Jennifer Fransson

Ana Isabel Gómez-Conde

Jesús Romero-Imbroda

Oscar Fernández

Laura Leyva

Fernando Rodríguez De Fonseca

Jerold Chun

Celine Louapre

Anne Baron Van-Evercooren

Violetta Zujovic

Guillermo Estivill-Torrús

Beatriz García-Díaz
email: beatriz.garcia@icm-institute.org

Beatriz García Díaz

Guillermo Estivill
email: guillermo.estivill@ibima.eu

Activation of Macrophages by Lysophosphatidic Acid through the Lysophosphatidic Acid Receptor 1 as a Novel Mechanism in Multiple Sclerosis Pathogenesis

Keywords: Lysophosphatidic acid, LPA 1 receptor, macrophages, multiple sclerosis, experimental autoimmune encephalomyelitis, inflammation central nervous system EAE: experimental autoimmune encephalomyelitis HD: healthy donors IMNC: infiltrating mononuclear cell oligodendrocyte precursor cell PBMC: peripheral blood mononuclear cell PP-MS: primary progressive multiple sclerosis

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Introduction

Multiple sclerosis (MS) is one of the most widespread neurological diseases in young adults affecting approximately 2.3 million people worldwide [START_REF] Rezapour | The impact of disease characteristics on multiple sclerosis patients' quality of life[END_REF]. MS pathogenesis consists of inflammation of the central nervous system (CNS), oligodendrocyte death and myelin damage, with many different immune cells exercising significant influence. Macrophages play a dual role in MS pathology; they can contribute to tissue damage and inflammation, but also exert a neuroprotective and regenerative effect [START_REF] Miron | M2 microglia and macrophages drive oligodendrocyte differentiation during CNS remyelination[END_REF]. Accordingly, classically activated ("M1") macrophages show proinflammatory characteristics, whilst alternatively activated ("M2"), macrophages display an anti-inflammatory phenotype [START_REF] Vogel | Macrophages in inflammatory multiple sclerosis lesions have an intermediate activation status[END_REF]. During remyelination, M1 macrophages phagocytize myelin debris and induce oligodendrocytes precursor cells (OPC) to proliferate and migrate to the lesion site. Next, a switch from the M1 to the M2 phenotype triggers the secretion of trophic factors that foster OPC differentiation into new myelin-forming oligodendrocytes [START_REF] Miron | Macrophages and CNS remyelination[END_REF]. However, it remains unclear to what extent macrophages intrinsically contribute to myelin destruction and repair in MS.

Lysophosphatidic acid (LPA) is a bioactive phospholipid that influences numerous cell responses and acts through a family of G-protein coupled receptors (LPA 1-6 ), widely distributed in peripheral organs and brain [START_REF] Choi | LPA receptors: subtypes and biological actions[END_REF][START_REF] Yung | LPA receptor signaling pharmacology, physiology, and pathophysiology[END_REF][START_REF] De San Román | Anatomical location of LPA1 activation and LPA phospholipid precursors in rodent and human brain[END_REF]. Among all LPA receptors, LPA 1 is the most studied in relation to biological action through of LPA [START_REF] Choi | LPA receptors: subtypes and biological actions[END_REF][START_REF] Yung | LPA receptor signaling pharmacology, physiology, and pathophysiology[END_REF].

LPA dysregulation has been implicated in different inflammatory diseases, such as atherosclerosis [START_REF] Bot | Atherosclerotic lesion progression changes lysophosphatidic acid homeostasis to favor its accumulation[END_REF], cancer [START_REF] Valdes-Rives | Autotaxin-Lysophosphatidic Acid: From Inflammation to Cancer Development[END_REF], and MS [START_REF] Schmitz | Dysregulation of lysophosphatidic acids in multiple sclerosis and autoimmune encephalomyelitis[END_REF] because of its effect on immune cells, of both the innate and adaptive immune systems [START_REF] Bagga | Lysophosphatidic acid accelerates the development of human mast cells[END_REF][START_REF] Goetzl | Cutting edge: differential constitutive expression of functional receptors for lysophosphatidic acid by human blood lymphocytes[END_REF][START_REF] Lee | Lysophospholipid regulation of mononuclear phagocytes[END_REF]. Although some studies showed a dysregulation of serum LPA levels in MS patients [START_REF] Schmitz | Dysregulation of lysophosphatidic acids in multiple sclerosis and autoimmune encephalomyelitis[END_REF][START_REF] Balood | Elevated serum levels of lysophosphatidic acid in patients with multiple sclerosis[END_REF][START_REF] Jiang | Elevated lysophosphatidic acid levels in the serum and cerebrospinal fluid in patients with multiple sclerosis: therapeutic response and clinical implication[END_REF] and suggested a role of LPA 2 in T cells homing [START_REF] Schmitz | Dysregulation of lysophosphatidic acids in multiple sclerosis and autoimmune encephalomyelitis[END_REF], the role of LPA 1 in MS, in particular through macrophage function, remains unclear.

LPA constitutes a major serum survival factor for murine macrophages [START_REF] Koh | Lysophosphatidic acid is a major serum noncytokine survival factor for murine macrophages which acts via the phosphatidylinositol 3-kinase signaling pathway[END_REF], in which it has been shown to upregulate the expression of proinflammatory cytokines, such as IL-1β and TNF-α [START_REF] Lee | Lysophospholipid regulation of mononuclear phagocytes[END_REF], while its role in human macrophages is largely unexplored. Interestingly, LPA 1 is expressed in peripheral blood monocytes and/or tissue macrophages in both mice and humans, and influences their activation, migration and infiltration in different murine disease models [START_REF] Hornuss | Human and rat alveolar macrophages express multiple EDG receptors[END_REF][START_REF] Gustin | LPA modulates monocyte migration directly and via LPA-stimulated endothelial cells[END_REF][START_REF] Miyabe | Necessity of lysophosphatidic acid receptor 1 for development of arthritis[END_REF].

Following these findings, we here aim to elucidate the role of LPA 1 in MS, by analyzing the disease course of experimental autoimmune encephalomyelitis (EAE) using both a genetic model (maLPA 1 -null) and a pharmacological model (LPA 1 antagonist). We provide evidence of a milder symptomatology in absence or antagonism of LPA 1 , suggesting a role of this receptor in the pathogenesis of the disease. This role was further strengthened with the analysis of LPA 1 expression levels in PBMC from EAE mice and from patients with relapsing-remitting (RR-), primary progressive (PP-) or secondary progressive (SP-) MS. We demonstrated that the initiation of relapses is accompanied by an increase of LPA 1 transcripts (Lpar1) in mouse PBMC. Finally, we provide in vitro data demonstrating that proinflammatory activation of human monocyte-derived macrophages includes increased expression of human LPA 1 transcripts (LPAR1) and that LPA is involved in LPA 1 -driven polarization of human macrophages towards a M1-like phenotype. These results evidence a role of LPA in the initiation of the inflammatory process during MS relapses via LPA 1 .

In short, our studies unveil a novel mechanism for LPA in the classical activation of macrophages through LPA 1 and suggest for the first time that targeting LPA 1 receptors represents a promising therapeutic strategy in MS as well as for other immune-related diseases.

Material and Methods

Mice

All experiments were performed on the Málaga variant of the LPA 1 -null mouse derived from the original colony of Contos et al [START_REF] Contos | Requirement for the lpA1 lysophosphatidic acid receptor gene in normal suckling behavior[END_REF] on a mixed C57BL/6J x 129X1/SvJ background, as previously described [START_REF] Estivill-Torrús | Absence of LPA1 signaling results in defective cortical development[END_REF][START_REF] García-Díaz | Loss of lysophosphatidic acid receptor LPA1 alters oligodendrocyte differentiation and myelination in the mouse cerebral cortex[END_REF]. Experiments were conducted on age-matched female wild-type and maLPA 1 -null homozygous littermates that were approximately 7 weeks old (n total =103).

All mice were housed in pathogen-free conditions on a 12-h light/dark cycle (lights on at 07:00 h) with water and food provided ad libitum. Experiments were conducted in accordance with the European guidelines and national laws on laboratory animal welfare and approved by the Experimentation Ethics Committees of the University of Malaga.

Induction of EAE.

Seven-week-old female mice were immunized according to a standard protocol [START_REF] Mendel | A myelin oligodendrocyte glycoprotein peptide induces typical chronic experimental autoimmune encephalomyelitis in H-2b mice: fine specificity and T cell receptor V beta expression of encephalitogenic T cells[END_REF] with subcutaneous injection of incomplete Freund's adjuvant containing 4 mg/mL Mycobacterium tuberculosis (strain H37Ra; Difco Laboratories, Detroit, MI, USA) and 200 μg of encephalitogenic myelin oligodendrocyte glycoprotein peptide ). The mice received intraperitoneal injections with 200 ng pertussis toxin (Sigma-Aldrich, St. Louis, Missouri, USA) at the time of immunization and 48 hours later. After 7 days, the mice received a half booster immunization with MOG, complete Freund's adjuvant (CFA) and pertussis toxin.

Control mice received identical injections without MOG . Clinical disease usually commenced around 15 days postimmunization (dpi). The mice were scored four times per week in the mornings as follows: limp tail or waddling gait with tail tonicity, defined score 1; waddling gait with limp tail (ataxia) as score 2; ataxia with partial limb paralysis as score 2.5; full paralysis of one limb as score 3; full paralysis of one limb with partial paralysis of second limb as score 3.5. Animals that maintained a score of at least 3.5 more than 3 days were euthanized with overdose of pentobarbital to minimize animal suffering. A total of 103 animals were immunized for this study.. DietGel® Recovery was put at their disposal on the bedding to provide a nutritionally fortified water gel and avoid dehydration during their clinical course. and AC-2012-1536). Written informed consent was obtained from all study participants. All patients fulfilled diagnostic criteria for MS, and individuals (MS patients and healthy donors) with any other inflammatory or neurological disorders were excluded from the study. The study was not pre-registered. No randomization was performed to allocate subjects in the study.

Isolation of PBMC for RNA extraction

PBMC were isolated from whole blood obtained from mice during the first minute of PBS perfusion (n = 42) by standard Ficoll®-Paque (Sigma-Aldrich) density gradient centrifugation.

