Tanguy Chaumette

Lauriane Delay

Julie Barbier

Ludivine Boudieu

Youssef Aissouni

Mathieu Meleine

Amandine Lashermes

Wassim Legha

Frédéric Sophie Antraigue

Antonio Carvalho

PhD Fabien Marchand
email: fabien.marchand@uca.fr

Frederic Antonio Carvalho

Alain Eschalier

Denis Ardid

Aziz Moqrich

Legha

Carvalho

c-Jun/p38MAPK/ASIC3 pathways specifically activated by nerve growth factor through TrkA are crucial for mechanical allodynia development

Keywords: NGF, TrkA, mechanical allodynia, ASIC3, p38MAPK NGF, TrkA, ASIC3, mechanical allodynia, p38MAPK

ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

We are very grateful for the constructive comments of the reviewers and section editor which have improved the quality of our manuscript. The manuscript has been modified according to the section editor comment. We look forward to hearing back from you on this revised submission. A response to the section editor comment is below in blue. Changes made in the manuscript appear in red.

Section Editor: I agree that the reviewers have addressed most of the previous comments, but I still think there is an issue that is not resolved. The authors state in the discussion that p38 and c-JUN are "essential" for NGF-induced allodynia, but they have not really tested this. No interventions were done for c-Jun, although others have done this for p38 inhibitors (e.g. the Ji lab). My major issue here is that ERK and mTOR are also very well known to be activated by mTOR and the authors seem to have ruled these out based on their PCR experiment. However, PCR is not really appropriate here because it does not matter if the RNA changes, what matters is the kinase activity. I think this was a point of the second review, who was unfortunately not available to re-review the paper. I do agree that this is a clever model to clearly delineate the signaling pathways that are necessary for NGFinduced mechanical allodynia, but I just am not sure that the authors can really conclude that they have discovered an "essential" pathway based on their experimental approach.

We agree that ERK and mTOR could be also activated by NGF in TrkAC mice and that PCR is definitely not the best approach to assess kinases activities. However, given the number of kinases that needed to be tested, it appeared to us at that time that it was the best approach. Here, we only privileged p38MAPK and c-Jun as important players in NGF induced mechanical allodynia but not allodynia/hyperalgesia in general. Actually, we even think that ERK and probably mTOR are crucial for NGF induced thermal hyperalgesia since ERK has been shown to modulate TRPV1 activity and mediated NGF induced heat hyperalgesia [START_REF] Zhuang | Phosphatidylinositol 3-Kinase Activates ERK in Primary Sensory Neurons and Mediates Inflammatory Heat Hyperalgesia through TRPV1 Sensitization[END_REF]. Importantly, we also showed previously that levels of pERK were significantly increased in TrkAC DRG neurons in culture 5 and 15 min after NGF stimulation compared to non-stimulated TrkAC neurons. Moreover, ERK activation was similar between TrkAC and wild type neurons, at least, 5 min after NGF stimulation (see Figure S2, [START_REF] Gorokhova | Uncoupling of molecular maturation from peripheral target innervation in nociceptors expressing a chimeric TrkA/TrkC receptor[END_REF]). Thus, it seems that the activation of this pathway is comparable between TrkAC and WT.

Since we did not test this hypothesis here and, it was a bit beyond the scope of the discussion, we did not discussed it.

Nonetheless, the wording "essential" has been down toned and replaced by "probably very important" in the discussion.

Introduction

Mechanical allodynia is the hallmark of several types of chronic pain disorders.

Following tissue injuries or chemical insults, several substances are released within the local tissue environment, ultimately changing the excitability of nociceptors and leading to different painful symptoms including mechanical allodynia. One of the most important substance is the nerve growth factor (NGF), a prototypic neurotrophin, which is critical for the development and maintenance of autonomic and sensory neurons, including nociceptors, in the developing nervous system [START_REF] Denk | Nerve Growth Factor and Pain Mechanisms[END_REF][START_REF] Lewin | Nerve growth factor and nociception: from experimental embryology to new analgesic therapy[END_REF]. In the adult, systemic or local administration of NGF in rodents and human results in profound and long lasting heat and mechanical hypersensitivity by binding mainly to TrkA receptors [START_REF] Petty | The effect of systemically administered recombinant human nerve growth factor in healthy human subjects[END_REF]. By contrast, blocking NGF effects by different strategies prevents the sensitization of nociceptors in several pain models (for review [START_REF] Denk | Nerve Growth Factor and Pain Mechanisms[END_REF][START_REF] Lewin | Nerve growth factor and nociception: from experimental embryology to new analgesic therapy[END_REF][START_REF] Miller | Nerve growth factor blockade for the management of osteoarthritis pain: what can we learn from clinical trials and preclinical models?[END_REF][START_REF] Mizumura | Role of Nerve Growth Factor in Pain[END_REF]). Following TrkA binding, NGF activates various intracellular pathways such as PLCγ/PKC pathway, phosphatidylinositol 3-kinase pathway (PI3-K) [START_REF] York | Role of phosphoinositide 3-kinase and endocytosis in nerve growth factor-induced extracellular signal-regulated kinase activation via Ras and Rap1[END_REF], mitogen-activated protein kinase kinase/mitogen-activated protein kinase pathways (MAPKK/MAPK) including Erks, JNKs, and p38 MAPK [START_REF] Klesse | Trks: signal transduction and intracellular pathways[END_REF][START_REF] Minden | Differential activation of ERK and JNK mitogen-activated protein kinases by Raf-1 and MEKK[END_REF][START_REF] Patapoutian | Trk receptors: mediators of neurotrophin action[END_REF][START_REF] Xing | Nerve growth factor activates extracellular signal-regulated kinase and p38 mitogen-activated protein kinase pathways to stimulate CREB serine 133 phosphorylation[END_REF].

Several of these downstream pathways such as MEK/ERK, PI3K, PKMzeta and PLCγ have been proposed as second messengers of NGF induced mechanical hypersensitivity [START_REF] Kays | Peripheral Synthesis of an Atypical Protein Kinase C Mediates the Enhancement of Excitability and the Development of Mechanical Hyperalgesia Produced by Nerve Growth Factor[END_REF][START_REF] Malik-Hall | Primary afferent nociceptor mechanisms mediating NGF-induced mechanical hyperalgesia[END_REF]. However, the role of NGF in both mechanical allodynia and thermal hyperalgesia has not been systematically assessed in these studies, and the injection of specific inhibitors of these pathways or NGF has been mostly performed into the hindpaw. Thus, the second messengers signalling pathways by which NGF acts specifically to sensitize nociceptors to mechanical stimulation and up regulates mechanically activated currents, still remain elusive. Therefore, it is crucial to better understand the precise mechanisms by which NGF leads to mechanical allodynia since anti-NGF drugs have the potential to make a major impact on chronic pain treatment in the near future but are associated with potential adverse events. Interestingly, we have previously shown that our generated knock-in mouse expressing a chimeric TrkAC receptor, formed by the extracellular part of TrkA and the functional transmembrane and intracellular parts of TrkC, the receptor of neurotrophin 3, seems to exhibit a specific deficit in mechanical allodynia but not thermal hyperalgesia following intraplantar CFA injection [START_REF] Gorokhova | Uncoupling of molecular maturation from peripheral target innervation in nociceptors expressing a chimeric TrkA/TrkC receptor[END_REF]. Importantly, while nociceptors survival and maturation are normal in the TrkAC adult mice, they present a reduction in peripheral, mostly peptidergic fibers, in the skin. However, the expression of both mechanosensory TrkB positive fibers and Ret in large diameter neurons, representing the ''early Ret'' population of rapidly adapting mechanoreceptors, remained unchanged in TrkAC mice [START_REF] Gorokhova | Uncoupling of molecular maturation from peripheral target innervation in nociceptors expressing a chimeric TrkA/TrkC receptor[END_REF]. The aim of our study was to use this particular deficit of mechanical allodynia development in TrkAC mice to investigate and better understand the role of NGF/TrkAactivated intracellular pathways in this phenomenon following somatic and visceral injuries. All experiments were performed in a randomized block design to control variability and avoid any unspecific effects across the experiment. The experimenter was blind regarding the genotype (WT or TrkAC mice) and treatment administered (i.e., NGF, surgery or cyclophosphamide).

Methods

Animals

Pain models

Systemic NGF injection induced pain

Systemic administration of NGF has been shown to induce thermal and long lasting mechanical hypersensitivity [START_REF] Kerr | A role for the TTX-resistant sodium channel Nav 1.8 in NGF-induced hyperalgesia, but not neuropathic pain[END_REF][START_REF] Lewin | Nerve growth factor-induced hyperalgesia in the neonatal and adult rat[END_REF]. Briefly, NGF (2.5S, Sigma Aldrich) was administered by subcutaneous route (0.1mL/10g) at the dose of 1mg/kg. Paw mechanical and thermal thresholds (see below) were assessed 24h before NGF injection and at 4h, 6h, 24h and 1h, 6h, 24h following NGF injection, respectively. n=8 animals in each group (WT + NGF and TrkAC + NGF).

Plantar incisional pain model

The post-operative pain model following plantar incision was used [START_REF] Brennan | Characterization of a rat model of incisional pain[END_REF]. Animals were anesthetized with 2-2.5% isoflurane and the skin of the operated paw was cleaned with povidone iodine 10% (Merck) and placed in the hole of a sterile drape. The glabrous skin and fascia of the left hind paw were incised longitudinally, starting 0.2 cm from the proximal edge of the heel, and the underlying flexor muscle was elevated, divided, and retracted. After haemostasis, the skin overlying the muscle was closed with two sutures of 5.0 polyamide monofilament (Ethicon, Somerville, NJ, USA). Animals were allowed to recover from anaesthesia. n=13-15 animals in each group (WT incision and TrkAC incision).

Cyclophosphamide induced-cystitis

Cystitis was induced by a single intraperitoneal injection of cyclophosphamide (CYP, Leancare Ltd, UK). Animals received either cyclophosphamide (150 mg/kg in 0.9% NaCl) or vehicle (0.9% NaCl) [START_REF] Lai | Anti-vascular endothelial growth factor treatment decreases bladder pain in cyclophosphamide cystitis: a Multidisciplinary Approach to the Study of Chronic Pelvic Pain (MAPP) Research Network animal model study[END_REF]. n=13-15 animals in each group (WT + saline, TrkAC + saline, WT + CYP and TrkAC + CYP).

Capsaicin-induced spontaneous behaviour

Following acclimation in individual acrylic glass boxes, mice were injected with a single intraplantar administration of capsaicin in the hindpaw (1.5μg/20μL/mice, Sigma-Aldrich) as described previously [START_REF] O'brien | Assessment of pain and itch behavior in a mouse model of neurofibromatosis type 1[END_REF][START_REF] Sakurada | The capsaicin test in mice for evaluating tachykinin antagonists in the spinal cord[END_REF]. Spontaneous nocifensive behaviours (i.e. licking, biting, lifting, and flinching) of the injected paw were measured for 5 minutes following capsaicin administration. n=6-7 animals in each group (WT + capsaicin, TrkAC + capsaicin).

Behavioural pain tests

Assessment of paw mechanical allodynia in systemic NGF and the plantar incisional pain models

Paw mechanical sensitivity in NGF and plantar incisional pain models was assessed using von Frey's hairs based on the up-down method developed by Chaplan [START_REF] Chaplan | Quantitative assessment of tactile allodynia in the rat paw[END_REF] and modified by Dixon [START_REF] Dixon | Efficient Analysis of Experimental Observations[END_REF]. The von Frey's hairs used were: 0.02, 0.04, 0.07, 0.16, 0.4, 0.6, 1, 1.4g. Animals were acclimatised for at least 1h in individual clear acrylic cubicles (22 × 16.5 × 14 cm) placed on the top of an elevated wire mesh. The filaments were applied on the hind paw with a 5 min interval between stimuli. Quick withdrawal or licking of the paw to the stimulus was considered a positive response. Threshold values were derived according to the method described by Chaplan [START_REF] Chaplan | Quantitative assessment of tactile allodynia in the rat paw[END_REF]. If the animal showed no response to the highest von Frey hair, the von Frey threshold of 1.4 g was assigned. If the animal showed response to all von Frey hairs, the von Frey threshold of 0.02 g was assigned. The 50% paw mechanical threshold was evaluated before and 2h, 24h and 48h following paw incision, and before and 4h, 6h, 24h following systemic NGF administration.

