidase activity was revealed by immunofluorescence with the same antibodies as in Fig. 2B. Vasohibins were probed with anti-GFP antibody. (B) Immunoblot of protein extracts from HEK293T cells expressing mCherry-α8-tubulin, each VASH and SVBP.Native or mutated versions of α8-tubulin, respectively ending with GEEF or GEEA, were used. Levels of de/tyrosinated tubulin were measured as in Fig.2A. Antibody to mCherry demonstrates same amounts of exogenous α-tubulin.

Fig. S5 .

 S5 Fig. S5. Down-regulation of VASH1 and VASH2 affects neuronal differentiation. (A, B) validation of shRNAs targeting the vasohibins. (A) RT-qPCR quantifications of neuronal vasohibins transcripts in the presence of control or VASH1 and VASH2 shRNAs. Mean + SEM.

Fig. S6 .

 S6 Fig. S6. Down-regulation of SVBP alters neuronal differentiation. (A, B) Validation of SVBP shRNAs. (A) RT-qPCR quantifications of neuronal Svbp transcripts in the presence of control or

Fig. S7 .

 S7 Fig. S7. Effect of vasohibins or SVBP down-regulation on Tau and ankyrin G staining of axons. Neurons were transfected by electroporation with shRNAs associated to turboGFP

  .22, 163.08, 155.63, 130.26, 126.90, 115.23, 62.43, 53.51, 52.44, 51.93, 36.64, 36.37, 31.46, 27.06, 13.80. MS (ESI) m/z: calcd: [M+H] + 453.14, measured: [M+H] + 453.18

	1 H NMR (400 MHz, Chloroform-d) δ 6.94 -6.87 (m, 2H), 6.65 (d, J = 8.1 Hz, 2H), 4.62 (t, J = 6.3
	Hz, 1H), 4.35 (d, J = 7.0 Hz, 1H), 4.19 (qt, J = 7.8, 4.0 Hz, 2H), 3.53 (dd, J = 22.0, 1.7 Hz, 2H),
	3.06 (dd, J = 14.2, 5.2 Hz, 1H), 2.89 -2.82 (m, 0H), 2.32 -2.21 (m, 2H), 2.01 -1.90 (m, 1H), 1.80

(dd, J = 14.5, 7.7 Hz, 1H), 1.25 (t, J = 7.1 Hz, 3H). 13C NMR (101 MHz, cdcl3) δ 178.

15, 174.41, 173.76, 167

Acknowledgements:

We would like to dedicate this manuscript to Dr Hector Barra, father of the de/tyrosination cycle who passed away on 2 nd december 2016. We are grateful to D. Job who strongly supported initiation of the project and for his constant encouragements. We thank M. Comte, E. Martin, C. Corrao, and C. Paoli for technical assistance. We thank members of the PIC-GIN platform for their help in cell imaging and zootechnicians of the Grenoble Institut des Neurosciences for animal care. We thank M. Steinmetz for providing purified TTL, A. Beghin for initial development of AutoNeuriteJ macro. We thank the support of the discovery platform and informatics group at EDyP. This work was supported by INSERM, CEA, University Grenoble Alpes, CNRS, and by La Ligue Contre le Cancer comités de l'Isère et de Savoie (to MJM), Association pour la Recherche sur le Cancer (SFI20111204053 to MJM; DOC20120605000 to AB; 20151203348 to LL), Fondation pour la Recherche Médicale FDT20160435356 (to CA), ANR-13-JSV2-0002/TYRTUB (to KR), INCA 2016-165 (to LL) and the National Institutes of Health grant R01 EB005011 (to MB). Proteomic experiments were partly supported by the ProFi grant (ANR-10-INBS-08-01).

A patent untitled "Methods and pharmaceutical compositions for treating tubulin carboxypeptidases associated diseases" has been filed by Inserm and UGA. Data described can be found in the main figures, supplementary materials and auxiliary table.

Supplementary materials:

Materials and Methods

Author contributions

Figs S1 to S7

Table S1 References [START_REF] Ramirez-Rios | Tau antagonizes end-binding protein tracking at microtubule ends through a phosphorylation-dependent mechanism[END_REF][START_REF] Argarana | Tubulinyl-tyrosine carboxypeptidase from chicken brain: properties and partial purification[END_REF][START_REF] Milbradt | Proteomic analysis of the multimeric nuclear egress complex of human cytomegalovirus[END_REF][START_REF] Pedelacq | Engineering and characterization of a superfolder green fluorescent protein[END_REF][START_REF] Arganda-Carreras | 3D reconstruction of histological sections: Application to mammary gland tissue[END_REF][START_REF] Barnat | Huntingtin-Mediated Multipolar-Bipolar Transition of Newborn Cortical Neurons Is Critical for Their Postnatal Neuronal Morphology[END_REF][START_REF] Khodiyar | A revised nomenclature for the human and rodent alpha-tubulin gene family[END_REF][START_REF] Aillaud | Evidence for new C-terminally truncated variants of alpha-and beta-tubulins[END_REF][START_REF] Paturle-Lafanechere | Accumulation of delta 2-tubulin, a major tubulin variant that cannot be tyrosinated, in neuronal tissues and in stable microtubule assemblies[END_REF][START_REF] Rogowski | A family of protein-deglutamylating enzymes associated with neurodegeneration[END_REF]

Supplementary Materials for

Vasohibins/SVBP are tubulin carboxypeptidases (TCP) that regulate neuron differentiation Chrystelle Aillaud, Christophe Bosc, Leticia Peris, Anouk Bosson, Pierre Heemeryck, Juliette Van Dijk, Julien Le Friec, Benoit Boulan, Frédérique Vossier, Laura E. Sanman, Salahuddin Syed, Neri Amara, Yohann Couté, Laurence Lafanechère, Eric Denarier, Christian Delphin, Laurent Pelletier, Sandrine Humbert, Matthew Bogyo, Annie Andrieux*, Krzysztof Rogowski*, and Marie-Jo Moutin*. *correspondence to: moutinm@univ-grenoble-alpes.fr, annie.andrieux@univ-grenoble-alpes.fr, or krzysztof.rogowski@igh.cnrs.fr.

This PDF file includes:

Materials and Methods

Author contributions

Figs S1 to S8

Table S1 and caption of Table S2 References [START_REF] Ramirez-Rios | Tau antagonizes end-binding protein tracking at microtubule ends through a phosphorylation-dependent mechanism[END_REF][START_REF] Argarana | Tubulinyl-tyrosine carboxypeptidase from chicken brain: properties and partial purification[END_REF][START_REF] Milbradt | Proteomic analysis of the multimeric nuclear egress complex of human cytomegalovirus[END_REF][START_REF] Pedelacq | Engineering and characterization of a superfolder green fluorescent protein[END_REF][START_REF] Arganda-Carreras | 3D reconstruction of histological sections: Application to mammary gland tissue[END_REF][START_REF] Barnat | Huntingtin-Mediated Multipolar-Bipolar Transition of Newborn Cortical Neurons Is Critical for Their Postnatal Neuronal Morphology[END_REF][START_REF] Khodiyar | A revised nomenclature for the human and rodent alpha-tubulin gene family[END_REF][START_REF] Aillaud | Evidence for new C-terminally truncated variants of alpha-and beta-tubulins[END_REF][START_REF] Paturle-Lafanechere | Accumulation of delta 2-tubulin, a major tubulin variant that cannot be tyrosinated, in neuronal tissues and in stable microtubule assemblies[END_REF][START_REF] Rogowski | A family of protein-deglutamylating enzymes associated with neurodegeneration[END_REF] Other Supplementary Materials for this manuscript includes the following:

Auxiliary Table (Table S2) cycles using the following primers: 5'-TACAAACCGCCCGCCTTCC and 5'-ACAGACCCTGACAGCTACCAACA for Vash1, 5'-GCAGCCTTCCATTGAGCGGT and 5'-CAGTCAACCCAGGGCTTTGCC for Vash2, 5'-CCAGCAGGAGCTGAAGCAAAGA and 5'-GCACCAGTTCCTCTGCCGGG for Svbp. Gapdh was amplified at 64°C by 25 cycles with 5'-TCAACGGGAAGCCCATCACCA and 5'-GTTTCTCCAGGCGGCACGTC primers. For RT-qPCR, cells were lysed using the SingleShot Cell Lysis kit (Bio-Rad) and analyzed using the iScript Reverse Transcription mix (Bio-Rad) and SsoAdvanced Universal SYBR Green Supermix (Bio-Rad). Products of 105 bp from mouse Vash1, Vash2, Svbp and Gapdh were amplified at 60°C by 40 cycles using the following primers:

5'-TGGCCAAGATCCACCCAGATG and 5'-TCGTCGGCTGGAAAGTAGGCAC for Vash1, 5'-AGGGGGAGAGATGGTAGGCGC and 5'-AGCCAGTCTGGGATCGTCATGG for Vash2,

5'-AACCAGCCTTCAGAGTGGAGAAGG and 5'-GCTCCGTCATGACTCTGTTGAGAGC for

Svbp, and 5'-CGTGCCGCCTGGAGAAAC and 5'-TGGGAGTTGCTGTTGAAGTCG for Gapdh.

Morphometric neuron analysis.

Mosaic images of 2DIV neuron fixed and stained with antitubulin antibody were acquired with a 20X N.A 0.5 objective on a DMI6000 Leica microscope with a motorized stand. Images were segmented after enhancement with a DoG filter. The cell bodies of single neurons were manually selected and used to process neurons individually with an homemade AutoNeuriteJ macro. Briefly the neuron images were skeletonized and neuritic loops were resolved using the "Analyze Skeleton 2D/3D ImageJ plugin" [START_REF] Arganda-Carreras | 3D reconstruction of histological sections: Application to mammary gland tissue[END_REF]. The ends of neurites were marked using the "BinaryConnectivity" imageJ plugin (developed by G. Landini, http://www.mecourse.com/landinig/software/software.html). Images of each neurite from their ends to the cell body were produced. Among the neurites with a path overlap, the longest was defined as primary neurite. The images of primary neurites were substracted to images of overlapping neurites, thus defining secondary neurites and number of branching. Neurite lengths were measured. Neurites hour. The cooling bath was then removed and the reaction mixture was stirred for further 2 hrs at room temperature. Afterwards the ethanol was removed under reduced pressure leaving an off white solid. This salt was then taken up with 5 mL of water and washed three times with dichloride methane. The aqueous phase was acidified by addition of concentrated hydrochloric acid to pH 3.0 and extracted four times with ethyl acetate. The combined organic phase was dried over magnesium sulphate and concentrated under reduced pressure. After freeze drying the pure product was obtained as a colorless solid (62 mg, 88% yield).

Synthesis of SD-161-PG (S)-tert-butyl 3-(4-hydroxyphenyl)-2-((2S,3S)-3-(prop-2-yn-1ylcarbamoyl)oxirane-2-carboxamido) propanoate:

To the solution acid SD-161 (60 mg, 0.17 (tGFP) cDNA just before plating and analyzed by immunofluorescence for tyrosinated tubulin and Tau contents at 3DIV (A) or for tyrosinated tubulin and ankyrin G levels at 10DIV (B). Levels of shRNA were imaged using an anti-tGFP antibody. S1. Vasohibin-1 peptides identified by mass spectrometry-based analysis (see Table S2 for detailed proteomic analyses). Numbering of start and stop amino acids is according accession number NP_796328. Numbers of spectral counts (SC) are indicated for each of the 3 independent fractions prepared using the clickable inhibitor (TCP-R1 to TCP-R3). Lower amounts of purified brain extract were used in R2 and R3 than in R1 (3-and 2-fold, respectively).


domain: scco sdv

text:
Marc Deffains

Marie-Hélène Canron

Margaux Teil

Qin Li

Benjamin Dehay

Erwan Bezard

Pierre-Olivier Fernagut
email: pierre.olivier.fernagut@univ-poitiers.fr

L-DOPA regulates -synuclein accumulation in experimental parkinsonism

Keywords: Parkinson's disease, levodopa, α-synuclein, protein aggregation, tau, MPTP, macaque Lewy bodies

Aims: Widespread accumulation of misfolded -synuclein aggregates is a key feature of Parkinson's disease (PD). Although the pattern and extent of -synuclein accumulation through PD brains is known, the impact of chronic dopamine-replacement therapy (the gold-standard pharmacological treatment of PD) on the fate of α-synuclein is still unknown. Here, we investigated the distribution and accumulation of α-synuclein in the 1methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) non-human primate of PD and determined the effect of chronic L-DOPA treatment on MPTP-induced α-synuclein pathology.

Methods:

We measured the density of -synuclein and tau immuno-positive neurons in the substantia nigra, putamen, hippocampal CA1 region, temporal cortex and dentate nucleus of control, MPTP and MPTP+L-DOPA-treated monkeys. Moreover, we also extracted and quantified Triton-X (TX) soluble and insoluble -synuclein in putamen and hippocampus samples from a separate cohort of control, MPTP and MPTP+L-DOPAtreated monkeys.

Results: MPTP-induced -synuclein accumulation in NHP model of PD was not limited to the substantia nigra but also occurred in the putamen, hippocampal CA1 region and temporal cortex. Tau was increased only in the temporal cortex. Moreover, increased intraneuronal TX insoluble -synuclein was truncated, but not in the structural form of Lewy bodies. The MPTP-induced increase in -synuclein levels was abolished in animals having received L-DOPA in all the brain regions, except in the substantia nigra.

Introduction

Widespread accumulation of aggregation-prone proteins is a key feature of neurodegenerative disorders. In Parkinson's disease (PD) and related-disorders designated as synucleinopathies, misfolded -synuclein accumulates either in Lewy bodies (LBs) in neurons in PD and dementia with Lewy bodies or in oligodendroglial cytoplasmic inclusions in multiple system atrophy. In addition to -synuclein, these cytoplasmic inclusions contain numerous components, including other aggregation prone proteins such as tau [START_REF] Ishizawa | Colocalization of tau and alphasynuclein epitopes in Lewy bodies[END_REF][START_REF] Wakabayashi | The Lewy body in Parkinson's disease and related neurodegenerative disorders[END_REF]. The spatial and temporal evolution of -synuclein accumulation over the course of PD has been described, suggesting an ascending progression from the brainstem, later invading subcortical and cortical territories in the most advanced stages of the disease [START_REF] Braak | Staging of brain pathology related to sporadic Parkinson's disease[END_REF]. Although this scheme has been confirmed in subsequent studies, deviation from this proposed pattern has also been reported (for review, see [START_REF] Jellinger | A critical reappraisal of current staging of Lewy-related pathology in human brain[END_REF]). Whether this relates to the intrinsic heterogeneity of the disease process, clinical subtypes associated with unconventional patterns of -synuclein accumulation, concurrent aging-related formation of LBs or external factors remains a matter of debate.