Briefly, heparinized blood was diluted with saline solution (1:1 dilution). Then, Ficoll®-Paque was covered with a layer of diluted blood. After 30 min of centrifugation (600 x g, room temperature (RT), without break), the PBMC could easily be collected. After two washing steps and counting, cells were resuspended in 1 ml of TRIzol™ Reagent (Invitrogen™, Thermo Fisher

Isolation of brain infiltrating mononuclear cells for RNA extraction

Infiltrated mononuclear cells (IMNC) were isolated from CNS of EAE mice according to Beeton and Chandy [START_REF] Beeton | Isolation of mononuclear cells from the central nervous system of rats with EAE[END_REF]. After dissecting and weighing brain and spinal cord from individual animals (n = 27), these were minced finely in phosphate buffer saline, centrifuged, and resuspended in 37% Percoll® (Sigma-Aldrich). This suspension was laid on a 70% Percoll® cushion and spun at 600 x g at room temperature for 25min. CNS IMNC were obtained from the 37-70% Percoll® interface, washed twice, and cell counted. Finally, cells were resuspended in 1 ml of TRIzol™ for RNA extraction.

RT-PCR from PBMC and IMNC

Total RNA was extracted from PBMC and IMNC using the TRIzol™ reagent as originally described by Chomczynski and Sacchi [START_REF] Chomczynski | Single-step Method of RNA Isolation by Acid Guanidinium Thiocyanate-Phenol-Chloroform Extraction[END_REF] . cDNA was synthesized using 1 µg of total RNA by the enzyme reverse transcriptase MMLV (Sigma-Aldrich) and random primers. Real-time PCR was performed by the LightCycler® System (Roche Molecular Systems, Inc., Pleasanton, CA, USA) following the manufacturer's specifications. The 10 µl final reaction volume consisted of 5.4 μl of RNAase-free distilled water, 1.3 μL of MgCl 2 , 0.2 μl of each forward and reverse primers, 1 μl of Fast SYBR™ Green Master Mix and 2 μl of cDNA. Reaction conditions were as follows: polymerase activation at 95°C for 15 min, 40 denaturation cycles of 95°C for 30 s, and annealing/elongation at 68°C (Lpar1 and GAPDH) for 5 s (Lpar1) or 10 s (GAPDH).

The primer sequences used in the amplification of Lpar1 and Lpar2 have been described previously [START_REF] Hama | Lysophosphatidic acid and autotaxin stimulate cell motility of neoplastic and non-neoplastic cells through LPA1[END_REF] (Lpar1 forward: GAGGAATCGGGACACCATGAT; and reverse:

ACATCCAGCAATAACAAGACCAATC, Gapdh forward: GCCAAGGTCATCCATGACAACT, and reverse: GAGGGGCCATCCACAGTCTT). A melting curve analysis was performed to assess primer specificity and product quality by step-wise denaturation of the PCR product at a rate of 0.1°C / sec to 98°C. The relative levels of receptor expression were quantified using the standard curve method.

Isolation of human primary monocytes and macrophage culture and activation.

Blood was sampled from all participants in acid citrate dextrose (ACD) tubes. PBMC were isolated using Ficoll®-Paque Plus and centrifugation (600 x g, 20 min without brake). Cells were washed in PBS (2x10 min at 1500 rpm) and RPMI 1640 with 10% foetal bovine serum (FBS) (Thermo Fisher). Monocytes were isolated with anti-CD14 microbeads (Miltenyi Biotec GmbH, Bergisch Gladbach, Germany) and plated in 12-well plates (500 000 cells/well) or in 24-well plates (200 000 cells/well) in RPMI 1640 with 10% FBS and granulocyte macrophage colonystimulating factor (GM-CSF) (500 U/ml, ImmunoTools GmbH, Friesoythe, Germany). After 72h, media was replaced with fresh media and one of the following: GM-CSF (500 U/ml); IFNβ (100 U/ml, ImmunoTools); IL4 (1000 U/ml, ImmunoTools); or combined IFNγ (200 U/ml, ImmunoTools) and ultra-pure LPS (10 ng/ml, InvivoGen, Inc., San Diego, CA, USA). Cell lysis and RNA extraction were performed 24h post-activation using Nucleospin RNA extraction kit (Macherey-Nagel GmbH & Co. KG, Dueren, Germany). Quality of RNA (RINe>8) was confirmed on Agilent TapeStation (Agilent Technologies, Inc., Santa Clara, CA, USA).

1-Oleoyl lysophosphatidic acid sodium salt, LPA (CAS number 325465-93-8; Tocris, Bio-Techne, Minneapolis, MN, USA 3854) and the LPA 1 antagonist Ki16425 (CAS number 355025-24-0; Sigma-Aldrich) were dissolved in 3% FFA-BSA and added to the medium at a final concentration of 1µM LPA and 400nM Ki16425 during 24h.

RNA sequencing

Transcriptome sequencing cDNA libraries of macrophage RNA were prepared using a stranded (http://rseqc.sourceforge.net/). Reads were mapped using STARv2.4.0 [START_REF] Dobin | STAR: ultrafast universal RNA-seq aligner[END_REF] on the hg19 Human genome assembly. Gene expression regulation study was performed as previously described [START_REF] Noli | Discordant Growth of Monozygotic Twins Starts at the Blastocyst Stage: A Case Study[END_REF]. Briefly, for each gene present in the FAST DB v2018_1 annotations, reads aligning on constitutive regions (that were not prone to alternative splicing) were counted. Based on these read counts, normalization was performed using DESeq2 in R (v.3.2.5) [START_REF] Love | Moderated estimation of fold change and dispersion for RNA-seq data with DESeq2[END_REF]. Genes were considered as expressed if their RPKM value was greater than 97.5% of the background RPKM value based on intergenic regions. The normalized data were used for all subsequent analysis.

mRNA

RT-PCR of monocyte-derived macrophages

RNA obtained from differentially activated macrophages were used as templates to synthetize cDNA using the Quantitect Reverse Transcription kit (QIAGEN GmbH, Hilden, Germany)

according

Results

LPA 1 deletion leads to milder EAE clinical course.

Recently, a role of LPA in the pathogenesis of MS and its animal model EAE has been suggested [START_REF] Schmitz | Dysregulation of lysophosphatidic acids in multiple sclerosis and autoimmune encephalomyelitis[END_REF], focusing on the contribution of LPA 2 -expressing T cells. Here, we question whether LPA 1 , also present in immune cells, could have a role in EAE. To answer this question, we first compared the EAE clinical course in presence and absence of LPA 1 using the Malaga variant of LPA 1 -null mouse (maLPA 1 -null mouse) [START_REF] Estivill-Torrús | Absence of LPA1 signaling results in defective cortical development[END_REF].

Analysis of MOG 35-55 induced-EAE clinical courses showed a relapsing-remitting clinical course in both wild-type and maLPA 1 -null animals but also highlighted important differences between the two genotypes. Notably, maLPA 1 -null mice showed a less severe clinical course compared to wild-type mice (Fig. 1a). Accordingly, maLPA 1 -null mice reduced the number of relapses and exhibited a significantly lower average clinical score and maximal clinical score reached during relapses, as well as a better recovery during remission (Fig. 1b).

Intravenous injection of an LPA 1 antagonist ameliorates EAE clinical score in wild-type mice.

LPA 1 is also expressed in oligodendrocytes and we previously demonstrated that its absence perturbs developmental myelination in maLPA 1 -null mice [START_REF] García-Díaz | Loss of lysophosphatidic acid receptor LPA1 alters oligodendrocyte differentiation and myelination in the mouse cerebral cortex[END_REF]. To exclude any interference of deficient myelin patterns in the EAE outcome in maLPA 1 -null animals, we analyzed EAE in wild-type mice that were intravenously injected with VPC32183, an LPA 1 antagonist that primarily blocks LPA 1 (10-100nM range), and partially blocks LPA 3 (10-fold lower; 100-1000nM range) [START_REF] Heasley | Initial structure-activity relationships of lysophosphatidic acid receptor antagonists: discovery of a high-affinity LPA1/LPA3 receptor antagonist[END_REF].

To maintain the levels of the antagonist, immunized mice were treated with repeated doses of VPC32183 every 5 days (Fig. 2a). Recurrent intravenous administration of the LPA 1 antagonist resulted in a milder EAE disease course characterized by a lower average clinical score, milder relapses, and better remissions (Fig. 2a,b), corroborating the requirement of LPA 1 activation to develop a normal EAE clinical course.

Lpar1 expression increases when mononuclear cells invade the CNS

Under normal physiological conditions, mononuclear cells are rarely found in the CNS.

However, in MS and EAE, activated immune cells infiltrate the CNS. Due to the reported role of LPA 1 in immune cell infiltration [START_REF] Schober | Lysophosphatidic acid in atherosclerotic diseases[END_REF] and its obvious impact in the clinical course, we analyzed whether the number of infiltrating mononuclear cells (IMNC) was altered in EAE-mice lacking LPA 1 . We quantified the number of IMNC from brain and spinal cord of wild type and maLPA 1null mice with similar clinical scores. We did not find significant differences in the number of infiltrates (Fig. 3a), suggesting that LPA 1 modulation of EAE course might intervene at another step of immune cell activation beside infiltration.

Despite LPA 1 not being essential for cell infiltration, we found that its expression still appeared to be related to immune presence in the CNS. When analyzing the Lpar1 expression in circulating immune cells (PBMC) and CNS-IMNC from wild-type EAE mice using RT-PCR, our results showed an increase of Lpar1 expression in immune cells in the CNS compared to the periphery (Fig. 3b). These data suggest that an increase of Lpar1 expression reflects immune cell activation.

Onset of EAE relapses correlates with increase expression of Lpar1 in PBMC.

Knowing that Lpar1 is expressed by immune cells, and that immune cells are critical for EAE development, we wondered whether Lpar1 expression in PBMC reflects disease activity. To this end, the expression of Lpar1 in PBMC along the EAE clinical course was evaluated in wild-type mice.

MOG-immunized animals showed a two-fold significant increase of Lpar1 expression compared to control animals (Fig. 4a). However, no significant correlation was found between Lpar1 expression and clinical score after sacrifice (EAE score 1: 0,459818 ± 0,123361 (n=9); EAE score 2: 0,681039 ± 0,151682 (n=7); EAE score 3: 0,648933 ± 0,144792 (n=8)).