Assessment of referred mechanical allodynia in the cystitis model

Abdominal mechanical sensitivity in the CYP induced cystitis model was assessed with von Frey's hairs using the up-down method developed by Chaplan [START_REF] Chaplan | Quantitative assessment of tactile allodynia in the rat paw[END_REF] and modified by Dixon [START_REF] Dixon | Efficient Analysis of Experimental Observations[END_REF], von Frey's hairs used were similar as those used for the paw.

Briefly, animals were acclimatised for 1h on an elevated wire mesh in individual clear acrylic cubicles (22 × 16.5 × 14 cm) placed on the top of an elevated wire mesh.

Abdominal stimulation was then realised starting with the middle 0.16g hair and followed by up and down stimulation. Quick withdrawal/licking of the abdomen or biting of the hair to the stimulus was considered a positive response. The 50% abdominal mechanical threshold was determined before and 4h, 24h and 48h following CYP administration.

Assessment of thermal hyperalgesia in systemic NGF and the plantar incisional pain models

Thermal hyperalgesia was assessed in the systemic NGF and plantar incisional pain models using the plantar test as previously described by [START_REF] Hargreaves | A new and sensitive method for measuring thermal nociception in cutaneous hyperalgesia[END_REF]. Each animal was placed in clear acrylic cubicles (22 × 16.5 × 14 cm) on top of a glass floor in a temperature controlled room (~22°C) and allowed to acclimatise for 1h before testing. The hindpaw was stimulated with a radiant heat source (Bioseb) placed under the glass floor.

Withdrawal latencies were averaged over three consecutive tests, at least 10 min apart, in response to the thermal challenge from a calibrated radiant light source (output of 190 mW/cm2). A cut-off of 20s was imposed to prevent any tissue damage. Thermal threshold was assessed 24h before and 2h, 24h and 48h following paw incision and before, 1h, 6h and 24h following NGF administration.

IL-6 ELISA

At two time points of the behavioural pain experiments (i.e., 2h and 48h following plantar incision and 4h and 48h following CYP administration), n=4-5 animals in each group at each time point were terminally anaesthetised using pentobarbitone. The incised paw skin and the bladder for the incisional pain model and the cystitis pain model, respectively, were quickly dissected out and snap frozen in liquid nitrogen. Tissue samples were subsequently homogenized in lysis buffer (137mM NaCl, 20mM TrisHCl, 1% NP40, 10% glycerol, 1mM PMSF, 10µg/mL aprotinin, 1µg/mL leupeptin, 0.5mM sodium vanadate) using a glass homogenizer. Homogenates were then centrifuged at 14 000g for 10min at 4C and supernatants were collected. Whole cell lysates were next titrated to determine their protein concentrations using a BCA Protein Assay kit (Pierce, UK).

Interleukin-6 (IL-6) content was measured using DuoSet Elisa (#DY406, R&D system, Minneapolis, USA). IL-6 content was determined using a quantitative sandwich enzyme immunoassay technique according to the manufacturer's guidelines. Samples were processed in triplicate and values were calculated from the standard sample curve in the linear range. IL-6 contents in the paw skin and bladder were assessed 2h and 48h and 4h and 48h, respectively, after injuries. Fluorescence emission at 510nm was acquired every 2 seconds using a CCD camera and recorded using MetaFluor® software. The ratio of F340/F380 fluorescence intensity was calculated for each cell and averaged for each conditions.

Calcium Imaging

DRG neurons primary culture

The calcium imaging has been done on 4 separate dishes with 2 dishes for each genotype (i.e., WT and TrkAC). During experiments neurons were continuously superfused with Krebs-Ringer solution. Cells were stimulated 5 times with capsaicin (100nM in Krebs-Ringer solution) for 15s with a 4min interval between each stimulation.

At the end of the experiment all excitable cells were identified by the application of high-KCl Krebs-Ringer solution (NaCl 93mM, KCl 50mM, MgCl2 1mM, CaCl2 2mM, D-Glucose 10mM and HEPES 10mM with pH adjusted at 7.4 and osmolarity at 300mOsm) for 1min.

For peak intensity calculation, the ratio value just prior to the start of the stimulus was subtracted from the maximum ratio value within the 80 seconds following capsaicin application or within 60 seconds after KCl application (50mM). Only cells displaying an increase of at least 15% of their peak intensity in response to KCl were included for analysis. Among them, cells were considered as positive to capsaicin if their peak intensity was increased by at least 15% upon the first capsaicin exposure. In total, 257 cells for the WT and 377 cells for the TrkAC conditions have been kept for analysis.

Quantitative PCR of NGF/TrkA signalling pathways in lumbar DRGs following plantar incision or cystitis in WT and TrkAC mice

To investigate changes in the NGF/TrkA signalling pathways in the plantar incision and cystitis models, we performed quantitative PCR (q-PCR) on DRGs 48h after incision or CYP administration. WT and TrkAC mice (n=3-5 per group for the paw incision model and n=4-6 per group for the CYP model) were terminally anaesthetised using pentobarbitone and ipsilateral and contralateral lumbar DRGs (L4, L5, L6 and L6, S1 for the plantar incision and cystitis models, respectively) were quickly dissected out, snap frozen in liquid nitrogen and stored at -80 °C until use. Total RNA was extracted using Quick-RNA_MicroPrep kit (ZymoResearch, Epigenetics, USA) according to the manufacturer's protocol. The concentration of RNA was quantified using Take3 nucleic acid quantification plate (Epoch, BioTek, USA). Equal amounts of RNA from each sample were reverse transcribed in a volume of 20μl to produce cDNA using High Capacity cDNA Reverse Transcription Kit (Applied Biosystems, USA) following the manufacturer's recommendation.

SYBR Green-based detection was carried out on a real-time PCR instrument (RealPlex, Eppendorf, GmBH) with thermal cycler conditions of: 95 °C for 30s, followed by 45 cycles (95 °C for 10s, 59 °C for 15s and 72°C for 15s). In each experiment, samples were assayed in triplicate. Data were analysed using the threshold cycle (Ct) relative quantification method. Relative quantities (RQ) were determined using the equation: RQ = 2 -ΔΔCt . β-Actin was used as housekeeping gene. Forward and reverse primers used are presented in Table 1.

Western immunoblotting

Forty eight hours after paw incision, WT and TrkAC animals (n=10 for each genotype) were terminally anaesthetized with pentobarbitone, ipsilateral and contralateral L4-L5-L6 DRGs were dissected out and snap frozen in liquid nitrogen. The ipsilateral or contralateral DRGs of two animals (i.e. 6 DRGs in total for each side) of the same genotype were pooled together and homogenized in RIPA (RadioImmunoPrecipitation Assay) buffer (50 mM Tris HCl pH 7.5, 150 mM NaCl, 1 mM EDTA, 1% NP-40, 0.1% SDS + 0.5% Deoxycholic acid containing complete protease inhibitor cocktail) using a glass homogenizer. Homogenates were then centrifuged at 14 000g for 10min at 4C and supernatants were collected. DRGs whole cell lysates were next titrated to determine their protein concentrations using a BCA Protein Assay kit (Pierce, UK).

For Western immunoblotting, Laemmli loading buffer was added to the samples and heated at 70°C for 30 min. The DRG homogenates (40µg protein) were separated using 4-20% SDS-PAGE pre-cast polyacrylamide gels. The separated proteins were then transferred to PVDF membranes using Trans-Blot Turbo blotting system (Bio-Rad, USA).

All membrane incubations were performed on a rotating plate. Washes were done in TBS-T (50 mM Tris-HCl and 6 mM NaCl containing 0.1% Tween 20). Blocking of unspecific binding sites was done by incubating the membranes in 5% non-fat dry milk in TBS-T or 5% BSA in TBS-T when is indicated for 1 hour at room temperature. The membranes were then incubated with the primary antibodies against the unphosphorylated form of Akt or p38MAPK (see below, Cell Signalling Technology) diluted in 5% non-fat milk in TBS-T or 5% BSA in TBS-T overnight at 4°C. After washing, the membranes were probed with appropriate Horseradish peroxidase conjugated secondary antibody diluted (1:10,000) with 5% non-fat dry milk in TBS-T or 5% BSA in TBS-T for 1 hour at room temperature. Following this first incubation and revelation, the blots were stripped twice with stripping buffer (0.2 M Glycine, 0.1% SDS, 1% Tween-20; pH 2.0), blocked and reprobed with the primary antibodies against the phosphorylated form of Akt or p38MAPK (see below).

Antibodies against phospho-p38 MAPK (Thr180/Tyr182, 1:2000; catalog #9216) and p38 MAPK (1:1000; catalog #8690) were diluted in 5% BSA in TBS-T. Antibodies against phospho-Akt (ser473, 1:1000; catalog #9271) and total Akt (1:1000; catalog #9272) were diluted in 5% non-fat milk in TBS-T. β-actin (1:5000; Sigma) was used as loading control.

The immunoreactive bands were quantified by densitometry analysis using Bio-Rad imaging software (Chemidoc). Phospho bands of p-Akt and p-p38 MAPK were normalized relative to total Akt and p38 MAPK, respectively, with all bands already normalized to β-actin. Results are expressed as the mean ± SEM of expression levels of contralateral and ipsilateral DRGs for each group (both ipsilateral and contralateral protein samples were run on the same gel).

Retrograde labelling of bladder DRG neurons

A retrograde labelling of DRG neurons specifically innervating the bladder for subsequent immunochemistry was realised one week before saline or CYP administration. Animals were anaesthetized with 2% isoflurane. Following a laparotomy, the bladder was carefully exposed. Eight µL of fluorogold (#39286; Hydroxystilbamidine bis (methanesulfonate), Sigma Aldrich) were very slowly administered in four different injection sites (2µL per site) in the dorsal bladder wall. Great care was taken to not go through the bladder wall. For each injection, the needle was left in the bladder wall for at least 1min to avoid leaking. The injection sites were then wiped and the bladder thoroughly washed with NaCl 0.9%. The bladder was then repositioned in the abdominal cavity and the abdominal muscles wound closed with 5.0 sutures (Ethicon, Somerville, NJ, USA). Wound skin was closed with 3.0 sutures (Ethicon, Somerville, NJ, USA).

Immunohistochemistry

DRG immunohistochemistry

At the end of the behavioural experiments (i.e. 48h after plantar incision or following CYP or saline administration), n=5 animals in each group and genotype (i.e. WT and TrkAC) were terminally anaesthetised using pentobarbitone and quickly perfused transcardially with saline followed by 4% paraformaldehyde (PFA). After perfusion, ipsilateral L4-L5-L6 DRGs for the incisional pain model and L6 DRGs and the bladder for the CYP model were excised, post-fixed 4h for DRGs and 12h for the bladder in the same perfusion fixative, cryoprotected in 30% sucrose in 0.1 M phosphate buffer (PB) for at least 48h at 4C, and then frozen in tissue freezing medium (O.C.T). Transverse DRG sections (10μm) were cut on cryostat, dried for 30 min at room temperature and then stored at -20°C before staining. DRG sections were stained as follow: after 3 washes in PBS containing 0.2% triton X-100 and 1% BSA, sections were incubated overnight at room temperature in a humid chamber with primary antibodies (i.e., ASIC3, CGRP, TrkA) diluted as indicated in Table 2 in antibody diluent (#S2022; Dako). Following 5 washes 5 min each in PBS, sections were then incubated with the appropriate secondary antibodies (Table 2) diluted in the same antibody diluent for 2h at room temperature. Sections were then washed 3 times in PBS and cover-slipped with fluorescent mounting medium (Dako).

Sections were visualised under a Nikon Eclipse Ni-E fluorescent microscope run with Nikon analysis software (NiS element). DRG neurons counts were done by a blind experimenter on L4-L5-L6 DRGs for the post incisional pain model and L6 DRG for the cystitis model (4-6 sections for each animal, n=5 per group and conditions (i.e. paw incision and CYP administration)).