Given its pivotal role in the pathogenesis of PD, -synuclein is considered as a promising biomarker [START_REF] Brooks | Imaging synucleinopathies[END_REF]. As such, it is critical to identify if symptomatic treatments used in PD may affect the pattern and extent of -synuclein accumulation. This remains an open question due to inherent limitations. Indeed, pathological studies on PD brains are unlikely to be able to reveal any effect of a given symptomatic treatment on protein aggregation due to clinical and pathological heterogeneity, combined with a large variety of drugs used over the course of the disease and incomplete or limited clinical records of drug intake.

Administration

of the parkinsonian neurotoxin 1-methyl-4-phenyl-1,2,3,6tetrahydropyridine (MPTP) is the gold-standard for modelling PD in non-human primates (NHPs), recapitulating many of the motor and non-motor clinical features of the disease [START_REF] Deffains | Subthalamic, not striatal, activity correlates with basal ganglia downstream activity in normal and parkinsonian monkeys[END_REF]7]. With regards to protein misfolding and accumulation, exposure to MPTP leads to a secondary accumulation of -synuclein in the substantia nigra [START_REF] Kowall | MPTP induces alphasynuclein aggregation in the substantia nigra of baboons[END_REF][START_REF] Mccormack | Pathologic modifications of alpha-synuclein in 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)treated squirrel monkeys[END_REF][START_REF] Purisai | Alpha-synuclein expression in the substantia nigra of MPTP-lesioned non-human primates[END_REF], that can still be detected over 10 years after administration of the toxin, suggesting that this neurotoxin leads to long lasting alterations of -synuclein homeostasis [START_REF] Halliday | No Lewy pathology in monkeys with over 10 years of severe MPTP Parkinsonism[END_REF]. These seminal studies have however focused on the substantia nigra pars compacta (SNpc) and did not investigate other regions of the central nervous system relevant to the pathophysiology of

Accepted Article

PD. Furthermore, these studies did not consider other aggregation prone proteins such as tau and did not investigate the potential effect of dopamine replacement therapy (DRT).

The aim of this study was to investigate in the NHP MPTP model of PD the possible occurrence of a widespread synucleinopathy as occurring in the human disease and to determine if the gold-standard pharmacological treatment (i.e., L-DOPA) may influence some neuropathological features such as neuronal loss in the substantia nigra, astrogliosis, and the fate of aggregation prone proteins such as α-synuclein and Tau.

Materials and Methods

Animals

Experimental protocol

Ten animals were kept as untreated controls (control group). The remaining 18 animals were treated daily with MPTP hydrochloride (0.2 mg/kg, i.v., Sigma, St Louis, MO) dissolved in saline according to a previously described protocol [START_REF] Bezard | Relationship between the appearance of symptoms and the level of nigrostriatal degeneration in a progressive 1-methyl-4-phenyl-1,2,3,6tetrahydropyridine-lesioned macaque model of Parkinson's disease[END_REF][START_REF] Bezard | A chronic MPTP model reproducing the slow evolution of Parkinson's disease: evolution of motor symptoms in the monkey[END_REF]. Following stabilization of the MPTP-induced syndrome, animals received either vehicle (MPTP,

Accepted Article

This article is protected by copyright. All rights reserved n=8), or were chronically treated twice daily with 20 mg/kg levodopa for 110 days (MPTP + L-DOPA, n=10). All animals were killed by sodium pentobarbital overdose (150 mg/kg, i.v.) 1 hr after the last dose of vehicle or L-DOPA, the brains were removed quickly after death and bisected along the midline. For the histopathological study (n=5 untreated controls, 3 MPTP, 5 MPTP + L-DOPA), brains, hemispheres were post-fixed in formalin before paraffin-embedding. For the western blot study (n=5 untreated controls, 5 MPTP, and 5 MPTP + L-DOPA), the two hemispheres were immediately frozen by immersion in isopentane (-45°C) and then stored at -80°C. Coronal 300 μm-thick sections were cryostat-cut and punches of brain tissue were taken for several brain regions including the striatum and the hippocampus [START_REF] Fernagut | Dopamine transporter binding is unaffected by L-DOPA administration in normal and MPTPtreated monkeys[END_REF][START_REF] Napolitano | Decreased Rhes mRNA levels in the brain of patients with Parkinson's disease and MPTP-treated macaques[END_REF][START_REF] Nuzzo | The levels of the NMDA receptor co-agonist D-serine are reduced in the substantia nigra of MPTP-lesioned macaques and in the cerebrospinal fluid of Parkinson's disease patients[END_REF][START_REF] Santini | Distinct changes in cAMP and extracellular signal-regulated protein kinase signalling in L-DOPAinduced dyskinesia[END_REF][START_REF] Stanic | Rabphilin 3A: A novel target for the treatment of levodopa-induced dyskinesias[END_REF].

Brain processing

Immunohistochemical studies were performed with procedures and antibodies used in human pathology, as described below. On 4μm thick coronal sections, a horseradish peroxidase method was used with the following antibodies : anti-tyrosine hydroxylase (TH), (monoclonal mouse antibody, 1:10000, Chemicon international, Temecula, California, USA), anti-human-specific α-synuclein clone LB509 (monoclonal mouse antibody, 1:100 ; Invitrogen by life technologies, France), anti-tau (polyclonal rabbit antihuman 1:1000; Dako, France), phosphorylation-dependant tau antibody (Tau AT8, PHFtau monoclonal, mouse anti-human, clone AT8, 1:100; Pierce Biotechnology, USA), anti-Glial Fibrillary Acidic Protein (GFAP, polyclonal rabbit, 1:4000; Dako, France).

Sections were deparaffinized in toluene and rehydrated in graded series of ethanol.

Antigen retrieval was not required for anti Tau or anti Tau AT8 labelling whereas sections labelled with GFAP were pre-treated with proteinase K (Dako, Trappes, France) and those labelled with LB509 α-synuclein were pressure-cooked in sodium citrate buffer (10 min in 0.01 M, pH 6.0). Non-specific binding was blocked with a universal blocking reagent (Biogenex) for LB509 α-synuclein, GFAP and Tau or with 5% normal goat serum in PBS for anti Tau AT8 for 30 min at room temperature. For TH labeling, non-specific binding was blocked with 3% normal horse serum in PBS for 60 min. Sections were incubated in primary antibodies overnight. Subsequently, sections were transferred in 3% H2O2 in PBS for 5 min to quench endogenous peroxidase activity and treated with a ready-to-use goat anti mouse or goat anti rabbit EnVision-HRP enzyme conjugate (Dako,

Accepted Article

This article is protected by copyright. All rights reserved

France) for 30 min. For TH staining, sections were incubated with the biotinylated horse anti-mouse/rabbit IgG (1:200) secondary antibody for 2 hours and then with the avidinbiotin-peroxidase complex (ABC Elite Kit, PK-6200, Vector Labs) for further 90 min.

Immunoreactions were revealed using the highly sensitive diaminobenzidine plus (DAB+) and the 3-amino-9 ethyl carbazol plus (AEC+) (both from Dako, France) as substrates chromogens. Finally, sections were counterstained with Mayer's hemalum and mounted in an aqueous medium for microscopy (Aquatex; Merck). Negative immunohistological control demonstrated the absence of signal when omitting the first antibody. Pictures were taken with a Leica microscope (Leica DM6000B) at x20 magnification.