To decipher whether Lpar1 expression in PBMC might reflect a different phase of the disease, Lpar1 expression was analyzed according to mice stratification based on whether animals were initiating a relapse or in remission/progressive course of the disease at the moment of the sampling. There was a significantly increased expression of Lpar1 during relapses when compared to control animals or animals in remission or progressive episodes (Fig. 4b). Of note, Lpar1 expression and clinical symptoms during the EAE relapses were significantly positively correlated during the clinical course of the disease (Fig. 4c).

LPAR1 expression increases during relapses in RR-MS patient PBMC.

The above observations in EAE mice suggest a modulation of LPA 1 in the first stages of the relapses during the inflammatory clinical course. To corroborate this observation in the context of MS, we compared the expression of LPAR1 in PBMC from RR-MS patients at the time of first relapse and compared with its expression in healthy donors (HD), matched in age and gender (Fig. 5a), and patients with progressive form of the disease (SP-MS and PP-MS). Like in EAE, LPAR1 expression was significantly higher in RR-MS patient PBMC than in HD or progressive patients (Fig. 5b). Thus, we provide evidence that alterations in LPAR1 expression associates with the inflammatory phase of MS.

LPAR1 expression correlates with a proinflammatory phenotype of human monocytederived macrophages

Circulating PBMC are mainly composed of lymphocytes and monocytes. We focused on the role of LPA 1 on monocytes/macrophages because of their dual role in MS pathology [START_REF] Vogel | Macrophages in inflammatory multiple sclerosis lesions have an intermediate activation status[END_REF], being both deleterious when endorsing a proinflammatory phenotype and beneficial under pro-regenerative activation [START_REF] Miron | M2 microglia and macrophages drive oligodendrocyte differentiation during CNS remyelination[END_REF][START_REF] Miron | Macrophages and CNS remyelination[END_REF].To elucidate the role of LPA 1 in macrophages polarization, we obtained naïve circulating monocytes from HD and RR-MS patients in remission. Naïve monocytes remain circulating in the blood stream for a short period before infiltrating the tissues [START_REF] Patel | The fate and lifespan of human monocyte subsets in steady state and systemic inflammation[END_REF], reducing the impact of other circulating factors before blood extraction. Blood CD14+ monocytes were differentiated into macrophages using GM-CSF. Then, monocyte-derived macrophages were exposed to: i) LPS+IFN, to activate a proinflammatory (M1) state; ii)IFNβ or IL4, to activate a pro-regenerative (M2) state; or iii) GM-CSF, to remain in a neutral state. We next evaluated the expression of membrane LPA receptors LPA 1 and LPA 2 , and the nuclear LPA-receptor PPAR.

Although the patients were recruited from a cohort of siblings with MS, the siblings did not show higher similarity in expression than random pairs of patients and, in consequence, they were considered as independent samples (data not shown).

Interestingly, while we could not detect a difference of LPAR2 expression (Fig. 6a) in any activation state, LPAR1 and PPAR receptors were differentially regulated, displaying an inverse pattern of across the macrophage activation states (Figs. 6b,c). In both MS and HD, LPAR1 expression was up-regulated in the M1 proinflammatory state, while PPAR expression was increased in the pro-regenerative state. This observation underlines the possible role of LPA 1 in the proinflammatory activation of human macrophages.

LPA mediates human macrophage polarization.

LPA levels are altered along the course of MS [START_REF] Schmitz | Dysregulation of lysophosphatidic acids in multiple sclerosis and autoimmune encephalomyelitis[END_REF][START_REF] Balood | Elevated serum levels of lysophosphatidic acid in patients with multiple sclerosis[END_REF] suggesting an important role of this phospholipid in the course of the disease. We therefore tested whether, in addition to observed M1-associated LPAR1 overexpression, LPA could promote an M1-phenotype in macrophages, as has been observed in murine microglia [START_REF] Plastira | 1-Oleyl-lysophosphatidic acid (LPA) promotes polarization of BV-2 and primary murine microglia towards an M1-like phenotype[END_REF].

LPS mediates time-dependent macrophage M1/M2 polarization, promoting the M1 phenotype over time [START_REF] Zhang | Lipopolysaccharide Mediates Time-Dependent Macrophage M1/M2 Polarization Through the Tim-3/Galectin-9 Signalling Pathway[END_REF]. We examined transcripts specific for proinflammatory or pro-regenerative profiles in human macrophages after an LPA treatment of 24h and compared the expression of different markers with the canonical M1 polarization by LPS (Fig. 7).

The levels of different M1 markers (CCL2, CCL20, CCL5, and TLR2), though to a lesser extent than after LPS treatment, increased after LPA incubation, indicating a role of LPA in the proinflammatory activation of human macrophages. Moreover, this M1 polarization was partially inhibited by addition of an LPA 1 inhibitor (Ki16425) revealing the mediation of LPA 1 in this LPAinduced response (Fig. 7). No significant alterations in the expression of M2 markers were observed after LPA incubation.

Discussion

In this study, we present evidence of a role of the LPA 1 receptor in the pathogenesis of the neuroinflammatory disease MS and its animal model, EAE. We also propose a mechanism through which LPA may exert this effect via macrophage activation.

After the discovery of the first receptor for LPA, the LPA 1 , in 1996 [START_REF] Hecht | Ventricular zone gene-1 (vzg-1) encodes a lysophosphatidic acid receptor expressed in neurogenic regions of the developing cerebral cortex[END_REF], this receptor has been implicated in a numerous process, with an outstanding importance in the physiology and pathology of the CNS [START_REF] Yung | Lysophosphatidic Acid signaling in the nervous system[END_REF]. In this context, the importance of LPA in MS pathogenesis has been suggested [START_REF] Schmitz | Dysregulation of lysophosphatidic acids in multiple sclerosis and autoimmune encephalomyelitis[END_REF][START_REF] Balood | Elevated serum levels of lysophosphatidic acid in patients with multiple sclerosis[END_REF][START_REF] Jiang | Elevated lysophosphatidic acid levels in the serum and cerebrospinal fluid in patients with multiple sclerosis: therapeutic response and clinical implication[END_REF]. However, the role of LPA 1 in the MS pathogenesis remains unclear. In the present study, we unveil a new aspect of LPA through the LPA 1 receptor in this neuroinflammatory disease.

Our results show, for the first time, the importance of the receptor LPA 1 in the EAE clinical course. The lack of LPA 1 , or its pharmacological inhibition by the repetitive intravenous injections of a LPA 1 antagonist (VPC32183), reduces the severity of the disease. The milder symptoms observed in the absence of LPA 1 signaling indicates that this pathway is involved in EAE pathogenesis. This is in contrast with a study of another LPA receptor, LPA 2 , of which reduction led to more severe disease [START_REF] Schmitz | Dysregulation of lysophosphatidic acids in multiple sclerosis and autoimmune encephalomyelitis[END_REF]. This indicates a complex role of LPA in MS and EAE, and that potential treatment strategies should target specific receptor pathways rather than LPA itself. In our study, the administration of VPC32183 confirmed the clinical course observed in LPA 1 -null EAE, albeit infiltrating mononuclear cells were only analyzed in LPA 1 -null mice. In this sense, we cannot ignore that VPC32183 is a selective LPA 1 /LPA 3 antagonist and the involvement of LPA 3 receptor in the endothelial cell interactions with monocytes during inflammation processes [START_REF] Gustin | LPA modulates monocyte migration directly and via LPA-stimulated endothelial cells[END_REF][START_REF] Lin | Lysophosphatidic acid regulates inflammation-related genes in human endothelial cells through LPA1 and LPA3[END_REF]. For this reason, the analysis of infiltrating mononuclear cells in EAE-mice was restricted to an LPA 1 -null scenario, to exclude any other receptor that could interfere the study.

Previous studies have described a role for LPA and autotaxin, its main synthesis enzyme, in inflammatory processes [START_REF] Bot | Atherosclerotic lesion progression changes lysophosphatidic acid homeostasis to favor its accumulation[END_REF][START_REF] Valdes-Rives | Autotaxin-Lysophosphatidic Acid: From Inflammation to Cancer Development[END_REF][START_REF] Lin | Lysophosphatidic acid regulates inflammation-related genes in human endothelial cells through LPA1 and LPA3[END_REF][START_REF] Srikanth | Lysophosphatidic acid and its receptor LPA(1) mediate carrageenan induced inflammatory pain in mice[END_REF]. In line with this, we found that the expression of To understand how LPA 1 exerts its influence on the inflammatory component of MS and EAE, we examined infiltration and activation of immune cells. In the case of LPA 2 , its effect on the EAE disease course appears to be reliant on its capacity to increase T-cell homing, thus reducing infiltration. While some studies have indicated a detrimental role of LPA 1 in bloodbrain-barrier (BBB) integrity [START_REF] Banks | Lysophosphatidic acid and amitriptyline signal through LPA1R to reduce P-glycoprotein transport at the blood-brain barrier[END_REF][START_REF] On | Rapid and reversible enhancement of bloodbrain barrier permeability using lysophosphatidic acid[END_REF][START_REF] Yu | Role of Rho kinase in lysophosphatidic acid-induced altering of blood-brain barrier permeability[END_REF] and potential to increase extravasation through induction of chemokine expression [START_REF] Lin | Lysophosphatidic acid upregulates vascular endothelial growth factor-C and tube formation in human endothelial cells through LPA(1/3), COX-2, and NF-kappaB activation-and EGFR transactivation-dependent mechanisms[END_REF], our results did not indicate a significant impact of the LPA 1 deletion on PBMC infiltration into the CNS. While this does not exclude an effect below statistical significance or an effect of BBB leakage independent of PBMC infiltration, we cannot explain the amelioration of clinical scores through reduced infiltration. Instead, the observation that infiltrating cells express Lpar1 to a higher degree than peripheral cells in EAE wild-type mice suggests that LPA 1 is involved in immune cell activation without necessarily affecting infiltration.