Bladder immunohistochemistry

Bladder cryostat cut sections (20μm) from the different groups were stained with the primary antibody against CGRP (rabbit anti-CGRP, 1:1000, Table 2), as described above for DRG sections, to assess peptidergic innervation. Bladder sections were visualised under a Nikon Eclipse Ni-E fluorescent microscope run with Nikon analysis software (NiS element). The percentage of CGRP staining was automatically quantified in a ROI including all the bladder area (n=5-6) for each animal in each group (n=4-5 per group).

Statistics

Results are expressed as mean ± standard error to mean (S.E.M). A one-way analysis of variance (ANOVA) was used to compare more than two groups, followed by Tukey test for multiple comparisons with repeated measures when appropriate. When treatment and time were compared, a two-way ANOVA was used. When the F value was significant, this was followed by Tukey test for multiple comparisons. For calcium imaging a two-way ANOVA followed by Holm-Sidak multiple comparison test was used. Neuronal DRG counting and q-PCR results have been analysed using a non-parametric Mann-Whitney test. Results were analysed with GraphPad prism 6 software (GraphPad Prism version 6.00 for Windows, GraphPad Software, La Jolla California USA). In all cases, the significance level was set at p<0.05.

Results

Specific lack of mechanical allodynia development in TrkAC mice following somatic and to a lesser extent visceral injuries.

To confirm that the lack of mechanical allodynia development previously observed in TrkAC animals following intraplantar CFA is directly mediated by NGF activation of TrkA receptors, NGF was delivered by subcutaneous route (1mg/kg) in both WT and TrkAC mice. Before NGF injection, 50% mechanical thresholds (i.e., baseline)

were not different between both genotypes (Fig. 1A). As expected, WT mice developed both mechanical allodynia from 4h to 24h (p≤0.0001 for each time point) and thermal hyperalgesia from 1h to 6h (p=0.002 and p=0.0009, respectively), following NGF administration compared to baseline (Fig. 1A&B). By contrast, TrkAC animals did not exhibit mechanical allodynia at any time point compared to WT (p=0.003, p=0.001 and p=0.034 at 4, 6 and 24h respectively). They only displayed significant thermal hyperalgesia from 1h to 6h (p=0.009 and p=0.002 respectively), similarly to WT (Fig.

1A&B).

We also assessed pain behaviours in the plantar incisional model. Before plantar incision, 50% mechanical thresholds (i.e., baseline) were not different between WT and TrkAC mice (Fig. 1C). Following plantar incision, WT animals exhibited mechanical allodynia from 2h to 48h (p=0.008, p=0.003 and p=0.006 at 2, 24 and 48h, respectively), characterized by a significant decrease of 50% mechanical thresholds compared to baseline (Fig. 1C). By contrast, TrkAC mice did not develop mechanical allodynia throughout the whole study with 50% mechanical thresholds similar to their baseline values but different from WT mice (p=0.006, p=0.0002 and p=0.0002 at 2, 24 and 48h respectively; Fig. 1C). Before plantar incision, baseline thermal withdrawal thresholds were not different between both genotypes (Fig. 1D). Following plantar incision, WT animals progressively exhibited significant thermal hyperalgesia from 24h to 48h post-incision characterized by a significant decrease of thermal withdrawal thresholds compared to baseline (p=0.0003, p=0.007 at 24 and 48h respectively; Fig. 1D). TrkAC animals also developed significant thermal hyperalgesia from 24h to 48h post-incision similarly to WT (p=0.025, p=0.042 at 24 and 48h respectively; Fig. 1D).

Finally, we assessed referred mechanical allodynia using abdominal von Frey test following intraperitoneal administration of cyclophosphamide (CYP) or saline in WT and TrkAC mice (Fig. 1E). Before CYP injection, 50% mechanical withdrawal thresholds (i.e., baseline) were not different between all groups and genotypes (WT and TrkAC). Saline administration did not significantly change mechanical thresholds in WT and TrkAC mice (Fig. 1E). However, following CYP administration, WT animals exhibited severe mechanical allodynia from 4h to 48h post-CYP which was characterized by a significant decrease of 50% mechanical threshold compared to WT saline group (p=0.029, p=0.002, p=0.037 at 4, 24 and 48h respectively; Fig. 1E). TrkAC mice treated with CYP also developed significant referred allodynia from 4h to 48h compared to WT saline treated mice, but to a smaller extent than WT CYP, since 50% mechanical threshold of TrkAC CYP mice was significantly different from WT CYP mice 4h and 24h following CYP administration (p=0.04, p=0.003 at 4 and 24h respectively; Fig. 1E).

To verify the development of cystitis, we evaluated bladder histology and cystometry parameters in WT and TrkAC mice following cystitis (see supplementary results). Overall, we did not observe any obvious anatomical difference of the bladder between WT and TrkAC saline treated mice (supplementary Fig. 1A). However, the bladder wall appeared thickened in WT CYP treated mice compared to WT saline group and was associated with partial loss of the urothelium (supplementary Fig. 1A). TrkAC CYP mice presented moderate mucosal ulceration, and mild to moderate oedema and haemorrhage compared to WT CYP group but were still significantly damaged compared to TrkAC and WT saline groups (supplementary Fig. 1A). We did not observed significant differences in the alterations of the cystometry parameters induced by CYP administration between TrkAC and WT mice (supplementary Fig. 1B, C&D and supplementary results for details).

Finally, since we observed this decrease of referred mechanical allodynia in TrkAC mice following cystitis, we assessed more directly mechanical visceral response to colonic distension in naive WT and TrkAC mice. TrkAC mice exhibited a significant decrease in visceromotor response to colonic distension at the highest volumes (i.e., 80 and 100µl) compared to WT mice (p=0.021, p=0.015 at 80 and 100 µl, respectively; supplementary Fig. 2A).

Similar development of inflammation in WT and TrkAC mice following plantar incision or cystitis

As NGF is released following an injury and participates to inflammation through TrkA activation, we assessed paw thickness, bladder weight ratio and IL-6 paw skin and bladder contents following plantar incision and cystitis, respectively. The thickness of the ipsilateral hindpaw, assessed with a vernier caliper, significantly increased compared to the contralateral paw in WT and TrkAC mice in a similar manner 2h and 48h following plantar incision (p≤0.0001 for each time point and each group; Fig. 2A). For the cystitis model, the bladder weight/total weight ratio was similar between WT and TrkAC at 4h and 48h following saline administration (Fig. 2B). CYP injection significantly increased bladder weight in both WT and TrkAC, in a similar manner, at 4h (p=0.0005) and 4h and 48h (p≤0.0001 and p=0.02), respectively, compared to their respective control groups (Fig. 2B).

The increase of IL-6 content in the hindpaw skin and its kinetic were similar between both WT and TrkAC mice with a significant increase compared to the contralateral side from 2h to 48h after plantar incision where IL-6 content was still significantly increased compared to the contralateral paw in both genotypes (p≤0.0001 for both genotypes at 6h; p=0.049 and p=0.016 for WT ipsi and TrkAC ipsi at 48h, respectively; Fig. 2C). We also observed a similar increase of IL-6 bladder content 4h following CYP injection in WT and TrkAC groups compared to their control groups (p=0.024 and p=0.0008, respectively), which reached basal levels 48h after CYP injection (Fig. 2D).

We also assessed CGRP bladder innervation since it has been shown that CGRP staining in the bladder is increased following CYP treatment [START_REF] Gonzalez | The effects of tempol on cyclophosphamide-induced oxidative stress in rat micturition reflexes[END_REF][START_REF] Vizzard | Alterations in neuropeptide expression in lumbosacral bladder pathways following chronic cystitis[END_REF] and we previously demonstrated a decrease of free nerve endings in the skin of TrkAC mice. As expected, CGRP staining was significantly increased in the detrusor as well as the urothelium of WT CYP group compared to WT saline group (lower and upper left panel, respectively, 3A). Taken together, these results suggest that TrkAC mice exhibit a specific decrease of mostly peptidergic free nerve endings compared to WT animals in visceral tissues as we previously described in the thick and thin glabrous skin.

Capsaicin-induced nocifensive behaviour and Ca 2+ imaging response of DRG cells is similar between WT and TrkAC mice

Since a deficit in fine pepdidergic fibers which largely expressed the prototypic heat sensing receptor TRPV1 was observed in TrkAC mice, we assessed nocifensive behavioural responses (i.e., licking, biting, lifting, and flinching of the left hindpaw) following intraplantar capsaicin administration (Fig. 3A). WT and TrkAC mice spent similar time in nocifensive behaviours (55.7 ± 5.5s and 59.8 ± 6s, respectively, p=0.621).

We also measured the [Ca 2+ ]i on cultured DRG neurons in response to capsaicin.

Interestingly, we observed a decrease of the number of capsaicin-responsive cells in

TrkAC mice compared to WT mice (51% vs 33%, Chi2 square value p ≤0.0001, Fig. 3B).

However, the peak intensity of the first calcium response following capsaicin was similar between WT and TrkAC DRG neurons while desensitization and probably tachyphylaxis phenomena following subsequent capsaicin applications seems significantly precocious in TrkAC mice compared to WT mice, at least following the second and third capsaicin applications (p ≤0.0001 and p=0.017, respectively, Fig. 3C&D).

Quantitative PCR of NGF/TrkA signalling pathways in DRGs following plantar incision or CYP administration in WT and TrkAC mice

Since NGF effect on genes expression in the DRG cell bodies is due to the trafficking of the NGF/TrkA complex from the periphery toward the DRG, we first assessed TrkA trafficking along the sciatic nerve 48h after plantar incision. The accumulation of TrkA receptors on the distal part of the ligature of the nerve 48h after plantar incision was similar between WT and TrkAC, suggesting normal trafficking of NGF/TrkA complex along the sciatic nerve in TrkAC mice (supplementary Fig. 3B).

Forty eight hours following plantar incision, we observed a significant increase of Akt, Mapk14 (i.e. p38 MAPK) and Jun mRNA levels in the ipsilateral lumbar DRGs of WT mice compared to the contralateral side (p≤0.0001, Table 3). By contrast, this increase of Akt, Mapk14 and Jun mRNAs levels was absent in the ipsilateral DRGs of TrkAC mice and their expression levels were similar to those observed on the contralateral side as well as the contralateral DRGs of WT mice (Table 3). The expression levels of the others pathways investigated (Erk1, Erk2, Pkc, Pkmzeta and Pka) were not different between the ipsilateral and contralateral DRGs in WT and TrkAC mice as well as between both genotypes (Table 3). Finally, we observed no significant difference of Asic3 mRNA expression in the contralateral DRGs of TrkAC mice compared to the contralateral DRGs of WT mice, indicating similar expression of Asic3 at basal level between TrkAC and WT mice. However, a significant decrease of Asic3 mRNA expression in ipsilateral DRGs of WT mice compared to the contralateral side 48h post incision (p=0.011) was observed.

In addition, we found a significant decrease of Asic3 mRNA level in the ipsilateral DRGs of TrkAC mice compared to the contralateral side and also to the contralateral DRGs of WT mice (p=0.034 and p=0.0002, respectively, Table 3).

By contrast to the plantar incision model, we did not observe any significant changes of Akt, Mapk14 (i.e. p38 MAPK),Jun, and Asic3 mRNA expression in the lumbar DRGs of WT CYP animals compared to WT control 48h following CYP administration (Table 3). The results were similar for the TrkAC mice with no significant increase in TrkAC CYP mice compared to TrkAC saline mice (Table 3).

Expression of phospho-AKT and phospho-P38 MAPK in the ipsilateral and contralateral lumbar DRGs of WT and TrkAC mice following paw incision

Our q-PCR data showed a significant increase of the levels of the transcripts especially encoding Akt and p38 MAPK, only in ipsilateral DRGs of WT mice following plantar incision. We therefore analysed by Western blotting, the expression of the activated form (i.e., phosphorylated) of at least, AKT and P38 MAPK proteins in the contralateral and ipsilateral DRGs of WT and TrkAC mice 48h following plantar incision (Fig. 4A,B,C&D). The expression of p-AKT/total AKT in the DRGs was unchanged between both sides in WT and TrkAC groups and between genotypes (Fig. 4A&B).