To assess the localization of α-synuclein, a double immunostaining was performed with anti-tyrosine hydroxylase (TH), (polyclonal rabbit antibody, 1:2000, Institute Jacques Boy, France) and anti α-synuclein, clone LB509, (monoclonal mouse antibody, 1:100 ;

Invitrogen by life technologies, France) antibodies. The same protocol for deparaffinization and antigen retrieval in sodium citrate buffer was used as above. Then, 4 µm midbrain sections were blocked with 5% normal goat serum in PBS and sequentially incubated overnight with the primary antibodies. Secondary antibodies were Alexa Fluor 488-labeled goat anti-mouse for α-synuclein, and Alexa Fluor 568-labeled goat anti-rabbit for TH (both from Invitrogen, France). To lower the intensity of autofluorescence, slides were incubated for 10 minutes with 0.1% Sudan Black B (Sigma-Aldrich, France) in 70% ethanol. After thorough washing in PBS, slides were mounted in fluorescence mounting medium (Agilent, USA). Immunofluorescence was visualized by using a Zeiss Axioplan 2 epifluorescent microscope at x40 magnification.

Quantitative analysis

To minimize the inherent variability in the immunochemical procedures, all sections from all animals were processed simultaneously for a given antibody and brain region. The number of immunopositive cells was analysed using a computerized image analysis system (Mercator V6.50, Explora Nova) linked to a Leica microscope type DM 6000B.

Quantitative analysis was carried out on the whole structure area and results were expressed as an average of immunopositive cells per mm 2 . α-Synuclein immunofluorescence intensity was assessed on 15 α-syn positive dopaminergic neurons in each animal. For cell fluorescence intensity, ImageJ software v1.52a was used to

Accepted Article

This article is protected by copyright. All rights reserved measure pixel intensity and area with respect to background intensity and cell surface respectively, according to the following formula: Intensity of stained cell -(sample of background/area of sample background) X area of stained cell.

Sequential α-synuclein extraction and Western blot analysis

Tissue was homogenized in Triton-X (TX) extraction buffer (50 mM Tris-base pH 7.6, 150 mM NaCl, 1% Triton-X-100, 2 mM EDTA) containing protease and phosphatase inhibitors [START_REF] Refolo | Progressive striatonigral degeneration in a transgenic mouse model of multiple system atrophy: translational implications for interventional therapies[END_REF]. The lysate was sonicated and then centrifuged (120,000 x g for 60 min at 4 °C) and the supernatant was collected (TX soluble fraction). The pellet was then washed 3 times with 1 M PBS/1% TX, centrifuged (13,000 x g for 15 min) and re-suspended in SDS extraction buffer (50 mM Tris pH 7.6, 150 mM NaCl, 1% Triton-X-100, 0.5% Nadeoxycholate, 1% SDS), sonicated, and centrifuged (120,000 x g for 60 min at 4 °C) and the supernatant was collected (TX insoluble fraction). The samples (30 ug) were run on 15% SDS-PAGE gels. Primary antibodies included antibodies against α-syn [monoclonal syn-1, (1:1000; BD Biosciences)] and β-actin (1:2000; Millipore Sigma A5441) as a loading control. The intensity of the immunoreactive bands was estimated by densitometric quantification using ImageJ (relative density, RD) and then normalized to the corresponding β-actin levels.

Statistical analysis

Data are presented as mean  SEM. Assumptions about normality of distribution and homogeneity of variances were analysed using the Shapiro-Wilk and Brown-Forsythe tests, respectively. Data were analysed using one-way ANOVA followed by post hoc Tukey tests for multiple comparisons or using Kruskal-Wallis followed by Dunn's multiple comparisons tests whenever appropriate. These analyses were completed using Graphpad Prism V 8.0. A probability level of 5% (p<0.05) was considered significant.

Data Accessibility

The data are available from the corresponding author upon request.

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Results

L-DOPA treatment does not affect MPTP-induced neurodegeneration and astrogliosis

We first aimed to determine if L-DOPA could have an impact on neuronal loss in the substantia nigra. As expected, MPTP intoxication induced a significant loss of TH-positive neurons in the substantia nigra (F (2,10) =46.40, p<0.0001, Fig. 1). The extent of THimmunoreactive cell loss was similar between MPTP (79.9%, p<0.0001) and MPTP + L-DOPA-treated monkeys (76.8%, p<0.0001). Counting of Nissl-stained neurons confirmed neurodegeneration (F (2,10) =12.46, p<0.01), both in MPTP (-66%, p<0.01) and in MPTP + L-DOPA-treated animals (-80.4%, p<0.01). All MPTP-treated monkeys exhibited a mild parkinsonian syndrome that was alleviated in animals receiving L-DOPA (data not shown), as previously reported [START_REF] Bezard | A chronic MPTP model reproducing the slow evolution of Parkinson's disease: evolution of motor symptoms in the monkey[END_REF][START_REF] Bézard | Attenuation of levodopa-induced dyskinesia by normalizing dopamine D3 receptor function[END_REF][START_REF] Engeln | Selective Inactivation of Striatal FosB/DeltaFosB-Expressing Neurons Alleviates L-DOPA-Induced Dyskinesia[END_REF][START_REF] Imbert | Comparison of eight clinical rating scales used for the assessment of MPTP-induced parkinsonism in the Macaque monkey[END_REF].

Because astrogliosis is a prominent feature of PD that is recapitulated in MPTP models

MPTP and L-DOPA differentially affect -synuclein and tau accumulation

MPTP intoxication and subsequent chronic L-DOPA treatment differentially affected synuclein immunoreactivity (Fig. 3). In the substantia nigra, there was a significant

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This article is protected by copyright. All rights reserved difference on the density of -synuclein immuno-positive neurons between groups (H = 6.76, df = 2, p<0.05, Fig. 3A). Post-hoc analysis indicated increased -synuclein staining in MPTP-treated monkeys compared to controls (p<0.05). A significant effect was found for the density of -synuclein positive fibres in the putamen (H=7.47, p<0.05, Fig. 3B),

where -synuclein immunostaining was significantly elevated in MPTP-treated monkeys compared with the MPTP + L-DOPA group (p<0.05). A similar effect was found in CA1 (H=11.76, p<0.01, Fig. 3C) and post-hoc analysis indicated that MPTP-treated monkeys displayed increased -synuclein immunoreactivity compared with control and MPTP + L-DOPA groups (p<0.01). In the temporal cortex the density of -synuclein-immunopositive fibres was also significantly different between groups (H = 8.366, df = 2, p<0.01, Fig. 3D), with the MPTP group displaying higher density of -synuclein positive fibres compared with the control and MPTP + L-DOPA groups (p<0.05). Conversely, no significant effect was found in the dentate nucleus (H=3.33, p=0.23, Fig. 3E). Remarkably, the marked synuclein immunostaining following MPTP was abolished in animals having received L-DOPA in all regions except in the substantia nigra (Fig. 3Q-T). Double immunofluorescence for -synuclein and tyrosine hydroxylase revealed no cytoplasmic accumulation of -synuclein in controls (Fig. 4A-C) and confirmed that -synuclein accumulation in the substantia nigra of MPTP and MPTP + L-DOPA-treated animals occurred in the remaining dopaminergic neurons (Fig. 4D-I). Quantification of -synuclein fluorescence intensity in remaining nigral dopaminergic neurons revealed that albeit L-DOPA was not able to clear -synuclein, immunofluorescence intensity was significantly reduced in MPTP + L-DOPA compared with the MPTP group (p<0.05, Fig. 4J).