Following the hypothesis that LPA 1 correlates with immune cell activation, we examined its expression in activated human macrophages. Our results show an increase of LPAR1 expression, but not LPAR2, in both HD and RR-MS patient macrophages when activated towards a proinflammatory phenotype. On the other hand, expression of the LPA nuclear receptor PPAR not only decreases when macrophages acquire M1 polarization but also shows a trend towards increasing after pro-regenerative activation. We did not identify a difference between MS patients and healthy controls, but this could be due to the fact that MS samples were taken during a remission phase. In this case, the modular expression of different LPA receptors after differential activation hints a complex role of LPA signaling in the homeostasis of macrophages during the disease and suggests that modulating the expression or saturating the activation of one receptor or another could be a mechanism of trans-differentiation of human macrophages. Although this aspect requires further study, the fact that LPA 1 is related to glycolysis [START_REF] Bernhart | Lysophosphatidic acid receptor activation affects the C13NJ microglia cell line proteome leading to alterations in glycolysis, motility, and cytoskeletal architecture[END_REF][START_REF] Ha | LPA Induces Metabolic Reprogramming in Ovarian Cancer via a Pseudohypoxic Response[END_REF][START_REF] Mukherjee | Lysophosphatidic Acid Up-Regulates Hexokinase II and Glycolysis to Promote Proliferation of Ovarian Cancer Cells[END_REF] (main source of energy for proinflammatory polarized macrophages) and PPARƔ induces oxidative phosphorylation [START_REF] Odegaard | Macrophage-specific PPARgamma controls alternative activation and improves insulin resistance[END_REF], and that these two metabolic processes are central in pro-and anti-inflammatory macrophage activation respectively [START_REF] Galvan-Pena | Metabolic reprograming in macrophage polarization[END_REF], suggests that the modulation of these LPA receptors could have major implications in the macrophage physiology and activation.

Knowing that LPA is dysregulated in MS relapses [START_REF] Balood | Elevated serum levels of lysophosphatidic acid in patients with multiple sclerosis[END_REF][START_REF] Jiang | Elevated lysophosphatidic acid levels in the serum and cerebrospinal fluid in patients with multiple sclerosis: therapeutic response and clinical implication[END_REF] and that LPAR1 expression is increased in macrophage activation, we hypothesized that LPA 1 could mediate LPA-induced proinflammatory activation in macrophages. This was confirmed through increased expression Although this work focused on peripheral immune cells, a similar effect of LPA on microglia and non-parenchymal macrophages could be predicted in view of reported LPA-dependent effects on microglia including chemokinesis, activation, oxidative stress response or proinflammatory cytokine production [START_REF] Plastira | 1-Oleyl-lysophosphatidic acid (LPA) promotes polarization of BV-2 and primary murine microglia towards an M1-like phenotype[END_REF]. Moreover, LPA 1 has been associated to proinflammatory microglial activation and their TNF-α production [START_REF] Kwon | Identifying lysophosphatidic acid receptor subtype 1 (LPA(1)) as a novel factor to modulate microglial activation and their TNF-α production by activating ERK1/2[END_REF]. Similarities between microglia and non-CNS macrophages are extensive, and their final phenotype is largely dependent on ontogeny [START_REF] Bennett | A combination of ontogeny and CNS environment establishes microglial identity[END_REF]. It is thus possible that the hypothesized feedback loop of LPA in inflammation is further exacerbated by resident immune cells.

In short, our study unveils for the first time a role of LPA 1 in the pathogenesis of MS and its animal model, EAE, and the importance of the regulation of the LPA signaling in the Analyses were performed by one-way ANOVA followed by Bonaferroni posthoc test (p = 0.04).

* p < 0.05.

LPA 1

 1 antagonist administration. VPC 32183 (S), (S)-Phosphoric acid mono-(2-octadec-9-enoylamino-3-[4-(pyridine-2ylmethoxy)-phenyl]-propyl) ester (Ammonium Salt) (CAS number 799268-75-0;Avanti Polar Lipids, Alabaster, AL, USA) was dissolved in 3% fatty-free acid BSA (FFA-BSA; Sigma-Aldrich) in saline. VPC32183 was diluted to a concentration of 5 µM and a volume of 100 µl was injected intravenously in the tail vein at the time-points described in the text. Non-treated control mice only received vehicle injections (3% FFA-BSA in saline). Subjects RNA analysis of total PBMC was performed on samples provided by the Biobank of our hospital, as part of the Andalusian Public Health System Biobank. All patients participating in the study gave their informed consent and protocols were approved by institutional ethical committees (Comite de Ética de la Investigación provincial de Málaga). The study was conducted according to international ethical principles contained in the Declaration of Helsinki, Spanish regulations on biomedical research (Law 14/2007, of July 3, on biomedical research) and the provisions of the European General Personal Data Protection Regulation (Royal Decree-Law 5/2018, of 27 July, and Regulation (EU) 2016/679, of April 27, 2016). Patient selection was based on the criteria of first diagnosis and under no MS treatment. For the RNA sequencing analysis, 22 MS patients (11 pairs of siblings from a cohort established for a parallel study) and 9 healthy controls were recruited for the macrophage experiments. The study was approved by the French Ethics committee and the French ministry of research (DC-2012-1535

LPA 1 was

 1 high during relapsesa period generally associated with high inflammatory activityin immune cells from both EAE and MS. The differential expression of LPAR1 in the different clinical forms of MS indicates a direct role of the LPA-LPA 1 pathway in the inflammatory component of the disease and suggests a potential use of LPAR1 expression as a biomarker of disease activity. These results are consistent with previous studies showing increased levels of LPA in blood or cerebrospinal fluid of RR-MS patients during relapse compared to HD or RR-MS patients in remission [14, 15] and suggest a broader dysregulation of LPA signaling than previously thought. We also found a significant positive correlation between the levels of Lpar1 expression during relapses and the severity of the EAE clinical course, encouraging future analysis of RR-MS patient clinical disability and LPAR1 expression. Preliminary results showed that LPAR1 expression in patients when first diagnosed and after two years of treatment decreased. Despite these promising observations, more clinical data and treatment-based sorting would strengthen the implication of LPA 1 in the disease course and potentially enable the use of its expression to estimate the individual patient's prognosis. Nevertheless, large cohorts and the inclusion of immune-modulatory treatments would be necessary to extract meaningful statements.

  of M1 markers following LPA incubation with partial correction by exposure to the LPA 1 antagonist Ki16425. Increased expression of LPA 1 in EAE and MS PBMC during relapse thus suggests both an activated state as well as a predisposition to further proinflammatory activation. The coordinated responses between the induced LPA 1 expression and the proinflammatory activation of LPA via the LPA 1 would promote a positive feedback loop that grants to LPA the role of boosting the inflammatory response and maintain the classical activation of macrophages. The milder EAE clinical course observed in LPA 1 -null and LPA 1antagonized mice, which present lower maximal and minimal scores in relapses and remissions respectively, could therefore be explained by a milder activation of immune cells, whereas the number or relapses and the onset of the disease unaffected as infiltration still occurs to the same extent.

Fig. 1 .

 1 Fig. 1. LPA 1 null mice exhibit a less severe EAE disease course than wild-type (WT).

Fig. 2 .Fig. 3 .

 23 Fig. 2. LPA 1 antagonist treatment lowers clinical scores in EAE mice.

Fig. 4 .Fig. 5 .

 45 Fig. 4. Lpar1 expression in mouse PBMC during EAE development

Fig. 6 .

 6 Fig. 6. LPA receptor LPA 1 and PPARƔ are differentially regulated in human macrophages after proinflammatory and pro-regenerative differentiation.

Fig. 7 .

 7 Fig. 7. LPA 1 antagonist directs human macrophages toward a more pro-regenerative phenotype.

Fig. 2

 2 Fig. 2.

*Fig. 5 .

 5 Fig. 5.

  Fig. 6.

  polyA selection (Truseq stranded mRNA kit, Illumina, Inc., San Diego, CA, USA). For each sample, we performed 60 million single-end, 75 base reads on a NextSeq 500 sequencer

	(Illumina).	RNA-Seq	data	analyses	were	performed	by	GenoSplice
	and	insert	size	estimation	were	performed	using	FastQC

technology(www.genosplice.com, Paris, France). Sequencing, data quality, reads repartition, (https://www.bioinformatics.babraham.ac.uk/projects/fastqc/), Picard-Tools (https://broadinstitute.github.io/picard/), Samtools (http://www.htslib.org/) and rseqc

  to the manufacturer's protocol. Quantitative RT-PCR was performed with the The sample size calculation was performed by the resource equation method, trying to minimize the sample size to follow the ARRIVE guidelines for reporting animal research. Each n represents one animal or sample in the experiment. All mouse studies were repeated a minimum of 3 times, and each experimental group included at least 4 samples. The values are expressed as mean ± SEM. The statistical analysis was done with GraphPad Prism (GraphPad Software, San Diego, CA, USA). Normality in the variable distributions was assessed by the D'Agostino & Pearson omnibus test and Grubbs' test was used to detect and exclude possible outliers. If the normality test was passed, the means were compared by two-tailed Student's t

	Hs_CD180_1_SG QuantiTect Primer Assay (200) Cat No./ID: QT00203574
	Hs_PDGFC_1_SG QuantiTect Primer Assay (200) Cat No./ID: QT00026551
	Statistical methods
	LightCycler® 1536 Instrument (Roche Molecular Systems), and the following primers:
	Hs_CD86_1_SG QuantiTect Primer Assay (200) Cat No./ID: QT00033915
	Hs_TLR2_1_SG QuantiTect Primer Assay (200) Cat No./ID: QT00236131
	Hs_CCL2_1_SG QuantiTect Primer Assay (200) Cat No./ID: QT00212730
	Hs_CCL5_1_SG QuantiTect Primer Assay (200) Cat No./ID: QT00090083
	Hs_CCL20_1_SG QuantiTect Primer Assay (200) Cat No./ID: QT00012971
	Hs_LPAR1_1_SG QuantiTect Primer Assay (200) Cat No./ID: QT00021469
	Hs_MRC1_1_SG QuantiTect Primer Assay (200) Cat No./ID: QT00012810
	Hs_CD163_1_SG QuantiTect Primer Assay (200) Cat No./ID: QT00074641

test. When one or both groups did not follow a normal distribution, means were compared by two-tailed Mann-Whitney U test. One-way ANOVA plus Bonferroni posthoc tests was used to compare different independent groups. Statistical significance was determined using the appropriate statistical test mentioned in each experiment. Values were statistically significant when p < 0.05. No blinding was performed in the analysis of the results.