However, the expression of p-P38/totalP38 in the DRGs was significantly increased in the ipsilateral DRGs of WT mice compared to the contralateral side and TrkAC ipsilateral side (p=0.004 and p=0.001, respectively). This increase of P38 MAPK was not observed in the TrkAC mice such as found at the transcript level (p=0.815, Fig. 4C&D).

ASIC3, TrkA, CGRP expression in ipsilateral DRGs of WT and TrkAC mice following paw incision or cystitis

Since increase expression of ASIC3 has been observed following plantar incision in rat [START_REF] Deval | Acid-sensing ion channels in postoperative pain[END_REF], NGF is a regulator of Asic3 gene expression through TrkA/JNK/p38 MAPK pathways [START_REF] Mamet | How nerve growth factor drives physiological and inflammatory expressions of acid-sensing ion channel 3 in sensory neurons[END_REF] and, ASIC3 may participate to mechanotransduction phenomenon [START_REF] Lin | Evidence for the involvement of ASIC3 in sensory mechanotransduction in proprioceptors[END_REF][START_REF] Page | Different contributions of ASIC channels 1a, 2, and 3 in gastrointestinal mechanosensory function[END_REF],

we assessed the expression of ASIC3, TrkA and CGRP in ipsilateral L4-L5-L6 DRGs neurons 48h following plantar incision (Fig. 5A,B&C). The representative photomicrographs showed, from left to right, ASIC3, TrkA, CGRP and merge staining in ipsilateral DRGs of WT (upper panel) and TrkAC (lower panel) 48h following plantar incision (Fig. 5A). Arrows on the pictures indicate DRG neurons expressing both ASIC3, TrkA and CGRP which was decreased in TrkAC mice compared to WT. The percentage of TrkA and CGRP positive neurons was similar between WT and TrkAC mice following plantar incision (29.8 ± 1.9% and 32.2 ± 3.2 % in WT vs 32.7 ± 3.1% and 26.7 ± 1% in TrkAC, respectively, Fig. 5B). However, the number of ASIC3 positive neurons was significantly decreased in TrkAC animals compared to WT following plantar incision (68 ± 4.5% vs 79.9 ± 5.1%, respectively, p=0.03 Fig. 5B). Importantly, this decrease of ASIC3 was found in CGRP (WT: 25.9 ± 4% vs TrkAC: 17.1 ± 1.7%, p=0.041) and TrkA/CGRP (WT: 21.1 ± 2.2% vs TrkAC: 14.6 ± 1.7%, p=0.028) positive neurons, respectively (Fig.

5C).

Since increase expression of ASIC3 has been observed following plantar incision, we also assessed its expression along with CGRP and TrkA in L6 DRG neurons 48h following saline or CYP. Representative photomicrographs showed an increase of the number of CGRP positive DRGs neurons specifically innervating the bladder only in WT CYP mice (Fig. 6A). Importantly, ASIC3/CGRP and ASIC3/TrkA/CGRP bladder coexpressing neurons were only increased in WT CYP mice but not in TrkAC CYP mice (Fig. 6A).

The average percentage of fluorogold positive neurons specifically innervating the bladder in L6 DRG was around 34% of the total number of DRG neurons which is like what has been reported previously [START_REF] Vizzard | Alterations in neuropeptide expression in lumbosacral bladder pathways following chronic cystitis[END_REF][START_REF] Vizzard | Increased expression of neuronal nitric oxide synthase (NOS) in visceral neurons after nerve injury[END_REF]. This percentage was similar between WT and TrkAC mice (34 ± 2% and 35 ± 1%) and stayed unchanged following CYP administration, as expected (32.7 ± 2% and 32 ± 1.6% for WT CYP and TrkAC CYP, respectively). Among fluorogold positive neurons (i.e., bladder innervating neurons) a vast majority, around 85%, expressed ASIC3 channels (Fig. 6B), which is similar to what has been observed previously [START_REF] Hughes | Localization and comparative analysis of acid-sensing ion channel (ASIC1, 2, and 3) mRNA expression in mouse colonic sensory neurons within thoracolumbar dorsal root ganglia[END_REF]. This global expression of ASIC3 was the same between WT and TrkAC mice injected with saline and unchanged following CYP administration. Also in fluorogold positive neurons, around 40% of bladder neurons expressed TrkA (Fig. 6B). This expression of TrkA was similar between WT and TrkAC saline mice and stayed unchanged following CYP or saline administration. The percentage of bladder neurons expressing CGRP was not different between WT and TrkA saline control groups (Fig. 6B). However, we observed a significant increase of the number of CGRP positive bladder neurons in WT CYP mice compared to WT saline group (50.5 ± 3% vs 40.5 ± 2.1%, respectively, p=0.037, Fig. 6B). We did not find such an increase in the TrkAC CYP group compared to TrkAC saline group (40.8 ± 6.5% vs 42.1 ± 6%, respectively, p=0.877,

Fig. 6B).

We also observed a significant increase of ASIC3/CGRP positive bladder neurons in WT CYP group compared to WT saline group (41.9 ± 3% vs 28.7 ± 1.2%, respectively, p=0.015, Fig. 6C). There was no increase of this subtype of neurons in the TrkAC CYP group compared to TrkAC saline group (32.4 ± 4.2% vs 32 ± 2.1%, respectively, Fig. 6C) which is also not different from WT saline group. The number of TrkA/CGRP double positive bladder neurons was similar between WT and TrkAC saline mice and was not modified following CYP administration (Fig. 6C). However, we found a significant increase of the number of ASIC3/TrkA/CGRP triple positive bladder neurons in the WT CYP group compared to WT saline group (30.4 ± 6% vs 22.6 ± 2.8%, respectively, p=0.031, Fig. 6C). We did not observe such increase of this type of neurons in the TrkAC CYP group compared to TrkAC saline group (21.3 ± 2.1% vs 22.7 ± 1.9%, respectively, Fig. 6C), which is also not different from WT saline group. TrkAC mice present a specific and profound deficit in mechanical allodynia development after systemic NGF injection and plantar incision. Following cystitis, TrkAC mice exhibited a slight but significant reduction of referred mechanical hypersensitivity compared to WT mice. By contrast, cystometry parameters were similarly decreased in both genotypes following CYP administration as described previously in WT mice [START_REF] Silva | Spinal blockage of P/Q-or N-type voltagegated calcium channels modulates functional and symptomatic changes related to haemorrhagic cystitis in mice[END_REF][START_REF] Studeny | Urinary bladder function and somatic sensitivity in vasoactive intestinal polypeptide (VIP)-/-mice[END_REF][START_REF] Wang | Effects of low energy shock wave therapy on inflammatory moleculars, bladder pain, and bladder function in a rat cystitis model[END_REF]. Moreover, colonic mechanical sensitivity assessed by colorectal distension was impaired in TrkAC mice compared to WT mice. Alterations in the development of peripheral inflammation cannot explain the mechanical allodynia deficit in TrkAC mice since a similar increase of different inflammatory parameters following somatic or visceral injuries was observed between TrkAC and WT mice as described previously [START_REF] Banik | Increased nerve growth factor after rat plantar incision contributes to guarding behavior and heat hyperalgesia[END_REF][START_REF] Coelho | Urinary bladder inflammation induces changes in urothelial nerve growth factor and TRPV1 channels[END_REF][START_REF] Guerios | Nerve growth factor mediates peripheral mechanical hypersensitivity that accompanies experimental cystitis in mice[END_REF][START_REF] Sahbaie | Role of substance P signaling in enhanced nociceptive sensitization and local cytokine production after incision[END_REF]. However, a decrease in the density of CGRP-positive fibres in the bladder and the colon of TrkAC mice, as well as an absence of an increase of CGRP bladder staining following CYP in TrkAC mice compared to WT was observed, suggesting a specific reduction in peripheral, mostly peptidergic, innervation in visceral tissues similar to what we have previously described in the skin [START_REF] Gorokhova | Uncoupling of molecular maturation from peripheral target innervation in nociceptors expressing a chimeric TrkA/TrkC receptor[END_REF]. Thus, TrkAC mice present specific developmental target innervation defects of mostly peptidergic terminal branches in both somatic and visceral tissues, well known to be highly dependent on NGF [START_REF] Denk | Nerve Growth Factor and Pain Mechanisms[END_REF][START_REF] Lewin | Nerve growth factor and nociception: from experimental embryology to new analgesic therapy[END_REF][START_REF] Mizumura | Role of Nerve Growth Factor in Pain[END_REF]. Therefore, an obvious explanation for the specific loss of mechanical allodynia in TrkAC mice could be this peripheral innervation defect.

Discussion

However, these peptidergic fibres, expressing mainly TRPV1, are dispensable for mechanically-evoked behaviours, but are essential for behaviours evoked by noxious heat [START_REF] Cavanaugh | Distinct subsets of unmyelinated primary sensory fibers mediate behavioral responses to noxious thermal and mechanical stimuli[END_REF]. Indeed, NGF-induced heat hyperalgesia is absent in TRPV1 knock-out mice or after TRPV1 blockade [START_REF] Caterina | Impaired nociception and pain sensation in mice lacking the capsaicin receptor[END_REF]. Nonetheless, thermal hyperalgesia was totally preserved in TrkAC mice at baseline and following systemic NGF administration or plantar incision. TrkAC mice could rather display a functional deficit of TRPV1. However, basal TRPV1 expression in lumbar DRG is unaltered in TrkAC mice [START_REF] Gorokhova | Uncoupling of molecular maturation from peripheral target innervation in nociceptors expressing a chimeric TrkA/TrkC receptor[END_REF], and they exhibit similar nocifensive behavioural responses following capsaicin and calcium imaging DRGs neuronal response after the first capsaicin application compared to WT mice.

Finally, despite significant reduction in the density of epidermal neuritis including peptidergic fibres, mice lacking p75 NTR , the pan receptor of neurotrophins, still developed mechanical and thermal hyperalgesia after systemic NGF delivery while they exhibited a decrease in basal mechanical and thermal thresholds [START_REF] Bergmann | Analysis of cutaneous sensory neurons in transgenic mice lacking the low affinity neurotrophin receptor p75[END_REF][START_REF] Bergmann | Nerve growth factor evokes hyperalgesia in mice lacking the low-affinity neurotrophin receptor p75[END_REF]. By contrast, TrkAC mice did not display any changes in basal mechanical and thermal thresholds despite reduction in the density of epidermal and visceral innervation but only a specific loss of mechanical allodynia development following somatic and, to a lesser extent, visceral injuries. We have also previously shown that the expression of some subtypes of important peripheral mechanosensory fibres (TrkB and early Ret positive fibres) were unaffected in TrkAC mice. Therefore, this marked decrease in peripheral innervation cannot explain by its own the lack of mechanical allodynia development in TrkAC mice, especially following somatic injury.