Because Tau pathology is frequent in PD [START_REF] Zhang | Tau Pathology in Parkinson's Disease[END_REF], we investigated the effects of MPTP and subsequent L-DOPA treatment on Tau accumulation. Contrary to the effect on the accumulation and extent of -synuclein, MPTP intoxication and subsequent chronic L-DOPA treatment led to only modest effects on tau immunoreactivity (Fig. 5). In the nigrostriatal pathway, MPTP and MPTP + L-DOPA induced a non-significant increase in the number of tau immunoreactive neurons in the substantia nigra (H = 3.14, p=0.21, Fig. 5A) and in the putamen (F (2,10) =1.95, p=0.19, Fig. 5B). No significant effects of MPTP and MPTP + L-DOPA were found in CA1 (F (2,10) =0.52, p=0.60, Fig. 5C) or in the dentate

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This article is protected by copyright. All rights reserved nucleus (H=1.80, df=2, p=0.43, Fig. 5E). Conversely, there was a significant effect in the temporal cortex (F (2,10) =4.7, p<0.05). Post-hoc analysis indicated a significantly increased number of tau immunoreactive neurons in MPTP-treated monkeys (p<0.05) compared with controls (Fig. 5D). Additional investigation of the effect of MPTP and chronic L-DOPA administration on tau was performed using the AT8 antibody that recognizes tau To further investigate the effect of chronic L-DOPA treatment on -synuclein homeostasis, putamen and hippocampus samples from a separate cohort of control, MPTP and MPTP + L-DOPA-treated monkeys were subjected to a sequential protein extraction protocol in order to quantify triton-X (TX) soluble and TX insoluble -synuclein by western blot (Fig. 6). -synuclein immunoblots performed on TX-soluble and TXinsoluble fractions revealed a signal at the expected weight of 14 kDa (full length synuclein) and a faint signal at 12 kDa (truncated -synuclein) that could be quantified upon overexposure. No high molecular weight assemblies of -synuclein were detected.

In the putamen, there was no significant effect of MPTP or MPTP + L-DOPA on fulllength -synuclein (F (2,12) =1.83, p=0.20, Fig. 6A,C), whereas a significant effect was found for the 12kDa band (F (2,12) =20.12, p<0.0001, Fig. 6A,D) in the TX soluble fraction.

Post-hoc analysis revealed a significant decrease of -synuclein in MPTP + L-DOPAtreated animals compared with controls and with the MPTP group (p<0.01). Interestingly, in the TX-insoluble fraction, there was a significant effect of treatment on full length synuclein (F (2,12) =10.05, p<0.01, Fig. 6B,E

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In the hippocampus, there was a significant effect on full length -synuclein in the TXsoluble fraction (F (2,12) =12.17, p<0.01, Fig. 6G,I), with MPTP + L-DOPA-treated monkeys showing significantly more -synuclein than their MPTP-treated counterparts (p<0.01) and controls (p<0.05). No significant effect was detected for the 12kDa band (F (2,12) =2.47, p=0.12). In the TX-insoluble fraction, no significant differences were found among the three groups for full length -synuclein (F (2,12) =1.49, p=0.26). However, there was a significant effect of 12 kDa -synuclein (F (2,12) =10.92, p<0.01). Post-hoc analysis revealed that 12 kDa -synuclein was significantly increased in MPTP-treated monkeys compared with controls (p<0.05) and MPTP + L-DOPA-treated animals (p<0.01, Fig. 5L).

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Discussion

The spatial and temporal evolution of -synuclein accumulation over the course of PD has been well-described [START_REF] Braak | Staging of brain pathology related to sporadic Parkinson's disease[END_REF]. However very little is known about the impact of long-term exposure to DRT (i.e., the gold-standard pharmacological treatment of PD) on the propagation/distribution/accumulation of α-synuclein. Since it has already been shown that intoxication with MPTP induces -synuclein aggregation in the SNpc of NHPs [START_REF] Kowall | MPTP induces alphasynuclein aggregation in the substantia nigra of baboons[END_REF][START_REF] Mccormack | Pathologic modifications of alpha-synuclein in 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)treated squirrel monkeys[END_REF][START_REF] Purisai | Alpha-synuclein expression in the substantia nigra of MPTP-lesioned non-human primates[END_REF][START_REF] Halliday | No Lewy pathology in monkeys with over 10 years of severe MPTP Parkinsonism[END_REF],

we therefore investigated in MPTP-treated monkeys the fate of -synuclein and tau in several regions of the central nervous system relevant to the pathophysiology of PD and examined the consequences of chronic DRT on the pattern and extent of these aggregation-prone proteins. Therefore, characterizing the impact of DRT on their fate could aid in developing and validating reliable biomarkers to assess the progression and severity of the disease.

As reported previously, MPTP-treated animals showed large neuronal loss and pronounced astrogliosis in the SNpc (Fig. 1&2) [START_REF] Charron | Astrocytosis in parkinsonism: considering tripartite striatal synapses in physiopathology?[END_REF]. Moreover, we showed that MPTPinduced astrogliosis was not limited to the substantia nigra but also occurred in the putamen (Fig. 2B) and the temporal cortex (Fig. 2D). So far, it has been demonstrated that MPTP -possibly due to its inhibitory action on the complex I of the mitochondrial respiratory chains [START_REF] Hartmann | Postmortem studies in Parkinson's disease[END_REF][START_REF] Langston | Chronic Parkinsonism in humans due to a product of meperidine-analog synthesis[END_REF] -induced -synuclein accumulation in the surviving midbrain dopaminergic neurons of NHPs [START_REF] Kowall | MPTP induces alphasynuclein aggregation in the substantia nigra of baboons[END_REF][START_REF] Mccormack | Pathologic modifications of alpha-synuclein in 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)treated squirrel monkeys[END_REF][START_REF] Purisai | Alpha-synuclein expression in the substantia nigra of MPTP-lesioned non-human primates[END_REF][START_REF] Halliday | No Lewy pathology in monkeys with over 10 years of severe MPTP Parkinsonism[END_REF] and mice [START_REF] Duka | Alpha-synuclein induces hyperphosphorylation of Tau in the MPTP model of parkinsonism[END_REF][START_REF] Vila | Alphasynuclein up-regulation in substantia nigra dopaminergic neurons following administration of the parkinsonian toxin MPTP[END_REF]. In line with these studies, we found a significant increase in the density of -synuclein immuno-positive neurons in the SNpc of the MPTP-treated monkeys (Fig. 3A, Fig. 4). Moreover, we demonstrate for the first time in MPTP-treated monkeys that the density of -synuclein increased in the putamen (i.e., a motor-related/subcortical structure, Fig. 3B) as well as in neurons of non-motor and cortical regions, such as the hippocampal CA1 (Fig. 3C) region and temporal cortex (Fig. 3D). In PD, LBs in which α-synuclein is a major constituent are widely distributed in the central nervous system, including brainstem, subcortical and cortical areas [START_REF] Wakabayashi | The Lewy body in Parkinson's disease and related neurodegenerative disorders[END_REF][START_REF] Braak | Staging of brain pathology related to sporadic Parkinson's disease[END_REF][START_REF] Braak | Cognitive decline correlates with neuropathological stage in Parkinson's disease[END_REF][START_REF] Wakabayashi | The Lewy body in Parkinson's disease: molecules implicated in the formation and degradation of alpha-synuclein aggregates[END_REF]. Moreover, their distributions are most likely correlated with the motor and non-motor symptoms of PD [START_REF] Wakabayashi | The Lewy body in Parkinson's disease and related neurodegenerative disorders[END_REF][START_REF] Braak | Staging of brain pathology related to sporadic Parkinson's disease[END_REF][START_REF] Wakabayashi | The Lewy body in Parkinson's disease: molecules implicated in the formation and degradation of alpha-synuclein aggregates[END_REF][START_REF] Lim | Overview of the extranigral aspects of Parkinson disease[END_REF]. A major difference between idiopathic PD and the MPTP-primate models of PD is the presence of insoluble, high molecular weight assemblies of α-synuclein in LBs in PD, but not in animal models of PD

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This article is protected by copyright. All rights reserved [START_REF] Kowall | MPTP induces alphasynuclein aggregation in the substantia nigra of baboons[END_REF][START_REF] Halliday | No Lewy pathology in monkeys with over 10 years of severe MPTP Parkinsonism[END_REF]. Here, and in line with previous results, we did not find LBs in the different brain regions of MPTP-treated animals. Nevertheless, we still demonstrated the accumulation of -synuclein in non-motor cortical regions, indicating that the widespread synucleinopathy classically observed in PD patients is somewhat recapitulated in the MPTP NHP model of PD.