Scientific Inc., Waltham, MA, USA) to extract RNA.

Acknowledgements. We also gratefully acknowledge IBIMA joint support structures for research (ECAI) of General Services, Microscopy and Animal Experimentation. For the ICM, we would like to acknowledge the center for clinical investigation, the IGENSEQ sequencing platform and the CELIS cell culture facility but also the fund raising, scientific affairs and the administrative department.

Funding. This work was supported by grants from Spanish Ministry of Science and Innovation, Institute of Health Carlos III, co-funded by European Regional Development Fund (ERDF, EU), (PI16/01510, to GET), Andalusian Regional Ministries of Economy, Knowledge, Business and University (Talent Hub Programme, co-funded by FP7,UE, Agreement Num. 291730, contract TAHUB-II-107, to BGD; CTS643 to GET) and of Health and Families (Nicolas Monardes Programme, and PI-0187/2008, PI-0234-2013 to GET), and Ramon Areces Foundation to BGD. This work was supported by the OCIRP foundation, the program "Investissements d'Avenir" ANR-10-IAIHU-06 and "Translational Research Infrastructure for Biotherapies in Neurosciences" ANR-11-INBS-0011-NeurATRIS.

Availability of data and material Not applicable

Code availability Not applicable

development of the disease. In addition to opening up to new avenues for immuno-modulatory treatment, this research also indicates a potential for LPA 1 as a biomarker of disease activity.

Further research on LPA in MS should therefore consider the exact pathways being targeted and the current level of disease activity in the patient, in order to develop strategies to better follow and treat these neurological patients.

Declarations


Eléonore Morisset

Aurélien Chardon

Jacques Rouden

Jérôme Blanchet

Phenysilane and Silicon Tetraacetate: Versatile Promotors for Amide Synthesis

Keywords: hydrosilane, amide, peptide, acetylation, coupling reagent

Phenylsilane was reevaluated as a useful coupling reagent for amide synthesis. At room temperature, a wide range of amides and peptides were obtained in good to excellent yields (up to 99%). For the first time, Weinreb amides synthesis mediated by a hydrosilane were also documented. Comparative experiments with various acetoxysilanes suggested the involvement of a phenyltriacyloxysilane. From this mechanistic study, silicon tetracetate was shown as an efficient amine acylating agent.

Introduction

Amide bonds are ubiquitous in chemical science. From natural products to polymers, amide is one of the most popular and useful functional groups in chemistry. [1] In biological systems, the importance of the amide bond lies in sustaining life by linking amino acids together thus forming proteins. [START_REF] Wieland | The World of Peptides: A Brief History of Peptide Chemistry[END_REF] Therefore, it is not surprising that amide bond formation is the most frequently performed transformations in the chemist's toolbox. [3] The use of chlorinating, diimide or onium based coupling reagents (SOCl2, DCC, EDCI, HATU…) are highly predictable methodologies to combine carboxylic acids and amines. [START_REF] Albericio | For recent reviews about peptides synthesis see: a)[END_REF] Nevertheless, some issues are still associated with these reagents, related to partial racemization, waste, toxicity or chemoselectivity. [5] Within this context, novel methodologies for amides and peptides synthesis are in great demand and stimulated global efforts in the area of amide synthesis. [START_REF] Lunberg | For recent reviews about amide bond formation see: a)[END_REF] Alongside tremendous progresses in metal-and boron-catalyzed methodologies [7,[START_REF] Ishihara | For key references in boron-catalysed amide synthesis see: a)[END_REF] , there has been a steep rise in the development and implementation of silicon-based reagents as a useful alternative to recently developed coupling reagents. [9] Pioneered by Chan in 1969, [10a,b] the use of silicon active reagents derived from SiCl4 was later investigated by Liskamp (Me2SiCl2) and Mukaiyama to culminate recently with the development of a 9-silafluorenyl dichloride by Charette (Figure 1). [10c-h] Although a useful scope was accomplished with the later, this methodology required a large excess of a Brønsted base as hydrogen chloride scavenger. This resulted in large chemical wastes and enantiomeric erosion of substrate, notably in the presence of sensitive phenylglycine derivatives. In 2018, Braddock reported a simple and efficient procedure however relying on highly toxic tetramethylorthosilicate (TMOS). [11] Within this framework, hydrosilanes are appealing considering low toxicity and that hydrogen gas and silanols are expected as only side products. However, the use of hydrosilanes appears underinvestigated: only two reports mention it so far. In 2006, phenylsilane (PhSiH3) was examined as a mild coupling reagent using a high-throughput screening of various amines and carboxylic acids. [START_REF] Ruan | or more amino carboxylic acid molecules[END_REF] Driven by initial success in homogeneous solution, further solid phase syntheses were carried out using large excess of phenylsilane at room temperature. [START_REF]Typically, 10 equiv of amines and 20 equiv of PhSiH3 were used[END_REF] Later, Charette documented diphenylsilane (Ph2SiH2) as an effective coupling reagent for amide and peptide synthesis. [START_REF] Sayes | [END_REF] Under the reported conditions (DMAP 0.5 equiv., DIPEA 1 equiv., acetonitrile, 80 °C), an array of dipeptides were synthesized albeit considerable epimerization was observed in the case of N-Boc-phenylglycine (e.r. 61:39). Moreover, Fmoc protecting group was found incompatible with the reported reaction conditions. Surprisingly, upon screening the nature of the hydrosilane, phenylsilane was found less reactive. Intrigued by this result, we decided to reassess phenylsilane potential as coupling reagent for peptide and Weinreb amide synthesis.

Results and Discussion

Our investigation began by examining a model reaction involving morpholine and phenylacetic acid. A stoichiometric amount of both partners in methylene chloride afforded a promising 50% yield after one hour at room temperature (Table 1, entry 1).

[a] E. Morisset, Dr A. Chardon, Pr. J. Rouden, Dr J. Blanchet Laboratoire de Chimie Moléculaire et Thio-organique, Normandie Univ, ENSICAEN, UNICAEN, CNRS, 14000 Caen, France. jerome.blanchet@ensicaen.fr. https://www.lcmt.ensicaen.fr/cv-jerome-blanchet/ Supporting information for this article is given via a link at the end of the document. [a] Determined using 1,3,5-trimethoxybenzene as internal standard. [b] Reaction carried out in MeCN.

In sharp contrast with previous study [START_REF] Sayes | [END_REF] , an extra amount of phenylsilane appeared detrimental for the amide bond formation (Table 1, entry 2). Longer reaction time associated to amine excess led to an improved conversion of 74% (Table 1, entry 4). However, a substantial acceleration was observed in the presence of a two-fold excess of phenylacetic acid and a quantitative conversion was reached in only two hours at room temperature (Table 1, entry 6). Additional solvent screening led to an even faster reaction in acetonitrile, giving a complete conversion in only 30 minutes (Table 1, entry 7). Control reactions with other hydrosilanes (Et3SiH, PhMe2SiH, PMHS, TMDS, Ph2SiH2) did not produce any amide 3. Only hexylsilane was found less reactive providing a 33% conversion under similar reaction conditions. Efforts to identify a more efficient coupling reagent prompted us to investigate the reactivity of phenylsilane derivatives 4a-4e, synthesized according to known procedures (Figure 2). [15] Figure 2. Considered phenylsilane derivatives.

Unfortunately, monitoring reaction progress using 1 H NMR, slightly lower performance were showed for hydrosilanes 4a and 4b and an absence of reactivity with 4c and 4d (see supporting information Figure S1). In our hands, 4e was found surprisingly too volatile to be evaluated. Using optimal reaction conditions, various aliphatic, aromatic and heteroaromatic carboxylic acids (1a-f) derivatives were smoothly coupled with primary amines or cyclic secondary amines (Scheme 1). As expected, electron rich 2-furancarboxylic acid and less nucleophilic aniline were found to be challenging substrates and moderate 64-61% yields were obtained after a prolonged reaction time. Also, a limitation was identified with highly hindered mesityl carboxylic acid that gave no detectable amount of amide product. More challenging peptides couplings were then examined using phenylsilane. Surprisingly, a solvent change to methylene chloride was required, as slightly lower yield of Boc-Phe-Phe-OMe 7a was obtained with acetonitrile. Additives such as DMAP, DMAPO [16] or sodium bisulfite [17] did not improve the yield. Only one equivalent of potassium cyanide was found to barely improve the yield of the reaction but the acute toxicity of such additive excluded further investigations. When α-amino ester hydrochloride was used as a substrate in the presence of added triethylamine, the yield of Boc-Phe-Phe-OMe 7a dropped to 29%. Therefore we investigated the scope of the reaction using α-amino esters as free amine. All the reactions proceeded smoothly to afford the desired dipeptides 7 in good to moderate yields. Boc-glycine was efficiently coupled to methyl α-amino esters of phenylalanine, proline, valine, methionine, aspartic acid and sarcosine (7b-g, Scheme 2). More significantly, other N-protected α-amino acids derived from proline, phenylalanine and tryptophan performed equally well with 62-83% isolated yields (7h-n, Scheme 2). Under our mild reaction conditions, usual protecting groups were well tolerated, including the versatile Fmoc group as illustrated by the synthesis of Fmoc-Phe-Phe-OMe in 75% yield (7m, Scheme 2). Strongly hindered O-protected Boc-threonine and valine delivered peptides 7o and 7p, albeit in moderate 55-56% yields (Scheme 2). In contrast, a control reaction with Boc-threonine bearing an unprotected hydroxyl group failed to deliver the corresponding peptide. Importantly, no epimerization was detected when using chiral N-protected α-amino acids even in the presence of sensitive Boc-phenylglycine as demonstrated with the synthesis of 7q in 57% yield (Scheme 2). Unfortunately, as the peptide chain extend yields plummeted and tripeptides 7r and 7s were obtained in moderate 48% and 55% yields, respectively. However, those yields are in line with Charette's study [START_REF] Sayes | [END_REF] while reactions were run at ambient temperature in our case.

As a case study, Weinreb amide synthesis was also investigated.