Thus, another possibility is that the remaining primary afferents fibres, important in the development of mechanical allodynia, present a specific functional silencing due to the absence of TrkA specific pathways. By contrast, an increase activation of TrkC specific pathways and/or TrkA and TrkC intracellular signalling pathways largely overlap, to mediate thermal hyperalgesia. Alternatively, it has been shown that acute noxious heat sensing in mice depends on a triad of transient receptor potential ion channels (TRPM3, TRPV1 and TRPA1), which are not expressed solely in TrkA positive neurons [START_REF] Usoskin | Unbiased classification of sensory neuron types by large-scale single-cell RNA sequencing[END_REF][START_REF] Vandewauw | A TRP channel trio mediates acute noxious heat sensing[END_REF]. Consequently, we assessed the mRNA expression of several common and specific TrkA/TrkC intracellular transduction pathways in the DRGs after plantar incision or CYP in WT and TrkAC mice. The basal mRNA expression of all the pathways investigated were similar between both genotypes, suggesting that the expression of TrkAC did not modified significantly the overall NGF/TrkA signalling of DRG cells in basal conditions. However, following plantar incision, there was a marked decrease of the up-regulation of Akt, p38 MAPK and c-Jun mRNA expression in the ipsilateral DRGs of TrkAC mice compared to WT. At the protein level, we also demonstrated an absence of increased phosphorylation of p38 MAPK in TrkAC mice compared to WT. Interestingly, the mRNA expression of the same pathways was not modified following CYP in WT mice as well as TrkAC mice, suggesting that these pathways are mainly involved in the development of somatic mechanical allodynia. These changes of some pathways, such as p38 MAPK, between WT and TrkAC mice may be due to few proteins, which bind differentially to the intracellular domains of TrkA and TrkC receptors [START_REF] Kato | Synectin in the nervous system: expression pattern and potential as a binding partner of neurotrophin receptors[END_REF][START_REF] Mortimer | Axon guidance by growth-rate modulation[END_REF][START_REF] Nakamura | Grit, a GTPase-activating protein for the Rho family, regulates neurite extension through association with the TrkA receptor and N-Shc and CrkL/Crk adapter molecules[END_REF], leading to the modulation of distinct downstream pathways. Alternatively, structural differences between TrkA and TrkAC receptors could lead to different activation levels of downstream effectors [START_REF] Markus | Raf and akt mediate distinct aspects of sensory axon growth[END_REF]. Some of these pathways (i.e, PI3K, p38 MAPK) have been already involved in the development of NGF induced hyperalgesia but typically following intraplantar administration of the specific inhibitors of these pathways and tested either on thermal or mechanical hyperalgesia [START_REF] Zhuang | Phosphatidylinositol 3-Kinase Activates ERK in Primary Sensory Neurons and Mediates Inflammatory Heat Hyperalgesia through TRPV1 Sensitization[END_REF]. Thus, changes of a set of intracellular pathways including p38 MAPK and probably c-Jun could be specifically involved in NGF-induced mechanical allodynia. Overall, changes of intracellular pathways and probably, genes expression in the DRG, seem essential for the development of mechanical allodynia induced by NGF while thermal hyperalgesia could be mainly due to peripheral sensitization of heat transducers in primary afferent terminals.

The expression/function of the mechanostransducer(s) involved in the development of mechanical allodynia at the periphery is largely dependent on NGF, and several candidates have been proposed [START_REF] Castro | Modulation of sensory neuron mechanotransduction by PKC-and nerve growth factor-dependent pathways[END_REF]. ASIC3, a proton gated channel, seems to be an important contributor of the nociceptor mechanosensory function and, importantly, its expression is regulated by NGF and particularly TrkA transduction pathways [START_REF] Mamet | How nerve growth factor drives physiological and inflammatory expressions of acid-sensing ion channel 3 in sensory neurons[END_REF]. Interestingly, we found a decrease in the up-regulation of ASIC3 protein expression in TrkA-positive DRGs neurons in TrkAC mice following both somatic and visceral injuries. At the mRNA level, we observed a decrease of Asic3 expression in the ipsilateral DRG of WT mice only following paw incision, which could indicate increased translation. A further decrease of Asic3 mRNA in the ipsilateral DRG of TrkAC mice was also found. Despite an increase translation of Asic3 in TrkAC mice such as in WT, the level of Asic3 mRNA in the ipsilateral DRG could be too low to observe a significant increase of ASIC3 protein expression. This decrease of ASIC3 expression could partially explain the lack of mechanical allodynia development in TrkAC mice, but we cannot exclude the involvement of others mechanotransducers.

Nonetheless, ASIC3 has been already involved in mechanical hypersensitivity in several types of somatic pain models [START_REF] Deval | Acid-Sensing Ion Channels in Postoperative Pain[END_REF][START_REF] Deval | ASIC3, a sensor of acidic and primary inflammatory pain[END_REF][START_REF] Rudan | Prevalence of rheumatoid arthritis in low-and middle-income countries: A systematic review and analysis[END_REF][START_REF] Walder | ASIC1 and ASIC3 Play Different Roles in the Development of Hyperalgesia Following Inflammatory Muscle Injury[END_REF], and recently, Zhou et al. [START_REF] Zhou | Inhibition of Nerve Growth Factor Signaling Alleviates Repeated Dural Stimulation-induced Hyperalgesia in Rats[END_REF] demonstrated that NGF-induced hyperalgesia following repeated dural infusions of an inflammatory soup, seems dependent on ASIC3 expression in the trigeminal nucleus caudalis. Several studies also support an important contribution of ASIC3 in visceromechanosensitivity but also TRPV1, contrasting to somatic mechanosensitivity [START_REF] Hughes | Localization and comparative analysis of acid-sensing ion channel (ASIC1, 2, and 3) mRNA expression in mouse colonic sensory neurons within thoracolumbar dorsal root ganglia[END_REF][START_REF] Kato | Synectin in the nervous system: expression pattern and potential as a binding partner of neurotrophin receptors[END_REF][START_REF] Page | Different contributions of ASIC channels 1a, 2, and 3 in gastrointestinal mechanosensory function[END_REF]. This differential contribution of TRPV1 between visceral and somatic mechanosensitivities could explain the partial decrease of referred mechanical allodynia in TrkAC mice following CYP. Finally, Mamet et al., demonstrated that ASIC3 overexpression by NGF was dependent on the cooperation of JNK (c-Jun)/p38 MAPK pathways [START_REF] Mamet | How nerve growth factor drives physiological and inflammatory expressions of acid-sensing ion channel 3 in sensory neurons[END_REF]. Strikingly, we observed a decreased expression of these two specific pathways in the DRGs of TrkAC mice associated with a decrease of ASIC3 expression, which could finally lead to mechanical allodynia deficit in chronic pain states especially following somatic injuries. Mechanotransduction is a complex phenomenon especially in mechanical allodynia. Consequently, others contributors of the nociceptor mechanosensory function could be also at play. Thus, we have demonstrated a specific deficit of mechanical allodynia development in TrkAC mice following somatic and to a lesser extent, visceral injuries. This deficit does not seem to be due to the peripheral innervation defects in the skin and visceral tissues of TrkAC mice but rather an alteration of TrkA specific transduction pathways. Changes in the expression of different transduction pathways, especially p38 MAPK, were observed, which could ultimately lead to a specific deficit of mechanical allodynia development through a decrease of, at least, ASIC3 expression, especially after somatic injury. Further studies are needed to better clarify this differential role of TrkA specific transduction pathways in mechanical allodynia and thermal hyperalgesia, where the TrkAC mice could be a very valuable tool.

Figures legends

  TrkAC knock-in animals and WT-littermates from C57BL/6J background were bred in our animal facility and housed in a controlled room with a 12-hour light/dark cycle, with free access to food and water. All procedures were approved by the local ethical committee (Comité Régional d'éthique en matière d'expérimentation animale Auvergne (CEMEA-Auvergne)) and in accordance with European Communities Council Directive for the care of laboratory animals (86/609/EEC). Experiments followed guidelines of the International Association for the Study of Pain and ARRIVE [41,57]. Only male TrkAC and WT-littermates (10-12 weeks old) have been used for somatic pain models (systemic NGF injection induced pain and plantar incisional pain model) except for intraplantar capsaicin where both female/male TrkAC and WTlittermates were used. Only female TrkAC and WT-littermates (10-12 weeks old) have been used for the visceral pain model induced by cyclophosphamide administration and both male and female for colorectal distension.

  Dorsal root ganglia from 12 weeks old TrkAC knock-in and WT littermate mice (n=1 per genotype) were quickly collected in ice-cold Hanks' balanced salt solution (HBSS) and incubated 45min at 37°C in 5% CO2 in a digestion enzyme mix containing: Collagenase type 3 (5mg/mL), Dispase (10mg/mL), Glucose 10mM and HEPES 5mM in Ca 2+ and Mg 2 free HBSS. After dissociation, DRGs were transferred in a tube containing Dulbecco's modified Eagle's medium (DMEM) supplemented with heat-inactivated Fetal Bovine Serum 10%, L-Glutamine 2mM, Minimum Essential Vitamins solution 1%, Penicillin/Streptomycin solution 1%, Sodium Pyruvate 1mM and Non-essential amino acids solution 1%. Ganglia were gently triturated using fire-polished glass Pasteur pipettes. After centrifugation, cells were resuspended in supplemented DMEM and plated on poly-L-lysine and laminin-coated glass coverslips. Neurons were kept at 37°C in 5% CO2 and used for calcium imaging experiments within 2 days following culture. Imaging DRG neurons were washed twice with DMEM and incubated for 45min at 37°C under gentle shaking in DMEM containing Fura-2 AM 4µM and Pluronic Acid 0.04%. DRG neurons were then washed twice in Krebs-Ringer solution containing NaCl 140mM, KCl 3mM, MgCl2 1mM, CaCl2 2mM, D-Glucose 10mM and HEPES 10mM with pH adjusted at 7.4 and osmolarity at 300mOsm. Coverslips were mounted on the recording chamber and placed on the platform of an inverted microscope (Zeiss). Intracellular calcium changes were recorded at x10 magnification by alternatively illuminating cells with 340nm and 380nm LED light sources with an exposure time of 200ms for each wavelength.

Fig

  Fig. 2E & inset). However, TrkAC saline mice already presented a significant decrease

  Here, we have confirmed and extended the specific lack of mechanical allodynia development in TrkAC mice following somatic and, to a lesser extent, visceral injuries. Thermal hyperalgesia development was unaffected, and despite a deficit of mostly peptidergic fibers innervation in somatic and visceral tissues, TrkAC and WT mice exhibited similar pain behaviours and calcium imaging response of DRG neurons following capsaicin. This specific loss of mechanical allodynia development in TrkAC mice was not associated with changes in different inflammatory parameters and NGF/TrkA trafficking. However, alterations in the expression/function of mainly two TrkA specific intracellular pathways (p38 MAPK and c-Jun) in the DRGs of TrkAC mice following plantar incision were observed, which ultimately lead to a reduction of, at least, ASIC3 channel expression in TrkA peptidergic neurons. Taken together, these results suggest that these two specific TrkA intracellular pathways, especially p38 MAPK, are probably very important for the development of mechanical allodynia at least following somatic injury and that ASIC3 might be a crucial player in both somatic and visceral pain conditions mediated by NGF/TrkA.

Figure 1 :

 1 Figure 1: Specific lack of mechanical allodynia in TrkAC mice especially

Figure 2 :

 2 Figure 2: No difference in inflammatory reaction following injuries between

Figure 3 :

 3 Figure 3: No changes in behavioural and Ca 2+ imaging responses of DRG

Figure 4 :

 4 Figure 4: Lack of increase of p-P38MAPK expression in the ipsilateral DRG of

Figure 5 :

 5 Figure 5: Decrease of ASIC3/TrkA/CGRP positive DRG neurons in TrkAC mice

Figure 6 :

 6 Figure 6: Lack of increase of ASIC3/TrkA/CGRP positive DRG neurons

(

  A) From left to right, representative photomicrographs of fluorogold-positive neurons retrogradely labelled from the bladder wall (blue), ASIC3 (green), CGRP (grey) and TrkA (red) positive neurons in L6 DRG, from top to bottom, of WT vehicle, TrkAC vehicle, WT CYP and TrkAC CYP mice. Merge image (right) shows ASIC3-positive neurons expressing or not expressing TrkA and CGRP. (B) Percentage of fluorogold positive neurons expressing ASIC3, TrkA and CGRP in WT and TrkAC mice 48h following saline or CYP. (C) Percentage of double or triple labelled fluorogold positive neurons of L6 DRG in WT and TrkAC mice. Results are expressed as a percentage of fluorogold positive neurons. 4-6 sections for each animal, n=5 per group (i.e., WT saline, TrkAC saline, WT CYP and TrkAC CYP). Non parametric Mann-Whitney test. * p ≤ 0.05 compared to WT saline treated animals.