Under physiological conditions, -synuclein is a soluble protein widely distributed in the brain and especially expressed in the presynaptic nerve terminals [START_REF] Wakabayashi | The Lewy body in Parkinson's disease and related neurodegenerative disorders[END_REF][START_REF] Iwai | The precursor protein of non-A beta component of Alzheimer's disease amyloid is a presynaptic protein of the central nervous system[END_REF]. However, during the disease process, α-synuclein changes its conformation to form oligomers and high molecular weight insoluble aggregates. Previous studies have shown that the clearance of -synuclein in the substantia nigra is mainly related to autophagy-lysosomal pathway (reviewed in [START_REF] Dehay | Lysosomal impairment in Parkinson's disease[END_REF]), in particular through the chaperone-mediated autophagy (CMA), as -synuclein is a CMA-substrate. Autophagy is impaired in the substantia nigra in experimental models of parkinsonism (including MPTP models) and in PD, as shown with deficits in several lysosomal proteins such as LC3, LAMP1, LAMP-2a, cathepsin-D, glucocerebrosidase or ATP13A2 [START_REF] Alvarez-Erviti | Chaperone-mediated autophagy markers in Parkinson disease brains[END_REF][START_REF] Chu | Alterations in lysosomal and proteasomal markers in Parkinson's disease: relationship to alpha-synuclein inclusions[END_REF][START_REF] Dehay | Pathogenic lysosomal depletion in Parkinson's disease[END_REF][START_REF] Dehay | Loss of P-type ATPase ATP13A2/PARK9 function induces general lysosomal deficiency and leads to Parkinson disease neurodegeneration[END_REF][START_REF] Murphy | Lysosomal-associated membrane protein 2 isoforms are differentially affected in early Parkinson's disease[END_REF][START_REF] Murphy | Reduced glucocerebrosidase is associated with increased alpha-synuclein in sporadic Parkinson's disease[END_REF].

Remarkably, post-translational modifications, such as phosphorylation, ubiquitination, nitration and truncation are possible enhancers for -synuclein aggregation [START_REF] Atik | Alpha-Synuclein as a Biomarker for Parkinson's Disease[END_REF][START_REF] Beyer | 237-51 Accepted Article aggregation and neurodegeneration in prototypical neurodegenerative diseases: Examples of amyloidopathies, tauopathies and synucleinopathies[END_REF][42]. In accordance with the aggregation propensity of truncated -synuclein, our biochemical results revealed an increase in the TX insoluble fraction of the truncated (not the fulllength) -synuclein in hippocampus samples of MPTP-treated monkeys. However, we did not find high molecular weight assemblies of -synuclein in our experimental conditions that might reflect aggregated forms of -synuclein as occurring in LBs.

Overall, these biochemical results corroborate the histopathological results that showed that MPTP-induced -synuclein accumulation in NHPs is not limited to the substantia nigra and that the increased intraneuronal -synuclein immunoreactivity was insoluble but not in the structural form of LBs. Furthermore, the lack of effect of MPTP on total synuclein levels (Fig. 6) together with the widespread increased immunoreactivity (Fig. 3)

suggest that an anatomical redistribution of the protein may possibly occur, leading to its accumulation in cell bodies or processes.

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Although -synuclein is a major constituent of LBs, other aggregation prone proteins such as tau, play a role in the pathogenesis of LBs [START_REF] Ishizawa | Colocalization of tau and alphasynuclein epitopes in Lewy bodies[END_REF][START_REF] Wakabayashi | The Lewy body in Parkinson's disease and related neurodegenerative disorders[END_REF]. Indeed, tau is also the major structural component of neurofibrillary tangles (NFTs). It has already been showed that tau and -synuclein co-exist in LBs, and that NFTs are present around LBs [START_REF] Ishizawa | Colocalization of tau and alphasynuclein epitopes in Lewy bodies[END_REF][START_REF] Arima | Cellular colocalization of phosphorylated tau-and NACP/alpha-synuclein-epitopes in lewy bodies in sporadic Parkinson's disease and in dementia with Lewy bodies[END_REF][START_REF] Duda | Concurrence of alpha-synuclein and tau brain pathology in the Contursi kindred[END_REF][START_REF] Yamaguchi | Abundant neuritic inclusions and microvacuolar changes in a case of diffuse Lewy body disease with the A53T mutation in the alpha-synuclein gene[END_REF],

thus indicating that tau aggregation and NFTs are also significant hallmarks of PD.

However, whether tau accumulation, tau aggregation and NFTs occur in the MPTP animal models of PD remains relatively unexplored. Here, we only observed a slight increase in the density of tau immuno-positive neurons in the temporal cortex of MPTPtreated monkeys (Fig. 5D), thus indicating that MPTP intoxication induced larger effects on -synuclein than tau accumulation. Importantly, earlier in vitro studies demonstrated that the hyperphosphorylation of tau may promote its aggregation [START_REF] Despres | Identification of the Tau phosphorylation pattern that drives its aggregation[END_REF]. Moreover, tau is usually phosphorylated in LBs and NFTs [START_REF] Arima | Cellular colocalization of phosphorylated tau-and NACP/alpha-synuclein-epitopes in lewy bodies in sporadic Parkinson's disease and in dementia with Lewy bodies[END_REF][START_REF] Duda | Concurrence of alpha-synuclein and tau brain pathology in the Contursi kindred[END_REF][START_REF] Yamaguchi | Abundant neuritic inclusions and microvacuolar changes in a case of diffuse Lewy body disease with the A53T mutation in the alpha-synuclein gene[END_REF]. Phosphorylation of tau at Threonine 231, Serine 202/Threonine 205 and Serine 396 has already been reported in Alzheimer disease [START_REF] Dávila-Bouziguet | Differential accumulation of Tau phosphorylated at residues Thr231, Ser262 and Thr205 in hippocampal interneurons and its modulation by Tau mutations (VLW) and amyloid-β peptide[END_REF][START_REF] Hu | Phosphorylation of Tau and α-Synuclein Induced Neurodegeneration in MPTP Mouse Model of Parkinson's Disease[END_REF]. Remarkably, in the MPTP mouse model of PD, MPTP-induced tau hyperphosphorylation -which was -synuclein-dependent -was found at Serine 396/404 [START_REF] Duka | Alpha-synuclein induces hyperphosphorylation of Tau in the MPTP model of parkinsonism[END_REF]. Moreover, an increase in tau phosphorylation at Serine 202/Threonine 205, which might occur through GSK3β pathway, was also recently observed in the MPTP mouse model of PD in which phosphorylated tau and -synuclein co-localized in SNpc and hippocampus and formed plaques in SNpc [START_REF] Hu | Phosphorylation of Tau and α-Synuclein Induced Neurodegeneration in MPTP Mouse Model of Parkinson's Disease[END_REF]. In contrast with this latter study, despite -synuclein accumulation, we did not find significant increases in the density of phosphorylated tau (detected using the AT8 antibody) immuno-positive neurons in the SNpc, putamen, hippocampal CA1 region, temporal cortex and dentate nucleus of the MPTP-treated monkeys. Therefore, our results suggest that although changes in synuclein homeostasis might be necessary for phosphorylated tau accumulation and aggregation in PD brains [START_REF] Duka | Alpha-synuclein induces hyperphosphorylation of Tau in the MPTP model of parkinsonism[END_REF], -synuclein accumulation does not necessarily lead to phosphorylated tau accumulation and aggregation.