To the best of our knowledge hydrosilane mediated synthesis of such useful intermediates is unreported and so might represent a useful alternative to well-known procedures. [18] As expected, the use of N,O-dimethylhydroxylamine was found unreliable since the free amine readily decomposed. As a consequence the reaction was optimized with the corresponding hydrochloride salt. Several aliphatic and electron depleted aromatic carboxylic acids gave the corresponding Weinreb amides in good to moderate 86-44% yields (8a-e, Scheme 3).

Scheme 3. Substrate scope of phenylsilane promoted Weinreb amide synthesis.

The less electrophilic 2-methoxybenzoic acid reacted sluggishly due to a combination of unfavorable electronic and steric factors (Scheme 3). The mechanism of amide synthesis involving hydrosilanes is still unclear. Charette conducted a brief mechanistic study and proposed a chemical ligation pathway requiring the addition of both amine and carboxylic acid at the silicon atom followed by the intramolecular addition of silylamine 9 to neighboring silylester. [10h,14] However, such mechanism involving the formation of a silanone was later determined thermodynamically unfavorable using DFT calculations. [19] A related study by Denton supported that phenylsilylester 10 (Figure 3) is effective for amide synthesis however in refluxing toluene and with a moderate yield of 64%. [20a] The observation that an excess of carboxylic acid significantly bolstered the reaction kinetic suggested than a putative polysilylester intermediate might be responsible for the high reactivity observed at room temperature. Therefore we hypothesized that diester 11 or triester 12, readily formed by a two-fold or three-fold dehydrogenative addition of carboxylic acid to phenylsilane, might be responsible for the reactivity observed at room temperature. However, in our hands any effort to isolate reactive esters 11 and 12 failed and systematically ended with polysiloxanes formation. As a surrogate, the acetylation of morpholine 2a was investigated with various commercially available silyl acetates 13a-c (Scheme 4). Interestingly, diacetoxydimethylsilane 13a was found not productive at all: even after 5 hours at room temperature, no trace of amide product was detected by 1 H NMR (Scheme 4). On the contrary, triacetoxy(methyl)silane 13b displayed a moderate conversion of 26% at 0 °C and more than two-fold increased at room temperature (Scheme 4). Finally, silicon tetraacetate 13c reacted quantitatively and almost immediately at 0 °C. While a direct comparison is arguable, such results suggest that silicon esters 10 and 11 are not responsible for the reactivity observed at room temperature. Indeed it appeared clearly that added acetate groups at the silicon significantly boosted the electrophilicity of the acetyl moiety. This prompted us to rationalize that phenylsilane mediated amide synthesis is likely to occur through putative ester 12. [20b] Scheme 5. Substrate scope of silicon tetraacetate promoted acetamide synthesis. Reaction times are not optimized for individual substrates. [a] e.e. > 99%. HPLC data available in supporting information.

To the best of our knowledge, silicon tetraacetate has never been considered so far as a general acetylating reagent of amines. Therefore, its versatility was demonstrated by the synthesis of a range of acetamides under mild conditions and in absence of any base (Scheme 5).

Most amines and anilines gave high yields at room temperature (14a-l, 75-99% isolated yields) and only electron poor or N-substituted anilines required mild heating to reach reasonable to excellent yields (14m-o, Scheme 5). Additional chiral alpha amino ester were also acetylated without noticeable racemization (14p-r, Scheme 5).

Conclusions

In conclusion, phenylsilane was reassessed as an efficient amide and peptide coupling reagent. A large scope of dipeptides and two tripeptides was achieved with good to moderate yields. It is worth noting that no side reaction such as epimerization was observed even with phenylglycine derivatives. Comparative experiments using various acetoxysilanes pointed towards phenyltriacyloxysilane as reactive intermediate. Finally, silicon tetraacetate was introduced as an interesting acetylating reagent for amines and anilines that operate in mild conditions and in absence of base.

Figure 1 .

 1 Figure 1. Reported silicon coupling agents.

Scheme 2 .

 2 Scheme 2. Substrate scope of phenylsilane promoted peptide synthesis. Reaction of N-protected α-amino acid 1 (2 mmol), α-amino ester 2 (1 mmol) and PhSiH3 (1.5 mmol) were carried out in dry CH2Cl2 (1 mL) for 16 hours at 20 °C. [a] d.e. > 99%. HPLC data available in supporting information.

Figure 3 .

 3 Figure 3. Possible reactive intermediate involved in hydrosilanes mediated amide synthesis.

Scheme 4 .

 4 Scheme 4. Silyl acetate reactivity in acetylation reaction. [a] NMR conversion determined using 1,3,5-trimethoxybenzene as internal standard. [b] Conversion after 5 h at room temperature. [c] Isolated yield.

Table 1 .

 1 Optimization of amide synthesis with phenylsilane.

	Entry	1 : 2 : PhSiH3	t	NMR conv.	[a]
	1	1:1:1	1 h	50 %
	2	1:1:3	1 h	39 %
	3	1:1:1	5 h	68 %
	4	1:1.5:1.5	5 h	74 %
	5	1.5:1:1.5	2 h	84 %
	6	2:1:1.5	2 h	100 %
	7	2:1:1.5	30 min	100 %	[b]

Acknowledgements

The authors thank the CNRS, Labex Synorg (ANR-11-LABX-0029) for AC fellowship, Normandie université, the Conseil Régional de Normandie and the European FEDER fundings for financial support.

Entry for the Table of Contents

COMMUNICATION

Phenylsilane as been reassed as useful coupling reagent for amides synthesis. Peptides and Weinreb amides are easily obtained under mild conditions in absence of epimzerization even with challenging substrates such as phenylglycine. Further investigation regarding the reactivity of stable acetoxysilanes supported phenylpolyacyloxysilane as key intermediate.

Amides synthesis

Eléonore Morisset [a] , Aurélien Chardon [a] , Jacques Rouden [a] and Jérôme Blanchet [a] Page No. -Page No. Phenysilane and Silicon Tetraacetate: Versatile Promotors for Amide Synthesis.


Isabelle Kruta
email: isabelle.kruta@sorbonne-universite.fr

Jérémie Bardin
email: jeremie.bardin@sorbonne-universite.fr

Christopher P A Smith
email: christopher.pierre.a.smith@gmail.com

Paul Tafforeau
email: paul.tafforeau@esrf.fr

Neil H Landman
email: landman@amnh.org

Enigmatic hook-like structures in Cretaceous ammonites (Scaphitidae)

Keywords: Ammonites, Scaphitidae, exceptional preservation, hooks, morphometrics

INTRODUCTION

The number of arms remains unknown for the Ammonoidea [START_REF] Klug | Soft part anatomy of ammonoids: reconstructing the animal based on exceptionally preserved specimens and actualistic comparisons[END_REF] and reconstructing the soft part anatomy of the brachial crown relies on phylogenetic bracketing [START_REF] Witmer | The extant phylogenetic bracket and the importance of reconstructing sort tissues in fossils[END_REF]. Knowledge of the internal anatomical features of ammonites is largely limited to the preservation and study of mineralized or sclerotized elements belonging to the buccal apparatus such as the upper and lower jaws and radula [START_REF] Tanabe | Ammonoid buccal mass and jaw apparatus[END_REF]. Nevertheless, some features interpreted as buccal elements are still enigmatic due to their unconventional morphology and could possibly belong to other parts of the animal. This is the case of large cuspidate structures documented by [START_REF] Kennedy | Jaws and radulae in Rhaeboceras, a Late Cretaceous ammonite[END_REF] in specimens of Rhaeboceras halli [START_REF] Meek | Descriptions of new species of Gasteropoda and Cephalopoda from the Cretaceous formation of Nebraska Territory[END_REF], a recoiled member of the Scaphitidae (Ancyloceratina) from the Upper Cretaceous (Campanian) Bearpaw Shale of Montana (Fig. 1, Table 1). These structures occur in the body chambers of many macroconch specimens in close association with the lower jaws and were interpreted as radular elements. However, some of the features of these structures are inconsistent with a radular interpretation, as pointed out by [START_REF] Kruta | The radula of the Late Cretaceous ammonite Rhaeboceras halli[END_REF]. The structures are extremely large, as much as four times larger than the teeth in modern nautilus. The radula interpretation was rejected by [START_REF] Kruta | The radula of the Late Cretaceous ammonite Rhaeboceras halli[END_REF] who discovered incontrovertible evidence of radulae in three specimens of Rhaeboceras halli. These radulae occur in situ and the size and morphology of the teeth are completely in agreement with radular teeth known from other aptychophoran ammonites [START_REF] Kruta | The radula in the Ammonoidea[END_REF].

Similar bicuspidate structures were also described by [START_REF] Landman | Scaphitid ammonites of the Upper Cretaceous (Maastrichtian) Fox Hills Formation in South Dakota and Wyoming[END_REF] in two other species of Scaphitidae from the Upper Cretaceous (Maastrichtian) Fox Hills Formation.

The structures occur in the body chambers of Hoploscaphites spedeni [START_REF] Landman | Scaphitid ammonites of the Upper Cretaceous (Maastrichtian) Fox Hills Formation in South Dakota and Wyoming[END_REF] and H. nicolletii (Morton, 1842). Since then, [START_REF] Landman | A new species of Hoploscaphites (Ammonoidea: Ancyloceratina) from cold methane seeps in the Upper Cretaceous of the U.S. Western Interior[END_REF] documented additional structures in association with H. gilberti [START_REF] Landman | A new species of Hoploscaphites (Ammonoidea: Ancyloceratina) from cold methane seeps in the Upper Cretaceous of the U.S. Western Interior[END_REF] from the Upper Cretaceous (Campanian) Pierre Shale in South Dakota (Fig. 1, Table 1).

In order to obtain more data to elucidate the function and origin of these bicuspidate structures, we used X-ray based imaging techniques (Synchrotron and CT-scan) to reveal 446 structures embedded in six specimens of Rhaeboceras halli. We documented the morphological variability of these structures to determine if they comprise a single complex apparatus. We present the first detailed reconstruction of the morphology of these structures in several specimens of Rhaeboceras halli.