Table 1 :

 1 List of primer sequences used for the q-PCR experiments.

Table 2 :

 2 List of primary and secondary antibodies, dilutions and manufacturers used for the immunohistochemistry experiments.

Table 3 :

 3 Figure1

Figure 2 Figure 3

 23 Figure 2

Figure 2 Figure 3

 23 Figure 4

  Figure 4

  Figure 5

Table 1

 1

Table 2

 2

	Antibodies	Dilution	Manufacturer
	Guinea pig anti-ASIC3	1/200	#AB5927, Merck Millipore (France)
	Rabbit anti-TrkA	1/2000	Generous gift from Dr Reichardt
	Mouse anti-CGRP	1/1000	#S57053, Santa Cruz (USA)
	Rabbit anti-CGRP	1/1000	#PC205, Calbiochem (France)
	Rabbit anti PGP 9.5	1/400	#E3340, Spring Bioscience (USA)
	Goat anti guinea pig-488	1/1000	Alexa fluor (Molecular Probes, USA)
	Goat anti rabbit-546	1/1000	Alexa fluor (Molecular Probes, USA)
	Donkey anti mouse-647	1/1000	Alexa fluor (Molecular Probes, USA)

Table 2

 2

Table 3

 3

	Incision	WT ipsi	WT contra	TrkAC ipsi	TrkAC contra
	Akt1	2.83 ± 0.19*	1.06 ± 0.19	0.69 ± 0.12	0.51 ± 0.04
	Mapk14 (P38)	3.35 ± 0.08*	1.05 ± 0.16	0.95 ± 0.14	0.93 ± 0.05
	Mapk3 (Erk1)	2.69 ± 0.77	1.07 ± 0.2	0.97 ± 0.12	0.97 ± 0.09
	Mapk1 (Erk2)	1.07 ± 0.06	1.15 ± 0.29	1.22 ± 0.08	0.95 ± 0.08
	Jun	8.74 ± 1.04*	1.49 ± 0.74	1.94 ± 0.33	1.47 ± 0.10
	Prkca (Pkc)	0.93 ± 0.01	1.01 ± 0.10	1.08 ± 0.10	0.97 ± 0.06
	Prkcz (Pkmz)	1.05 ± 0.05	1.09 ± 0.07	1.10 ± 0.09	1.10 ± 0.11
	Prkaca (Pka)	1.04 ± 0.31	1.41 ± 0.32	1.42 ± 0.25	1.88 ± 0.21
	Accn3	0.67 ± 0.07*	1.01 ± 0.06	0.47 ± 0.02*°	0.76 ± 0.06
	Cystitis	WT	WT CYP	TrkAC	TrkAC CYP
	Akt1	1.08 ± 0.17	0.69 ± 0.12	0.88 ± 0.05	0.81 ± 0.10
	Mapk14 (P38)	1.04 ± 0.15	0.63 ± 0.1	0.78 ± 0.05	0.73 ± 0.11
	Jun	1.37 ± 0.38	0.60 ± 0.10	0.74 ± 0.04	0.90 ± 0.34
	Accn3	1.01 ± 0.1	0.89 ± 0.13	0.55 ± 0.07	0.58 ± 0.16

Table 3 B C

 3C

		WT	TrkAC	WT CYP	TrkAC CYP
	Maximum bladder
	pressure	42.7 ± 2.4	40.4 ± 8.3	43.7 ± 6.3	35.5 ± 3.9
	(cmH 2 O)
	Contraction threshold
	(cmH 2 O)	24.2 ± 1.5	22.5 ± 5.8	19.0 ± 3.4	20.2 ± 2.0

Resting pressure (cmH 2 O) 16.4 ± 1.8 15.5 ± 4.8 12.7 ± 3.8 14.9 ± 2.1 Contraction amplitude (cmH 2 O) 18.6 ± 1.3 17.9 ± 2.6 24.7 ± 2.8 15.3 ± 2.3

Acknowledgements

This work was supported by Inserm, Conseil Régional Auvergne-Rhône-Alpes (project Ressourcement S3, Arth-Innov) and Feder as well as the French government IDEX-ISITE initiative 16-IDEX-0001 (CAP 20-25).

.

Declaration of Interests

All authors have read and agreed to the contents of the manuscript and have no competing financial interests.

Supplementary legends

Supplementary methods

Supplementary Figure 1 Bladder histology

In supplementary Fig. 1A, bladder sections from each group (CYP or saline)

and genotype (WT or TrkAC), were stained with Hematoxilin/Eosin for histology.

Representative photomicrographs of bladder sections were taken using a Zeiss Scope A1 microscope running Axiovision 4.8 image analysis software.

Cystometry

In supplementary Fig. 1B, forty eight hours after CYP or saline administration in WT and TrkAC mice, animals were anesthetized with a mixture of xylazine hydrochloride (Rompun ® , 0.4 mg/kg) and ketamine (Imalgene 500 ® , 100 mg/kg) and placed in a supine position. Abdominal skin was shaved, cleaned with povidone iodine 10% (Merck) and the abdomen carefully opened to expose the bladder. A PE10 catheter with a collar was then inserted through the apex of the bladder and secured with 5.0 sutures (Ethicon, Somerville, NJ, USA). The bladder was then emptied.

Perfusion of NaCl 0.9% was realized at the rate of 20µL/min. After bladder instillation started, the system was allowed to stabilize to achieve a stable contraction/voiding pattern prior to data collection for analysis. Recording of intra-bladder pressure was then realized for at least 1h with a pressure transducer (Harvard Apparatus, USA) connected to a catheter with a three way tap with the fluid filled pressure at one end, and injection pumps (Harvard Apparatus, USA) to the other.

Supplementary Figure 2

Assessment of colonic sensitivity using colorectal distension

In supplementary Fig. 2A

2B.

Following colorectal distension, the remaining 4 WT and 4 TrkAC mice were also terminally anaesthetised using pentobarbitone and caecum, colon and spleen were freshly dissected out and weighted. Total length of the colon was also measured.

There was no significant difference between WT and TrkAC mice in these different anatomical parameters (data not shown).

Supplementary Figure 3

Assessment of TrkA trafficking

To assess if NGF/TrkA complex trafficking from the periphery toward the DRG cell bodies was not affected in TrkAC mice, at the same time at the left hind paw incision was made, we carefully exposed the left sciatic nerve and tightly ligated the nerve with one 5.0 suture in 2 WT and 2 TrkAC mice. 48h after the surgery, mice were terminally anaesthetised using pentobarbitone and quickly perfused transcardially with NaCl 0.9% followed by 4% PFA. After perfusion, the left sciatic nerve were excised, post-fixed for 4h in the same perfusion fixative, cryoprotected in 30% sucrose in 0.

Spinal cord immunohistochemistry

Two animals of each genotype (i.e. WT and TrkAC) were terminally anaesthetised using pentobarbitone and quickly perfused transcardially with saline followed by 4% paraformaldehyde (PFA). After perfusion, lumbar spinal cord were excised, post-fixed 12h in the same perfusion fixative, cryoprotected in 30% sucrose in 0.1 M phosphate buffer (PB) for at least 48h at 4C, and then frozen in tissue freezing medium (O.C.T). Transverse spinal cord sections (20μm) were cut on cryostat, dried for 30 min at room temperature and then stored at -20°C before staining.

Spinal cord sections were stained as follow: after 3 washes in PBS containing 0.2% triton X-100 and 1% BSA, sections were incubated overnight at room temperature in a humid chamber with the following primaries antibodies: rabbit anti-CGRP (1/1000, Calbiochem) and Isolectin B4-FITC (1/1000, L2895, Sigma-Aldrich) in antibody diluent (#S2022; Dako). Following 5 washes 5 min each in PBS, sections were then incubated with the secondary antibody for CGRP (1:1000, goat anti rabbit Alexa fluor 546) diluted in the same antibody diluent for 2h at room temperature.

Sections were then washed 3 times in PBS and cover-slipped with fluorescent mounting medium (Dako). Sections were visualised under a Nikon Eclipse Ni-E


Kevin Moreau

Aurélia Le Dantec

A Rachid Rahmouni
email: rahmouni@cnrs-orleans.fr

Kévin Moreau

Deciphering the Dynamic Landscape of Transcription-Associated mRNP Quality Control Components

Keywords: mRNP biogenesis, mRNP quality control, ncRNAs, yeast, RNA-seq, ChIP-seq

HAL is

1-Introduction

In the nucleus of eukaryotic cells, nascent pre-mRNA molecules are sequentially coated with a large set of processing and binding proteins that mediate their transformation into export-competent ribonucleoprotein particles (mRNPs) ready for translation in the cytoplasm. Genetic and biochemical studies, using the yeast Saccharomyces cerevisiae as a model organism, have suggested that the various mRNA processing and packaging reactions, known as mRNP biogenesis, are physically and functionally coupled to transcription elongation by RNA polymerase II (RNAP II). The co-transcriptional maturation and assembly of export-competent mRNPs is facilitated by the C-terminal domain (CTD) of the largest subunit of RNAP II that serves as a platform for sequential recruitment of various acting factors [START_REF] Bentley | Coupling mRNA processing with transcription in time and space[END_REF][START_REF] Luna | Biogenesis of mRNPs: integrating different processes in the eukaryotic nucleus[END_REF].

The production of mature export-competent mRNPs is also under the surveillance of quality control (QC) steps that are interconnected with transcription elongation and mRNP biogenesis. Defective mRNPs resulting from suboptimal processing and packaging reactions are targeted by the surveillance system leading to their retention in the nucleus with ensuing elimination of their mRNA component by the RNA degradation machinery [START_REF] Doma | RNA quality control in eukaryotes[END_REF][START_REF] Fasken | Process or perish: quality control in mRNA biogenesis[END_REF][START_REF] Schmid | Transcription-associated quality control of mRNP[END_REF].

A decade of extensive investigations using yeast mutants with defects in the THO-Sub2 complex, which mediates the assembly and export of a subset of mRNPs, allowed the identification of the nuclear degradation machinery involved in the mRNP QC process [START_REF] Rougemaille | Dissecting mechanisms of nuclear mRNA surveillance in THO/sub2 complex mutants[END_REF][START_REF] Villa | Nuclear quality control of RNA polymerase II ribonucleoproteins in yeast: Tilting the balance to shape the transcriptome[END_REF]. It was shown that the mechanism of nuclear retention and degradation of defective mRNPs depends on the presence of the nucleus specific exosome-associated exonuclease Rrp6 and its cofactor Rrp47 as well as components of the exosome activating complex TRAMP (Trf4-Air2-Mtr4). However, these analyses were unable to provide an integrated view of the QC system especially regarding the co-transcriptional recognition of mRNP aberrancies and its role in the subsequent triggering of mRNA decay. [Figure 1 near here] The difficulty to decipher the actual mechanism of action underlying the QC process resides in the scarcity of mRNP biogenesis defects occurring under physiological context. In effect, the putative frequency of mRNP aberrancies occurring under physiological conditions does not allow a direct study of the QC mechanism within the cellular context. To circumvent this limitation, we recently implemented a new experimental approach based on global perturbation of mRNP biogenesis by the RNA-dependent helicase/translocase activity of the bacterial Rho factor. The heterologous expression of Rho in yeast induces a dose-dependent growth defect that stems from its interference in the co-transcriptional recruitment of mRNA processing and packaging factors yielding mRNPs that are recognized as aberrant and eliminated by the nuclear QC system [START_REF] Mosrin-Huaman | Expression of bacterial Rho factor in yeast identifies new factors involved in the functional interplay between transcription and mRNP biogenesis[END_REF]. In this approach, sufficient amounts of defective mRNPs can be produced by Rho action in the cell nucleus to serve as substrates for the QC, which facilitates deep investigations of the surveillance process [START_REF] Mosrin-Huaman | Bacterial Rho helicase: a new tool to dissect mRNP biogenesis and quality control in yeast[END_REF]. Using this methodology, we have shown that the QC process is coordinated by Nrd1, a component of the NNS (Nrd1-Nab3-Sen1) complex known to mediate termination and processing of the non-coding RNAs (ncRNAs) and which is recruited by the CTD of RNAP II through its CTD-interacting domain (CID) [START_REF] Vasiljeva | The Nrd1-Nab3-Sen1 termination complex interacts with the Ser5-phosphorylated RNA polymerase II C-terminal domain[END_REF]. The Rho-mediated production of aberrant mRNPs stimulates the recruitment of Nrd1 with its partner Nab3 to the transcription complex, which promotes the attachment of Rrp6 along with its co-activating components Rrp47 and Trf4 [START_REF] Honorine | Nuclear mRNA quality control in yeast is mediated by Nrd1 co-transcriptional recruitment, as revealed by the targeting of Rho-induced aberrant transcripts[END_REF][START_REF] Stuparevic | Cotranscriptional recruitment of RNA exosome cofactors Rrp47p and Mpp6p and two distinct Trf-Air-Mtr4 polyadenylation (TRAMP) complexes assists the exonuclease Rrp6p in the targeting and degradation of an aberrant messenger ribonucleoprotein particle (mRNP) in yeast[END_REF].