Earlier in vitro studies have showed that dopamine, L-DOPA, and other catecholamines (e.g., adrenaline and noradrenaline) inhibit α-synuclein fibrillation [START_REF] Conway | Kinetic stabilization of the alphasynuclein protofibril by a dopamine-alpha-synuclein adduct[END_REF][START_REF] Li | Dopamine and L-dopa disaggregate amyloid fibrils: implications for Parkinson's and Alzheimer's disease[END_REF][START_REF] Mazzulli | Cytosolic catechols inhibit alpha-synuclein aggregation and facilitate the formation of intracellular soluble oligomeric intermediates[END_REF] and its degradation through chaperone-mediated autophagy [START_REF] Martinez-Vicente | Dopamine-modified alpha-synuclein blocks chaperone-mediated autophagy[END_REF]. In aerobic conditions, these catecholamines tend to oxidize and their oxidative products appear to be even more

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effective for inhibiting α-synuclein fibrillation [START_REF] Li | Dopamine and L-dopa disaggregate amyloid fibrils: implications for Parkinson's and Alzheimer's disease[END_REF]. In line with these results, it has been demonstrated that the capacity of dopamine and L-DOPA to prevent the formation of αsynuclein fibrils was cancelled by the addition of antioxidants in the growth medium [START_REF] Conway | Kinetic stabilization of the alphasynuclein protofibril by a dopamine-alpha-synuclein adduct[END_REF].

Since quinones and aminochromes are two of the most stable classes of oxidative products derived from dopamine and L-DOPA, they could be at the source of α-synuclein fibril inhibition induced by dopamine and L-DOPA in these studies [START_REF] Li | Dopamine and L-dopa disaggregate amyloid fibrils: implications for Parkinson's and Alzheimer's disease[END_REF]. Indeed, it has been proposed that inhibition α-synuclein fibrillation is due to a covalent modification of αsynuclein by dopamine-derived orthoquinone [START_REF] Conway | Kinetic stabilization of the alphasynuclein protofibril by a dopamine-alpha-synuclein adduct[END_REF].

Although the relationship between distinct conformations of α-synuclein and neurodegeneration is still unclear and debated, if α-synuclein fibrillation process is involved in neuronal death [START_REF] Soto | Protein misfolding, aggregation, and conformational strains in neurodegenerative diseases[END_REF][START_REF] Wong | alpha-synuclein toxicity in neurodegeneration: mechanism and therapeutic strategies[END_REF], then disaggregating α-synuclein fibrils may reverse or slow down PD progression. However, despite the unique value of NHP models in translational research, the effect of DRT (L-DOPA+ carbidopa) on propagation/distribution/accumulation of pathological insoluble α-synuclein in the brain of the MPTP NHP model of PD was still unexplored. In the current study, we found that synuclein immunostaining was no longer detected in the putamen, hippocampal CA1 region, temporal cortex and dentate nucleus of the MPTP-treated monkeys with L-DOPA, thus indicating that DRT wiped out MPTP-induced alterations of α-synuclein immunoreactivity in several brain regions of the central nervous system relevant to the pathophysiology of PD (Fig. 3B-E). However, -synuclein immuno-positive SNpc neurons were still detected in the MPTP+L-DOPA group (Fig. 3A), even though the intensity of -synuclein immunofluorescence was reduced in animals treated with L-DOPA (Fig. 4). The marked deleterious effect of MPTP on autophagy in the SNpc may have contributed to the reduced clearance of -synuclein in this region. In contrast, we did not find any effect of L-DOPA on the density of the tau immuno-positive neurons in any brain regions investigated (Fig. 5). In line with these results, our biochemical results revealed that L-DOPA mainly affected the TX insoluble fraction of -synuclein (Fig. 6) from both putamen and hippocampus samples, so that the TX insoluble fraction of the full-length and the truncated -synuclein decreased in putamen samples (Fig. 6E andF, respectively), while only the truncated -synuclein decreased in the hippocampus samples (Fig. 6L). Accordingly, it has been shown that catecholamines (probably via

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their oxidative product as discussed above) not only inhibited α-synuclein fibril formation but also disassembled existing fibrils generated in vitro and α-synuclein deposits in tissue samples from SNpc of mice treated with the herbicide paraquat [START_REF] Li | Dopamine and L-dopa disaggregate amyloid fibrils: implications for Parkinson's and Alzheimer's disease[END_REF]. Similarly, it has been recently showed that administration of L-DOPA reduced phosphorylated αsynuclein accumulation in the SNpc of mice injected with preformed synthetic -synuclein fibrils in the striatum [START_REF] Shimozawa | Effect of L-DOPA/Benserazide on Propagation of Pathological α-Synuclein[END_REF]. Together with these studies performed in mice, our results obtained in NHPs reveal the ability of L-DOPA to mitigate α-synuclein pathology, either triggered by neurotoxins [START_REF] Li | Dopamine and L-dopa disaggregate amyloid fibrils: implications for Parkinson's and Alzheimer's disease[END_REF] or by -synuclein fibrils [START_REF] Shimozawa | Effect of L-DOPA/Benserazide on Propagation of Pathological α-Synuclein[END_REF]. Although further experiments are needed to understand why DRT might be less effective on α-synuclein pathology in the SNpc compared to others brain regions, our results strengthen earlier in vitro and in vivo reports. Moreover, our study is the first to demonstrate in vivo that DRT can dramatically ameliorate α-synuclein pathology in the MPTP NHP model of PD.

Our results may also have implications regarding the possible effects of DRT on the natural course of the disease that are tremendously difficult to apprehend in clinical trials.

The earlier vs. later L-DOPA (ELLDOPA) trial that evaluated the effects of DRT on the progression of the disease suggested a possible neuroprotective effect of L-DOPA [START_REF] Fahn | Levodopa and the progression of Parkinson's disease[END_REF].

However, in the Levodopa in Early Parkinson's Disease (LEAP) trial, no evidence for a disease-modifying effect of L-DOPA was found [START_REF] Verschuur | Randomized Delayed-Start Trial of Levodopa in Parkinson's Disease[END_REF]. What these trials show, in light of our results, is that L-DOPA does not protect from synucleinopathy as a primum movens. In the MPTP monkey model, the observed synucleinopathy is secondary to the neurotoxin insult and this secondary reaction is sensitive to L-DOPA. In idiopathic PD, one can assume that the primary synucleinopathy is a triggering factor and no evidence for sensitivity to L-DOPA has been shown so far.

The histopathological hallmark of Parkinson's disease (PD) is the presence of fibrillar aggregates referred to as Lewy bodies (LBs), in which α-synuclein is a major constituent [START_REF] Wakabayashi | The Lewy body in Parkinson's disease and related neurodegenerative disorders[END_REF][START_REF] Wakabayashi | The Lewy body in Parkinson's disease: molecules implicated in the formation and degradation of alpha-synuclein aggregates[END_REF]. Accordingly, -synuclein is regarded as a promising biomarker of the disease [START_REF] Brooks | Imaging synucleinopathies[END_REF][START_REF] Atik | Alpha-Synuclein as a Biomarker for Parkinson's Disease[END_REF]. Indeed, α-synuclein can be measured in peripheral tissue and body fluids (e.g., cerebrospinal fluid, plasma/serum, blood and saliva) [START_REF] Atik | Alpha-Synuclein as a Biomarker for Parkinson's Disease[END_REF]. Although we are still unable to directly image aggregates of -synuclein, their structural and functional consequences can be detected by current imaging strategies (e.g., MRI, SPECT, PET) [START_REF] Brooks | Imaging synucleinopathies[END_REF]. In this context and given the translational significance of the NHP studies, our results are

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fundamental and indicate that patient's dopaminergic medication should be systematically taken in consideration when assessing α-synuclein as a biomarker for accurate diagnosis, monitoring disease progression and response to treatment. In fact, special attention should be paid to the patient's full medical record and not only DRT since other drugs widely used in PD therapy (e.g., MAO-B inhibitors) may also influence α-synuclein homeostasis.