MATERIAL AND METHODS

Material

The specimens of Rhaeboceras halli studied are from the upper Campanian Baculites jenseni Zone of the Bearpaw Shale, Garfield County, Montana, USA (Fig. 1, Table 1). Over 30 specimens containing hook-like structures are deposited at the American Museum of Natural History (AMNH). A total of 12 specimens were CT-scanned in order to investigate the quality of preservation. All of the specimens consist of fragments of the body chambers of macroconchs, with only pieces of the outer shell wall preserved. The shell wall retains the original nacreous microstructure, indicating excellent preservation. Moreover, three of the specimens contain the lower jaw in the anterior end of the body chamber and one specimen (AMNH 51334) contains both lower and upper jaws and the radula in the posterior part of the body chamber [START_REF] Kruta | The radula of the Late Cretaceous ammonite Rhaeboceras halli[END_REF]. Six of these specimens (AMNH 64405,66448,66350,66351,66433 and 66434,) were very well preserved and were selected for more detailed investigations of all of the hooks inside the body chambers, using µCT-scanning and propagation phase-contrast X-ray synchrotron Microtomography (PPC-SR-µCT). In the majority of specimens, the structures occur on the outer surface of the body chamber against the flanks or venter (Fig. 2). Because of the presence of cusps, we refer to these structures as hooks. We use this as a general term, in full recognition of the fact that these structures do not curve backward as in coleoid hooks.

Data acquisition

Data acquisition was performed using µCT-scanning at the AMNH and PPC-SR-µCT scanning at the Synchrotron ESRF (proposal es-859). Specimens AMNH 64405,66351,66433,66434, were scanned at the AMNH with a GE PHOENIX v|tome|x s 240, while AMNH 66350 and 66448 were scanned at ESRF on ID19. Scan parameters for this study are available in the Dryad Digital Repository [START_REF] Kruta | Data from: Enigmatic hook-like structures in cretaceous ammonites (Scaphitidae)[END_REF].

3-D segmentation was performed using VG Studio MAX 3.0 (Volume Graphics, Heidelberg, Germany). 3-D reconstruction of the structures was performed manually or using threshold tools.

In X-ray examination, the low-density material comprising the hooks appears darker than the surrounding mineral matrix, facilitating reconstruction. When the material composing the hooks was missing due to taphonomic factors, the void corresponding to the hook was used for reconstruction. Many of the hooks are located on the exposed surface of the body chamber with the black layer eroded away, making 3-D reconstruction impossible in those areas. As a result, only 52% of the hooks are preserved as complete structures, and can be used for quantitative analysis.

Composition

The composition of the hooks (specimen AMNH 66349) was determined using EDX and micro X-ray diffractometry (Texray Laboratory Service, Brucker D5000 X-ray diffractometer).

Morphometric analysis

In order to analyze the overall morphology of the hooks, landmarks were positioned on the bestpreserved structures in VG Studio Max 3.0 for a total of 183 bicuspidate hooks. The base of the hooks is convex and forms a circular to ovoid opening on one side. Therefore, nine landmarks were used to describe the morphology of the hooks, four of which were located around the opening (Fig. 3).

The coordinates of the landmarks were exported and analyzed using R software (R Core Team 2016). A procrustes transformation was performed to remove the effects of size, position, and rotation. Exploratory analyses such as PCA were used to gain insights into the structure of the data in multivariate morphometric space and clustering was performed using a Gaussian mixture model [START_REF] Everitt | Applied multivariate data analysis[END_REF] with functions from the R package mclust [START_REF] Fraley | Mclust Version 4 for R: Normal Mixture Modeling for Model-Based Clustering, Classification, and Density Estimation[END_REF].

RESULTS

Composition and general description

In all the studied specimens, the hooks are composed of a layer of black material 0.15 mm thick (Fig. 4.). EDX and XRD analyses performed on this black layer (Fig. 4) indicate two different phases [START_REF] Kruta | Data from: Enigmatic hook-like structures in cretaceous ammonites (Scaphitidae)[END_REF]. The main crystalline phase identified is brushite (Ca (PO 3 OH) x H 2 O). Two amorphous regions were also identified, one of them characterized by longer bonds typical of organic compounds. The shape of the hooks forms a cavity subsequently filled in with the matrix sediment. The main crystalline phase identified for the matrix in XRD is calcite.

The number of hooks varies among the specimens from 40 in AMNH 66351 to 171 in AMNH 66350. These differences could be related either to differences in preservation (different size fragments of body chamber, parts of the body chamber exposed or eroded) or to variation in the original number of hooks. The specimen with the largest number of hooks in the body chamber (AMNH 66350) is 1) among the largest specimens based on estimates of body chamber size and 2) the specimen with the smallest number of hooks exposed in the body chamber.

The structures are generally bicuspidate although some are unicuspidate, tricuspidate, and one is rounded (Fig. 5). The morphological variation of the hooks is described with the opening facing us and the cusps pointing to the top (Fig. 3A). This orientation is used to define the left and right sides of the hooks.

The hook with a rounded tip (Fig. 5G) was identified in AMNH 66350. This hook is almost 8 mm long and the rounded tip is flattened. In the same specimen, three unicuspidate hooks less than 1 mm in length were also found (Fig. 5K). Tricuspidate hooks (Fig. 5O) were found in three specimens (AMNH 64405, 66448, and 66350) for a total of six hooks. Their size approaches 1 mm and the middle cusp is slightly longer compared to the two others, similar to the dimensions of a rachidian tooth in a radula. Approximately 98% of the remaining hooks recorded are bicuspidate. Their morphology varies from elongate (e.g., Fig. 5C-D) to short (e.g., Fig. 5A) with variation of cusp length and opening size. During the segmentation, several types of hooks were identified, based on their overall morphology and size (Fig. 5). In order to have better insights into the morphological disparity among these bicuspidate hooks, quantitative analyses were performed on the best-preserved specimens through the use of landmarks. One group of bicuspidate hooks (Fig. 5F) with length less than 1 mm was too small for precise landmark placement on the 3D program. This small morphotype was encountered in almost all specimens (except AMNH 64405), for a total of 43 hooks. The cusps are almost sub-equal in sizes with the tips of the cusps pointing slightly inward (Fig. 5F).

Bicuspidate hooks description

PCA analyses. Most hook shape variation is accounted for by a relatively small number of axes (Fig. 6). The three first PC-axes represent 88.9% of the variance. The main morphological trend on PC1 (49.8% of the variance) is associated with variation from a long slender hook with the right cusp longer than the left cusp, to a larger form with the left cusp longer than the right. PC2 (31.7% of the variance) reflects the variation in the size and position of the opening. The positive part of the axis reflects long and slender hooks with the opening in the lower part, and the negative part of the axis reflects shorter hooks with a larger opening. Variation on PC3 (7.3% of the variance) represents a trend where the cusps are either short with an acute angle between them or long with an obtuse angle between them.

Gaussian mixture analyses. Gaussian mixture analyses [START_REF] Everitt | Applied multivariate data analysis[END_REF] were performed focusing on the three first PC-axes. A 5-group solution (Fig. 7) emerged with the highest BIC (Bayesian Information Criterion). Among the 5 groups, two pairs (G1-G5 and G2-G4) are mirror images (Fig. 7). The groups G1 and G5 (Fig. 7) represent a slender morphology with a small opening. The right or left cusp predominates. Groups G2 and G4 (on the right and left sides of the graph) represent wider hooks with larger openings. The last group (G3) reflects hooks with nearly symmetric cusps.

Size of the hooks. The distance between landmarks 3 and 5, corresponding to the maximum linear length of the hooks ranges from 1.64 mm to 25.41 mm. The size variation of the hooks was studied by plotting the centroid size for each morphogroup per specimen (Fig. 8). The overall distribution is similar for all specimens (Fig. 8A) and will be described only for AMNH 66350 (the most complete specimen). The chiral groups G1 and G5 represent the most common bicuspidate structures. The box plot for these structures is comparatively tall with similar median values indicating a similar range of sizes. Within these two groups, the size of the largest hook is two to four times the size of the smallest hook. The size range of the chiral groups (G2) and (G4) only partially overlaps. Although a higher number of hooks are preserved in G4 group, the boxplot is relatively shorter with a median lower than in the red group. The G3 group exhibits the lowest values of centroid sizes in AMNH 66350 and all the other specimens where it is recorded.

Following this first analysis indicating chiral structures, we decided to transform (flip) all the hooks from those with left cusps dominant into hooks with right cusps dominant.

PCA on flipped hooks. The five first PC-axes summarize 89.1% of the variance. The main morphological trends are associated with variation in the angle between the plane of the opening and the plane of the cusps. PC1 (60.5% of the variance) is associated with variation from a long slender hook with a small opening, to a larger, almost equally sized cuspidate hook, with the opening extremely large. PC2 (18.8% of the variance) reflects the variation between hooks with long dominant cusps forming an open angle with the second cusp, a large opening, and a very narrow angle between the opening and the tips of the cusps (the hook is flat) to hooks with cusps almost equal in size, an opening in the lower part of the hook, and a large angle between the opening and the tips of the cusps. Variation on PC3 (4.2%) represents a trend where the cusps are either short with an acute angle between them or long with an obtuse angle.

Gaussian mixture analyses on flipped hooks. The analysis was performed on the four first PCaxes. A 5-group solution (Fig. 9) emerged with the highest BIC (Bayesian Information Criterion). The groups G2F and G3F are similar with a small opening in the lower part and a slender morphology. Both G1F and G5F hooks present large openings and cusps. In the last group G4F, the cusps are almost equal in size with a relatively large opening. Although groups were identified, the data distribution indicates a continuum in shape.

Size of the hooks vs estimated size of the ammonite. In order to visualize the relationship between the size of the hooks and the ammonites in which they occur, we plotted a boxplot of GPA centroid size of each morphogroup per specimen sorted by their whorl height at the base of the body chamber (Fig. 10). Ammonite size estimates expressed by the whorl height at the base of the body chamber must be regarded with caution as the specimens are incomplete and sometimes partially crushed. For AMNH 66334, AMNH 66350, AMNH 66433, as only parts of the body chamber are preserved, the estimates are based on more complete specimen of comparable size (e. g. AMNH 95898). From figure 10, no relationship can be identified and this is confirmed by the Spearman correlation test (rho=-0.075, p value = 0.31).