In this chapter, we provide a full description of our Rho-based experimental approach and outline its use to investigate the mRNP QC activities over the whole yeast genome. High-throughput RNA sequencing (RNA-seq) was used to survey a broad collection of mRNPs whose biogenesis is affected by Rho action (Figure 1). This genome-wide perspective of affected mRNPs was extended by generating highresolution maps (ChIP-seq) of the distribution of the QC components along the yeast chromosomes before and after perturbation of mRNP biogenesis by Rho. As illustrated in Figure 2, the perturbation of mRNP biogenesis redistributes the binding of QC components over the genome with a large shift of Nrd1 and Nab3 from genomic loci producing ncRNAs prone to processing and decay such as CUTs (Cryptic Unstable Transcripts), SUTs (Stable Unannotated Transcripts) and snoRNAs (small nucleolar RNAs) to protein-coding genes. Nrd1 and Nab3 are apparently titrated out from ncRNAs genomic features by a large recruitment to Rho-affected mRNA gene loci, leading to transcriptional termination defects of ncRNAs [START_REF] Moreau | Perturbation of mRNP biogenesis reveals a dynamic landscape of the Rrp6-dependent surveillance machinery trafficking along the yeast genome[END_REF]. This approach can be adapted to any protein potentially involved at the chromatin level in mRNA surveillance and/or processing. It can also be extended to the study of ncRNAs whose processing relies on the same machineries as mRNA.

[Figure 2 near here] 3. Liquid media for the growth of yeast cells, prepared from a mix of 3 different powders. For 900 ml of media, weigh 18 g of glucose, 6.03 g of YNB (Yeast Nitrogen Base) containing ammonium sulfate and 0.67 g of CSM -Trp (Complete Synthetic Medium without Tryptophan) dissolved in 900 ml of distilled water (see Note 2). After dissolution, dispatch into 3 portions of 300 ml in 500 ml bottles and autoclave. Store the autoclaved media for up to 6 months at room temperature.

2-Materials

Yeast strains, plasmids and growth media

4. 1,000x stock solution of Doxycycline at 5 mg / ml. Dissolve 125 mg of doxycycline in 25 ml of 50% ethanol/water (v/v), store at -20°C. The Tet promoter is repressed by addition of doxycycline to the growth medium at a final concentration of 5 µg/ml just before use.

Specific instruments and labware:

-Precision balance for weighting the powder for media preparation -Vortex for homogenization of solution -Magnetic plate for agitation (with heating function) -Centrifuge for pelleting the yeast cells when needed -Glass or plastic flasks for cell growth -Optical density measurement device to monitor cell growth -Rotary shaker (250-300 rpm) for cell growth at controlled temperature. -Dedicated set of pipettes and special RNase free tips.

RNA extraction solutions

-Fume hood to avoid inhaling phenol:chloroform vapes.

-Nanodrop spectrophotometer or equivalent.

Chromatin ImmunoPrecipitation (ChIP)

1. TBS buffer: 20 mM Tris-HCl, pH7.5, 150 mM NaCl.

37 % formaldehyde solution in water. We use a commercial solution from

Sigma, which also contains 10-15% methanol as stabilizer. 6. 2.5 M Glycine in water. Sterilize with a 0.22 µm filter and store as aliquots at -20 °C. 12. 10 mg/ml Proteinase K in water. [START_REF] Moreau | Perturbation of mRNP biogenesis reveals a dynamic landscape of the Rrp6-dependent surveillance machinery trafficking along the yeast genome[END_REF]. anti c-MYC sc-40X antibody at 200 µg/ml (Santa Cruz Biotechnology).

14. Protein G PLUS-agarose beads (Sigma).

Specific instruments and labware:

-Centrifuge with temperature control (must be maintained at 4°C).

-Heating bloc.

-RNase/DNase free tips with barrier filter.

-Fastprep device (MB Biomedicals) for cell breakage or an equivalent apparatus.

-Quick PCR purification kit (see Note 5).

-Sonication apparatus (see Note 6).

Libraries preparations and NGS data treatment

For the preparation of sequencing libraries from immunoprecipitated DNA fragments and inputs, different kits and specific tools are needed:

3-Methods

The first parts of each subsection are purely experimental and explain how we used Rho expression in yeast, how we extract RNA to perform high-throughput sequencing and how we perform chromatin immunoprecipitation to reveal the recruitment of QC components over the genome. The subsequent parts are dedicated to the use of bioinformatics to analyze the genome-wide sequencing results.

Cell Growth and Rho-induction for perturbation of mRNP biogenesis.

The expression of Rho in yeast is repressed by growing the cells in the presence of the tetracycline analogue, Doxycycline (Doxy), at a final concentration of 5 µg/ml, whereas the omission of Doxy in the growth medium allows maximum expression.

The cell growth is monitored by measuring the optical density at 600 nm (OD 600 ).

1. Inoculate the S. cerevisiae strain of interest transformed by the plasmid pCM185-Rho-NLS (TRP1 Marker) from the -80 °C stock or from a streaking on a fresh plate into 5 ml of liquid medium minus Tryptophan and with Doxy at a final concentration of 5 µg/ml. Then, let the cells grow in a rotating incubator at 25 °C overnight.

2. The following morning, use 800 µl of the culture to inoculate 10 ml of fresh medium containing 0.5 µg/ml of Doxy and grow the cells at 25 °C for approximately 6 hours until an OD 600 of 0.5.

3. Wash the cells three to four times with fresh medium lacking Doxy by sequential centrifugations at 3500 g for 5 min at 5 °C and removal of the supernatant (see Note 8). [START_REF] Fasken | Process or perish: quality control in mRNA biogenesis[END_REF]. Dilute the cells and allow them to grow overnight in 200 ml flasks containing 40 to 45 ml of the medium lacking Doxy to achieve maximum Rho expression.

In parallel, grow similar cultures with medium containing 5 µg/ml Doxy for the non-induced samples. After an overnight growth (14-16 h), the Rho-induced cells are typically at an OD 600 of 0.25-0.3, whereas the non-induced samples are at an OD 600 between 0.8 and 1 (see Note 9). 5. To analyse the samples, dilute the non-induced cultures with fresh medium to adjust the OD 600 to the induced cultures before harvesting the cells (RNA extraction) or crosslinking (ChIP).

RNA extraction

Total RNA extraction is performed using the acidic hot phenol/chloroform method according to [START_REF] Schmitt | A rapid and simple method for preparation of RNA from Saccharomyces cerevisiae[END_REF] with some modifications.

1. After adjusting the non-induced and induced cultures to the same lowest OD 600 , put back the yeast liquid cultures in the agitating incubator at 25 °C for 20 min.

2. During this 20 min meantime prepare all the necessary buffers and stand ready to perform all the following manipulations on ice.

3. Transfer the yeast cultures to 50 ml tubes, then centrifuge at 4 °C, 10 min, 3500 g. Remove the supernatant and wash the pellet with 1 ml of DEPCtreated water. ethanol, and precipitate at -80 °C for two hours followed by centrifugation at 4°C for 20 min at 15000 g. Remove the supernatant and wash the pellet with 75 % ethanol. Dry the pellet briefly and resuspend it in 30 µl of RNase-free water. Then, the RNA can be quantified from a ~1µL aliquot with a Nanodrop (or equivalent) spectrophotometer (assuming an extinction coefficient at 260 nm for RNA of 0.025 (μg/ml) -1 cm -1 ).

RNA-seq libraries preparation and sequencing

cDNA libraries preparations from isolated RNA were made by I2BC high-throughput sequencing platform (Gif sur Yvette) using Illumina ScriptSeq protocol after RiboZero treatment and followed with sequencing on an Illumina NextSeq 500 instrument. We always outsource the preparation of RNA-seq libraries to NGS platforms because they usually have high expertise and are better equipped for the task, especially regarding the quality checks of the samples. For each condition (Rho-repressing or Rho-inducing conditions), two biologically independent samples are used for libraries preparation and sequencing.

RNA-seq data processing and computational analysis

In this section, we provide an overview of the bioinformatics packages and manipulations used for the sequencing analysis and data processing pipeline (see

Note 10):

1. Run a quality control analysis of the reads using FastQC v 0.11.5 package.

2.

Remove the adaptor's sequences from the reads using Cutadapt 1.15.

3. Use the bwa 0.7.12 software package to map the reads on the reference genome (Saccharomyces cerevisiae genome V64.1.1). The genome annotations can be downloaded from SGD (https://www.yeastgenome.org/) and specific ncRNAs annotations are taken from [START_REF] Xu | Bidirectional promoters generate pervasive transcription in yeast[END_REF] for SUTs and from [START_REF] Neil | Widespread bidirectional promoters are the major source of cryptic transcripts in yeast[END_REF] for CUTs. [START_REF] Fasken | Process or perish: quality control in mRNA biogenesis[END_REF]. Quality control the alignment to the genome with Samtools 0.1.19.

5. Process the data by counting the number of reads per genomic feature using the package featureCounts V 1.5.0-p1, with -s 1 option.

6. Isolate a working pool of mRNAs from the Wt strain that are affected by Rho action by comparing the mRNA levels between the -Rho and + Rho conditions. This determines which mRNAs from the data sets of the Wt strain are becoming down-regulated under Rho action. For these differential expression analyses use DESeq2 package under R environment (V 3.4.4).

7. Perform the same differential analysis on the isolated mRNA pool using the data sets collected from the rrp47Δ strain, which lacks the exonuclease Rrp6.

8. Using the two sets of results obtained above (Wt and rrp47Δ strain), sort the mRNAs by their sensitivity to Rho action and which can be rescued under Rrp6 depletion (Figure 1A). 9. To visualize the effect of Rho as reads coverage, use the same subsets of the reads results obtained for the selected pools of mRNAs to generate Metatranscript profiles variations of mRNAs between Wt and the rrp47Δ strain in the absence or presence of Rho (Figure 1B). To perform these analyses, generate BigWig files using bedtools V2.25.0 (genomecov and bigwigCompare), and UCSC tool bedGraphToBigWig. After computing genome coverage with genomecov, convert the bedgraph outputs to bigwig files using bedGraphToBigWig. Meta-profiles plots are then made using bedtools 24 V2.25.0 with computeMatrix (scale-regions mode, with a bin size of 1 base) on BigWig files and plotProfile for graphical output.

Chromatin ImmunoPrecipitation (ChIP)

Each protein of interest within the cross-linked chromatin complex is immunoprecipitated via the MYC tag present at its C-terminus using an antibody recognizing the MYC epitope (anti c-MYC sc-40X antibody).

1. At the end of the overnight growth, as for RNA extraction, adjust the OD 600 of the non-induced cells by dilution with medium in order to obtain the same number of cells in each culture/condition. 2. To perform protein-nucleic acids crosslinking within the chromatin add 1.2 ml of 37% formaldehyde solution to the ~45 ml of yeast culture (from Section 3.1) directly into the culture flask and incubate for 20 min at room temperature on a rotating platform within a fume hood.

3. Stop the cross-linking reaction by adding 2.25 ml of 2.5 M Glycine and incubate 5 min at room temperature, then put the samples on ice (see Note 11).

4.

Transfer the culture into a 50 ml conical tube and wash the cells three times with cold TBS by applying centrifugation steps at 3500 g for 5 min at 4 °C and carefully removing all supernatant (see Note 12) 5. Resuspend the washed cell pellet in 1 ml of lysis buffer 140 by pipetting up and down and add the cell suspension directly to a 2 ml screw cap microtube, containing glass-beads (same volume of beads than the cell suspension).