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  Brain tissue samples were obtained from the non-human primate brain bank at the Institut des Maladies Neurodegeneratives (Bordeaux, France). Experiments were carried out in an AAALAC-accredited facility and in accordance with the European Union Directive (2010/63/EU) on the protection of animals used for scientific purposes.Experiments were conducted on 28 female rhesus monkeys (Macaca mulatta, Xierxin, Beijing, China, mean age = 5.4 ± 1 years, mean weight = 3.8 ± 0.4 kg). Animal cares were supervised by veterinarians skilled in NHP healthcare and maintenance. Animals were housed in individual primate cages under controlled conditions of humidity (50 ± 5%), temperature (24 ± 1°C) and light (12h light/12h dark cycles, lights on 8:00 am), food and water were available ad libitum. Even though animals were housed individually, the disposition of cages allowed each animal to have visual contacts and interact with monkeys housed in the adjacent cages. Stimulations for play were provided including rubber toys and mirrors that the monkeys used to view themselves and to get a greater look around the room. A radio was played daily from 8.00am-10.00am and 3.00pm-5.00pm to provide stimulation.

[ 23 ]

 23 , we next investigated if L-DOPA could affect the number of reactive astrocytes.MPTP intoxication induced significant astrogliosis in the nigrostriatal pathway compared with controls, as shown by increased densities of GFAP-positive astrocytes in the substantia nigra (F (2,10) =10.42, p<0.01, Fig.2A) and putamen (H=9.11, df=2, p<0.01, Fig.2B). Astrogliosis was also significant in MPTP + L-DOPA-treated monkeys compared with controls in the substantia nigra (p<0.01) but not in the putamen. In the temporal cortex, MPTP + L-DOPA-treated animals displayed increased counts of GFAP-positive astrocytes compared with controls (F (2,10) =6.54, p<0.05, Fig.2D). No significant effects of MPTP or MPTP + L-DOPA were found in CA1 (H=3.53, df=2, p=0.17, Fig.2C) or in the dentate nucleus (H=2.34, df=2, p=0.32, Fig.1E). The density of GFAP-positive astrocytes was not significantly different between MPTP and MPTP + L-DOPA-treated monkeys in any of the regions investigated, indicating that chronic L-DOPA treatment had no effect on MPTP-induced astrogliosis. Microscopic illustrations of the density of GFAPpositive astrocytes in the different regions of interest are depicted for control (Fig.2F-J), MPTP (Fig.2K-O), and MPTP + L-DOPA-treated (Fig.2P-T) monkeys.

  phosphorylated at serine 202 and threonine 205 (data not shown). Counts of AT8immunopositive neurons revealed no significant effects of MPTP and MPTP + L-DOPA in the substantia nigra (H=3.10, p=0.22), CA1 (F (2,10) =0.21, p=0.81), temporal cortex (F (2,10) =0.58, p=0.57), and dentate nucleus (H = 2.75, p=0.27), while in the putamen, there was a trend toward an increase of tau AT8-positive neurons in MPTP and MPTP + L-DOPA-treated animals (H = 4.94, p= 0.07).

  ), with MPTP + L-DOPA-treated monkeys showing significantly less -synuclein than their MPTP-treated counterparts (p<0.05) and controls (p<0.01). A significant effect was also observed on 12 kDa -synuclein (F[START_REF] Wakabayashi | The Lewy body in Parkinson's disease and related neurodegenerative disorders[END_REF][START_REF] Bezard | Relationship between the appearance of symptoms and the level of nigrostriatal degeneration in a progressive 1-methyl-4-phenyl-1,2,3,6tetrahydropyridine-lesioned macaque model of Parkinson's disease[END_REF] =8.95, p<0.01, Fig.6B, F), with reduced 12 kDa -synuclein in the MPTP + L-DOPA compared with the MPTP (p<0.01) and control (p<0.05) groups.

Figure 1 :Figure 2 :

 12 Figure 1: Effects of MPTP and L-DOPA on neuronal loss in the SNpc. Counting of TH-immunopositive neurons in the SNpc. (A) Representative images of TH immunostaining in the SNpc of control (B), MPTP (C) and MPTP + L-DOPA (D) treated monkeys. Scale bar = 200 m. ****, p<0.0001

Figure 3 :

 3 Figure 3: Effects of MPTP and L-DOPA on α-synuclein accumulation α-Synuclein accumulation, as determined by the density of α-synuclein immunopositive neurons in the SNpc (A), fibers in the putamen (B), CA1 (C), and temporal cortex (D) and neurons dentate nucleus (E). Representative images of α-synuclein immunostaining in the SNpc (F, K, P), putamen (G, L, O), CA1 (H, M, R), temporal cortex (I, N, S) and dentate nucleus (J, O, P). Scale bar = 50 m. *, p<0.05 vs control group; **, p<0.05 vs MPTP group, ****, p<0.01 vs control and MPTP+L-DOPA groups.

Figure 4 :

 4 Figure 4: Phenotypic identity of α-synuclein accumulating neurons in the SNpc Double immunofluorescence for α-synuclein (A, D, G) and tyrosine hydroxylase (B, E, H) and overlay (C, F, I), in control (A-C), MPTP (D-F) and MPTP+L-DOPA (G-I) treated monkeys. Scale bar = 30 m. Quantification of α-synuclein fluorescence intensity in remaining nigral dopaminergic neurons of MPTP and MPTP + L-DOPA treated animals (J). *, p<0.05.

Figure 5 :

 5 Figure 5: Effects of MPTP and L-DOPA on tau accumulation Accepted Article

Figure 6 :

 6 Figure 6: Effects of MPTP and L-DOPA on α-synuclein homeostasis and insolubility α-Synuclein immunoblot levels following sequential protein extraction of putamen (A, B) and hippocampus (G, H), in triton soluble (A, G) and insoluble fractions (B, H) and quantification of full-length (14 kDa) and truncated (12 kDa) α-synuclein in the putamen (C-F) and hippocampus (I-L). n=5 per group. Data represent mean ± SEM. *, p<0.05 ; **, p<0.01.

Acknowledgements

The Université de Bordeaux, Université de Poitiers, Centre National de la Recherche Scientifique and Institut National de la Santé et de la Recherche Médicale provided infrastructural support.

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Author Contributions

EB, POF, BD designed the research; MHC, MT, QL performed the research; POF, MT, MD analyzed the data; MD, POF, EB wrote the paper; All authors read and approved the final version of the manuscript.

Ethical approval

Experiments were carried out in an AAALAC-accredited facility and in accordance with the European Union Directive (2010/63/EU) on the protection of animals used for scientific purposes. Experiments were performed after acceptance of the study design by the Institute of Lab Animal Science (Chinese Academy of Science, Beijing, China) IACUC for experiments on non-human primates.

Conflict of interest

The authors declare no conflict of interest.

Accepted Article