Length of hooks versus length of lower jaw. For comparative purposes, ratios were calculated between the length of the lower jaws and the length of the radular teeth for other ammonites in which both structures were preserved in situ and were measurable: Baculites (Ancyloceratina, Aptychophora) (AMNH 55901) and Cravenoceras (Goniatitina) (AMNH 66407). In Baculites (AMNH 55901), the longest radular element (the marginal tooth) is 2 mm tall and is 5% of the maximum length of the wing of the lower jaw. In Cravenoceras (AMNH 66407), the length of the marginal tooth (0.8 mm) is 6% of the maximum length of the wing of the lower jaw. In contrast, the hooks in R. halli are a magnitude of size larger. In AMNH 66433 with the lower jaw (aptychus) in situ the largest hook element (12.6 mm long) is 30% of the maximum wing length of the lower jaw (43.9 mm).

DISCUSSION

With the exception of a few mono, tri, and non-cuspidate hooks (2% of the hooks reconstructed), the vast majority of hooks exhibit two cusps. Results from quantitative analyses indicate that fewer morphotypes among the bicuspidate hooks exist than it first appeared during 3D segmentation. Two pairs of groups with the same morphologies but with either the right or left cusp dominant were identified as chirals; one less abundant group contains hooks almost subequal in size. Besides chirality, shape variation includes slender or more robust morphologies, changes in opening size, and variation of the angle between the two cusps. Although discrete groups were identified, a continuum in shape was actually observed and might indicate the presence of intermediate forms in better-preserved specimens, probably linked to morphological gradients along structures. Important size variation occurs within groups (sizes double or triple) and between groups with some elements < 1 mm and others >20 mm.

The chirality of the hooks is reminiscent of radulae. Rows of teeth on a radular ribbon comprise lateral and marginal teeth symmetrically arranged around the rachidian central tooth, which is often tricuspidate [START_REF] Kruta | The radula in the Ammonoidea[END_REF]. The small tricuspidate morphogroup (1 mm long) is consistent in shape and size with the rachidian tooth of a radula. The recovery of this central tooth of the radular ribbon was missing in the description of [START_REF] Kruta | The radula of the Late Cretaceous ammonite Rhaeboceras halli[END_REF]. However, aside from this single structure, all of the other elements that we describe are inconsistent with the shape and size of radular teeth. Indeed, size variation itself is inconsistent with what is observed in radulae. Once secreted by the odontoblasts in the radular sac [START_REF] Messenger | The radular apparatus of cephalopods[END_REF], the radular teeth do not continue to grow. Therefore, within the same morphotype, the size is homogeneous contrary to what is observed in the hooks (Fig. 5). Moreover, no combination of small (< 1 mm) and large (> 10 mm) elements appears on any radula and the ratio between the size of the hooks and the size of the body chamber is inconsistent to what is known from other ammonite radulae. In addition, the radular teeth lack openings because the teeth are solid chitinous elements. Therefore, the hook-like structures in R. halli described herein cannot be interpreted as radular elements as was suggested by [START_REF] Kennedy | Jaws and radulae in Rhaeboceras, a Late Cretaceous ammonite[END_REF].

In extant cephalopods, palatine teeth are also present on palps located on either side of the radula in the buccal cavity (e.g., Figs 33-35 O' Shea et al. 2007, Boldstad 2008). These teeth are not homogeneous in size, bear one narrow cusp, and are irregularly embedded on the palps.

Neither their size (≈ 2 mm), nor their shape is similar to what we describe in Rhaeboceras. Based on the number and size of the hook like structures in Rhaeboceras, if they were palatine teeth, they would hardly fit on the palps in the buccal cavity. Therefore, we discard the palatine teeth interpretation as a possible explanation for the hook like structures in Rhaeboceras.

The lack of a relationship between the size of the ammonites (approximated by the size of the body chamber) and the size of the hooks (Fig. 10) could indicate that these structures do not belong to these individuals. The superficial resemblance of the hooks to claws (chelipeds) or crustacean appendages evokes a crustacean origin. Examples of inquilinism of ammonite shell by crustaceans are known in the literature (e.g. Fraye & Jaeger 1995, [START_REF] Landman | Inquilinism of a baculite by a dynomenid crab from the Upper Cretaceous of South Dakota[END_REF].

Nonetheless, in such cases, several parts of the crustaceans are preserved including parts of the telson, abdomen and feet. In Rhaeboceras no such structures are preserved. The anatomical features of the hooks as well as the high number of elements of this single type of structure (171 hooks in AMNH 66350) also rule out this hypothesis.

The fact that these structures are only found in Rhaeboceras halli and no other co-occurring ammonites (e.g., Baculites) strongly suggests they are related to this species and do not represent random infilling of the body chamber. Moreover, the preservation of the jaws in situ in AMNH 66433 indicates, at least for this specimen, that other ammonite structures are preserved as well.

Our hypothesis is also supported by the fact that similar structures are present in the same family Scaphitidae in Hoploscaphites spedeni, H. nicolletii, and H. nebrascensis from the Maastrichtian of the U.S. Western Interior, and are closely associated with H. nodosus, H. brevis, and H. gilberti from the Campanian of the same basin.

A possible explanation could be that the hooks are structures related to mating, such as hectocotyli (modifications of the arm for reproduction in males). This would explain the lack of a relationship between the size of the ammonites and the size of the hooks. The hooks would have belonged to the males and would have been inserted into the pallial cavity of the females during mating. Modification of the arm suckers -either through enlargement or reduction-together with enlargement of the trabeculae in a variety of combinations is known in several Ommastraphida [START_REF] Roeleveld | Generic interrelationships within the Ommastrephidae (Cephalopoda). 277-314[END_REF]. Although the hook like structures in Rhaeboceras are very different, the distribution of these structures vaguely resemble those in Notodarus, with larger modified elements on the base and smaller modified elements on the distal portion of the arm (Dunning & Fork, 1998). Because the distribution of the hook like structures in Rhaeboceras have been modified due to taphonomic processes, the original distribution of these structures is difficult to determine. Therefore, the hectocotylus hypothesis is still speculative, although the organic origin of the material comprising the hooks is consistent with an armature hypothesis. This hypothesis will be explored in further research aiming to study the relative position of the hooks in the body chamber in order to reconstruct the position of the morphogroups along a possible arm.

CONCLUSION

This first quantitative analysis of the exceptionally preserved hook-like structures found in Rhaeboceras halli allows us to identify the presence of chirality and explore the shape variation. The structures seem to present a plastic morphology dominated by forms with two cusps. Shape description could be improved by using finer morphological descriptors such as sliding landmarks or Fourier analysis to capture curvatures of the hooks. Nonetheless, the data already gathered invalidates the interpretation that they are radular elements (contra [START_REF] Kennedy | Jaws and radulae in Rhaeboceras, a Late Cretaceous ammonite[END_REF]. Only further analysis will allow a better idea of the organization of the structures with respect to each other. So far, the most plausible hypothesis is that these structures are related to mating in the Scaphitidae. If this were the case, these structures would be the first fossil remains of brachial crown elements in Ammonoidea and would therefore constitute a milestone in our comprehension of the paleobiology of this extinct group. Table 1. Scaphitid species in which hook-like structures occur. * : attributed to this species by close association, the structures are not preserved in the body chamber.

Figure 1 .

 1 Figure 1. A. Ammonite zonation of the U.S. Western Interior (modified from Cobban et al., 2006) showing the zones containing hook-like structures. B. Map showing localities at which hook-like structures occur. 1. Baculites reesidei-B. jenseni Zones, Bearpaw Shale, Garfield County, Montana. 2. Hoploscaphites nicolletii-H. nebrascensis Zones, Fox Hills Formation, Dewey, Ziebach, and Corson counties, South Dakota. 3. B. scotti-Didymoceras

Figure 2 .

 2 Figure 2. Overall views of Rhaeboceras halli with hooks. A-C AMNH 64405. D-E AMNH 66433. A, D. Photographs with the hooks exposed on the flank. The black arrow indicates the beginning of the phragmocone. The white arrow indicates the lower jaw. B, E. 3D rendering of the hooks with the body chamber in transparency. C, F. Sketch of the hooks in the body chamber. Hooks embedded in the matrix and revealed with X-rays are in dashed lines. The white arrows indicate the lower jaw, and the dashed lines the portion of the jaw embedded in the matrix. Scale bar 1cm.

Figure 3 .

 3 Figure 3. Schematic drawing of the hook morphology and landmarks. A. View with the opening. B. Other side view. Scale bar 2 mm.

Figure 4 .

 4 Figure 4. SEM micrographs of the black layer comprising the hooks. A. Cross section of the hook showing the black material (brushite) underlined by white dashed lines and indicated by an arrow. The internal portion of the hooks forms a cavity infilled with sediment during the fossilization process. B. Surface of the black material. Scale bar 400 µm.

Figure 5 .

 5 Figure 5. Hook diversity with outer and opening views represented. 3-D rendering VG Studio MAX 3.0. Scale bar 2 mm.

Figure 6 .

 6 Figure 6. PCA of the hooks with landmarks for all specimens.

Figure 7 .

 7 Figure 7. Gaussian mixture analysis and shape of the center of gravity for each morphogroup.

Figure 8 .

 8 Figure 8. A. Centroid size per group for all the specimens. B. Boxplot of the centroid size per group for AMNH 66350.

Figure 9 .

 9 Figure9. Gaussian mixture analysis on the flipped hook dataset and shape of the center of gravity for each group.

Figure 10 .

 10 Figure 10. Boxplots of the centroid sizes of hooks scattered by specimens and groups in relation with the shell whorl height (mm). Specimen numbers are indicated next to each boxplot, the numbers of hooks are written in brackets. For AMNH 66350, 66433 and 66434 only parts of the body chamber are preserved, the estimates are based on more complete specimen of comparable size (e.g. AMNH 95898).

Acknowledgments. We thank ESRF for proposal acceptance es-859. We also thank Alexandre Lethiers (CR2P) for helping with figures as well as Stephen Thurston (AMNH). We also thank the reviewers C. Klug and anonymous for improving the manuscript.

DATA ARCHIVING STATEMENT

Data for this study are available in the Dryad Digital Repository: https: https://doi.org/10.5061/dryad.1bg1c1f.