6. Break the cells with a Fastprep device in a cold room (4 cycles of 30 sec at 6000 rpm with a 1 min pause between each cycle). 7. To recover the cell extract without the glass-beads, drill a hole in the bottom of the microtube with an incandescent needle, then introduce it into a 15 ml conical tube. After removing the screw cap, centrifuge 1 min at 1500 g to recover the extract. 8. After recovering the extract and transferring it to a 2 ml microtube, adjust the volume to 1.2 ml with Lysis buffer 140, then shear the cross-linked chromatin by sonication on ice to reduce average DNA fragments to an approximate size of ~400-500 base pairs (see Note 13). 9. Centrifuge the samples for 5 min at 5000 rpm and 4 °C then recover the supernatant (approximately 1 ml) and put it in a new microtube. 10. Take 20 µl of supernatant genomic DNA which will be used as Input sample and mix the remaining part with 4 µL anti c-MYC antibody solution for the IP sample. Fixe the tubes on a rotating plate and incubate overnight at 4 °C.

11. The next morning, add 37 µL of the protein G PLUS-agarose beads slurry and let the samples rotate for 2 hours at 4 °C.

12. Perform successive washing steps by 2-3 min centrifugation at 4000 g and supernatant elimination at 4 °C (the agarose beads are pulled at the bottom of the tube): 2 times with 1 ml lysis buffer 140, 2 times with 1 ml lysis buffer 360, 1 time with 1 ml washing buffer and 1 time with 1 ml TE. 15. Add to each sample 15 µl of 10 mg/ml proteinase K and incubate for digestion 1.5 h at 37 °C. Recover the DNA samples using a PCR Purification Kit (see Note 5), following manufacturer's instructions.

DNA libraries preparations and sequencing for ChIP-seq

ChIP-seq libraries preparations in duplicates are done following the NEBNext Ultra II DNA Library Prep kit for Illumina from New Englands Biolabs as outlined by the supplier. Briefly, the kit workflow can be divided in three major steps (see Note 14).

1. The first step is applied directly on the DNA fragments from the ChIP samples and is based on the enzymatic preparation of both ends of the DNA fragments.

2. The second step consists of adaptors ligation at each end.

3. The third step uses the ligated adaptors as a platform for hybridization of primers (possessing unique barcodes used for multiplexed sequencing) and subsequent amplification of the DNA fragments by PCR. 4. A Qubit assay is then performed following the manufacturer's instructions to evaluate the amount of DNA obtained at the end of the PCR amplification (see

Note 14).

5. The libraries are sent to the pre-selected high-throughput sequencing platform that runs an Illumina NextSeq 500.

ChIP-seq data analysis

Upon completion of the high throughput sequencing, download the results and perform the data analyses according to the following steps (see Note 15):

1. Run a quality control of the reads using FastQC v 0.11.5 package.

2. Process the trimming of the adaptor's sequences from the reads using Cutadapt 1.15 package.

4-Notes

1. Each strain carrying a C-terminally MYC-tagged protein for the ChIP experiments was constructed by integration of appropriate DNA fragments encoding the Tag at the protein gene locus following homologous recombination. The DNA fragments were obtained by PCR amplification with appropriate primers on plasmid template pYM19 [START_REF] Janke | A versatile toolbox for PCR-based tagging of yeast genes: new fluorescent proteins, more markers and promoter substitution cassettes[END_REF]. The strains can be obtained from us upon request. Alternatively, similar strains derived from the wild-type background strain BY4741 are available from the Euroscarf yeast collection (http://www.euroscarf.de/).

2. The synthetic complete medium is without Tryptophan to maintain within the yeast cells the centromeric plasmid pCM185-Rho-NLS that has the TRP1 marker.

3. Stock solutions of PMSF in 100% isopropanol do not need any sterilization and they are stable for at least 9 months at 15 to 25 °C. 7. We recommend to have an early contact with the NGS outsourcing platform to discuss the specific needs (single end reads or paired reads, number of reads per library), the experimental design and the schedule for the sequencing operations.

8. Cell washing is an important step to remove all the residual Doxy and thus to allow full expression of Rho during growth for the induced samples. 9. The difference of the growth density between the cells grown under repressing conditions and those grown under inducing conditions results from the growth defect induced by Rho expression.

10. All the bioinformatics data processing packages are freely available and can be downloaded from respective web sites and run with Linux Ubuntu operating system. Alternatively, the Galaxy webserver (https://usegalaxy.org/) which is under a graphical user interface can also be used for data processing. But this alternative shows less flexibility in terms of option and data manipulations. 11. From this step, try to work on ice so the samples are kept as close as possible to 4 °C.

12. If necessary for timing purpose, the processing of the samples can be stopped at this step by storing the cell pellets at -80 °C.

13. As specified in Note 6, the shearing of the chromatin can be done with a BIORUPTOR or a usual sonicator with a slim probe that fits into a 2 ml tube.

With our sonicator device (Autotune Model 750 watts), we use the following parameters: 20 % amplitude, 7 sec of pulse ON and 3 sec of pulse OFF for a total time of 4 min with the probe diving at 2/3 inside the microtube which is maintained on ice. These parameters should be calibrated for each specific sonication device by analysing the size of the DNA fragments on agarose gel.

14. It is good to know that this kit can be used for a broad range of initial DNA quantity (from 500 pg to 1 µg). Another advice when using this kit is to avoid numerous PCR amplification cycles as far as possible in order to circumvent PCR duplicates that can make analysis bias during bioinformatics processing of the sequencing data.

15. All the bioinformatics data processing packages are freely available and can be downloaded from respective web sites. Note that the data processing of the ChIP-seq results can also be run on the Galaxy webserver (https://usegalaxy.org/) as for RNA-seq.

1 .

 1 All Saccharomyces cerevisiae strains used for the present work are listed below. They are derived from the wild-type strain BMA41 (MATa or MATα ade2-1 ura3-1 leu2-3,112 his3-11,15 trp1Δcan1-100) [14] and were constructed by homologous recombination (see Note 1) -rrp47Δ, like BMA41 with rrp47Δ::KAN -NAB3-MYC, like BMA41 with Nab3-MYC::HIS3 -NRD1-MYC, like BMA41 with Nrd1-MYC::HIS3 -RRP6-MYC, like BMA41 with Rrp6-MYC::HIS3 -TRF4-MYC, like BMA41 with Trf4-MYC::HIS3 2. pCM185-Rho-NLS plasmid, a derivative of the pCM185 vector (CEN, TRP) from Euroscarf (http://www.euroscarf.de) expressing bacterial Rho factor in yeast with a C-terminal NLS (Nuclear Localization Signal) and under the control of a Doxycycline-regulated (Tet-off) promoter [8].

2 .

 2 1X Phosphate buffered saline (PBS), pH 7.4. 3. PBS/BSA mixture: dissolve 125 mg Bovine Serum Albumin (BSA) in 25 ml 1X PBS. 4. 0.1 M PMSF solution. Dissolve 430 mg of protease inhibitor Phenylmethylsulfonyl fluoride (PMSF) in 25 ml of isopropanol and store as 1 ml aliquots at room temperature until use (see Note 3).

7 .

 7 1X TE buffer: 10 mM Tris-HCl and 1 mM EDTA, pH 8. 8. Lysis buffer 140: 50 mM HEPES pH 7.5, 140 mM NaCl, 1 mM EDTA, 1 % (v/v) Triton X-100, 0.1 % (w/v) sodium deoxycholate. Right before use, add 1:100 volume of 0.1 M PMSF (see Note 4). 9. Lysis buffer 360: 50 mM HEPES pH 7.5, 360 mM NaCl, 1 mM EDTA, 1 % (v/v) Triton X-100, 0.1 % (w/v) sodium deoxycholate. Right before use, add 1:100 volume of PMSF 0.1 M (see Note 4). 10. Washing buffer: 10 mM Tris-HCl pH 8, 0.25 M LiCl, 0.96 % (v/v) NP-40, 0.5 % (w/v) SDS, 1 mM EDTA. 11. Elution buffer: 50 mM Tris-HCl pH 8, 10 mM EDTA, 1 % (w/v) SDS.

1 .

 1 NEBNext Ultra II DNA Library Prep for Illumina from New England Biolabs or equivalent from other suppliers. 2. Qubit assay kit (Thermo Fisher Scientific) 3. Magnetic rack device for washing steps of the NEBNext Ultra II kit. 4. Magnetic beads (specific brands are suggested in the NEBNext Ultra II kit) 5. Spectrophotometer for Qubit assays measurement. 6. Academic core facility or commercial service for high-throughput nucleic acid sequencing, e.g. on an Illumina NextSeq 500 (see Note 7). 7. 64 GB RAM, 12 cores computer, equipped with Linux Ubuntu operating system and R software. 8. Software for NGS data treatment, e.g. as provided by the web-based Galaxy platform (https://usegalaxy.org/).

4 . 5 . 5 (

 455 Re-suspend the cell pellet in 1 ml of DEPC-treated water by vortexing and transfer the suspension to 1.5 ml microtubes. Centrifuge the tubes in a benchcentrifuge at 4000 g for approximately one minute. Remove the supernatant and add 400 µl of AE buffer and 40 µl of 10% SDS, then vortex to suspend the cells. Transfer the suspensions into 1.5 mL microtubes previously filled with micro glass beads (~1/5 of the tube). The next steps have to be performed under a fume hood.[START_REF] Rougemaille | Dissecting mechanisms of nuclear mRNA surveillance in THO/sub2 complex mutants[END_REF]. Add an equivalent volume (440 µl) of hot phenol/chloroform pH 4.previously warmed up to 65 °C).7. Vortex the tubes for 1 min, then incubate them at 65 °C for 1 min. This is repeated 3 times to optimize the mechanical breakage of the cells.8. Put the samples in ethanol/dry-ice bath for 2 min (alternatively, they can be stored 2 hours at -80 °C). Put back the samples to the 65 °C incubator for 3 min, vortex, and repeat the process twice. 9. Centrifuge the tubes at 4 °C, 12 min, 15000 g in a bench-centrifuge then carefully pipette the aqueous phase and transfer it in a new tube. After treatment with DNase I (as recommended by the supplier) to remove possible DNA contaminants, add an equivalent volume of phenol/chloroform pH 6.7, vortex and centrifuge at 4 °C for 10 min at 13000 rpm. 10. Carefully pipet the aqueous phase and transfer it in a new tube, then add sodium acetate to a final concentration of 0.3 M. Add 3 volumes of 100%

13 .

 13 Elute the DNA from the agarose beads twice with 75 µl of elution buffer during 10 min incubations at 65 °C, each time recovering supernatant. 14. The samples (150 µl of IP and the 20 µl of Input adjusted to 150 µl by addition of elution buffer) are reverse crosslinked by incubation at 65 °C overnight.

3 .

 3 Map the whole reads on the reference Saccharomyces cerevisiae genome (version V64.1.1) with the bwa 0.7.12 software package.

4 .

 4 Quality control the alignment of the reads to the reference genome using Samtools 0.1.19 package. 5. Proceed to a specific peak-calling step using the PePr V1.1.21 package by calculating reads ratios between IP and Input in a comparative mode where only peak signals detected simultaneously in the two replicates are considered. The piled-up peak signals obtained for the perturbed condition (+Rho) and non-perturbed condition (-Rho) are subsequently subjected to a step of differential analysis where only peaks appearing for one condition but not the other are scored (see Note 16).

4 .

 4 Upon addition of 1:100 volume of 0.1 M PMSF, the final working concentration of PMSF is 1 mM. 5. We use the GeneJET PCR purification kit (ThermoFischer) but other brands such as Quiaquick PCR purification kit (Qiagen) work as well. 6. Depending on what is available, use either a probe sonicator device while maintaining the sample on ice or use a water-bath sonicator such as the BIORUPTOR (Diagenode), which has the advantage of keeping the samples at 4 °C during sonication.

16 .FiguresFigure 1 :

 161 Figure 1: Fate of subpopulations of mRNAs down-regulated by Rho action in yeast

Figure 2 :

 2 Figure 2: Rho activity mediates the stimulation of massive recruitment of the QC

peaks distributions were taken with the IGV browser (Integrative Genomics Viewer).