mTOR activation by constitutively active serotonin6 receptors as new paradigm in neuropathic pain and its treatment

Chronic neuropathic pain is a highly disabling syndrome that is poorly controlled by currently available an- algesics. Here, we show that painful symptoms and associated cognitive deficits induced by spinal nerve ligation in the rat are prevented by the administration of serotonin 5-HT 6 receptor inverse agonists or by the mTOR inhibitor rapamycin. In contrast, they are not alleviated by the administration of 5-HT 6 receptor neutral antagonists. Likewise, activation of mTOR by constitutively active 5-HT 6 receptors mediates allodynia in ox- aliplatin-induced peripheral neuropathy in rats but not mechanical nociception in healthy rats. Furthermore, both painful and co-morbid cognitive symptoms in neuropathic rats are strongly reduced by intrathecal delivery of a cell-penetrating peptide that disrupts 5-HT 6 receptor/mTOR physical interaction. Collectively, these findings demonstrate a deleterious influence of non-physiological mTOR activation by constitutively active spinal 5- HT 6 receptors upon painful and cognitive symptoms in neuropathic pains of different etiologies. They suggest that targeting the constitutive activity of 5-HT 6 receptors with inverse agonists or disrupting the 5-HT 6 receptor/ mTOR interaction might be valuable strategies for the alleviation of neuropathic pain and cognitive co-mor- bidities.


Introduction
Neuropathic pains refer to an heterogeneous group of pains arising as a direct consequence of a lesion or disease affecting the somatosensory system (Treede et al., 2008). Neuropathic pains affect 5-10 % of the general population depending on the country and the screening tools used (Bouhassira et al., 2008;Chenaf et al., 2018; van Hecke et al., 2014). Due to their severity and chronicity, neuropathic pains often coexist with co-morbid symptoms such as anxiety, depression, sleep disturbances and cognitive deficits (Radat et al., 2013). Chronic neuropathic pain and co-morbidities are especially problematic because of the limited efficacy of first-line treatments. For instance, the number of patients who needed to be treated (NNT) to obtain a 50 % pain relief in one patient is 3.6 for the tricyclic antidepressant amitriptyline, one of reference treatments in neuropathic pain (Finnerup et al., 2015). Furthermore, reference treatments induce frequent adverse effects, with a safety profile, evaluated by the number needed to harm (number of patients who need to be treated for one patient to drop out because of adverse effects), in the range of 11.8-13.9 (Finnerup et al., 2015). Collectively, these observations underscore the need for new therapeutic strategies for the treatment of neuropathic pain. Pain processing at the spinal level is finely controlled by serotonergic projections originating from the midbrain and brain stem (Millan, 2002). Given the multiplicity of serotonin receptors expressed in dorsal spinal cord and the resistance of neuropathic pain to selective serotonin reuptake inhibitor (SSRI) antidepressants, one possibility is that certain serotonin receptors induce pro-algesic effects. These include the 5-HT 6 receptor, which is expressed in excitatory interneurons of the dorsal spinal cord, especially in a region below lamina II inner to lamina IV outer called "low-threshold mechanoreceptor-recipient zone" and involved in tactile perception (Abraira et al., 2017). The 5-HT 6 receptor is coupled to the Gs-adenylyl cyclase pathway and stimulates cAMP production, which leads to depolarization of neurons. Consistent with these excitatory effects, 5-HT 6 receptor blockade with arylsulfonamide derivatives of pyrazolo [3,4]pyridine-7-ones and 1-aryl-5-isopropyl-pyrazoles or SB258585 produces anti-allodynic or anti-hyperalgesic effects in rodent models of traumatic (Devegowda et al., 2013;Hong et al., 2017;Pineda-Farias et al., 2017) and metabolic (Sari et al., 2019) neuropathic pain. 5-HT 6 receptor blockade also improves cognition in a wide range of cognitive impairment paradigms in rodents (Codony et al., 2011;Hirst et al., 2006;Loiseau et al., 2008;Vanda et al., 2018;Zajdel et al., 2016). Several 5-HT 6 receptor antagonists have entered clinical trials for the indication of cognitive deficits in various neuropsychiatric disorders, including schizophrenia and dementia (Khoury et al., 2018), underscoring their potential for the treatment of cognitive co-morbidities associated to neuropathic pain. An important feature of the 5-HT 6 receptor is its high level of constitutive activity, which was established not only for recombinant receptors expressed in transfected cells but also for native receptors present in cultured neurons or mice brain (Deraredj Nadim et al., 2016;Duhr et al., 2014;Grychowska et al., 2016;Kohen et al., 2001). Whether 5-HT 6 receptor constitutive activity contributes to neuropathic pain and cognitive co-morbidities and whether 5-HT 6 receptor inverse agonists could be useful for their clinical management have never been explored.
Using proteomics strategies, we previously showed that 5-HT 6 receptors physically interact with several proteins of the mechanistic Target Of Rapamycin (mTOR) pathway (Meffre et al., 2012). Further studies revealed that non-physiological activation of mTOR by prefrontal 5-HT 6 receptors underlies cognitive deficits in rodent development models of schizophrenia (Meffre et al., 2012). Interestingly, an increasing body of evidence indicates a role of mTOR signaling in neuropathic pain sensitivity: central administration of the mTOR inhibitor rapamycin reduces mechanical hypersensitivity in spared nerve injured rats (Geranton et al., 2009) and mice (Obara et al., 2011), as well as mechanical allodynia and thermal hyperalgesia in rats with impact-induced spinal cord injury (Wang et al., 2016). Rapamycin administration also attenuates mechanical allodynia induced by chronic nerve constriction injury in mice (Zhang et al., 2013), reduces mechanical allodynia and cold hypersensitivity in bortezomib-induced peripheral neuropathy in rats (Duan et al., 2018) and mechanical hyperalgesia in streptozotocin (STZ)-induced diabetic neuropathy (He et al., 2019).
In light of these observations, we investigated, in the present study, the role of 5-HT 6 receptor constitutive activity and mTOR signaling under the control of constitutively active 5-HT 6 receptors in neuropathic pain and co-morbid cognitive symptoms induced by spinal nerve ligation (SNL) or oxaliplatin (OXA) administration in the rat. We show in both models that activation of mTOR by constitutively active spinal 5-HT 6 receptors mediates allodynia and co-morbid cognitive symptoms. These findings suggest that 5-HT 6 receptor inverse agonists currently under clinical evaluation for the treatment of cognitive impairment in dementia, and mTOR inhibitors already used to prevent graft rejection in transplanted patients or for the treatment of breast and kidney cancer, might be repositioned for the alleviation of neuropathic pains of various etiologies and cognitive co-morbidities.

Animals
All animal procedures described here were reviewed and approved by the Auvergne Animal Experiment Ethics Committee, CE2A and by the French Ministry of Higher Education and Innovation (authorization N°2018111617333273V5) and conducted in accordance with the NC3R ARRIVE rules (Kilkenny et al., 2010). Sprague-Dawley adult male rats were purchased from Janvier Labs (Le Genest-Saint-Isle, France). Male rats were chosen for all behavioral experiments to avoid any influence of ovarian hormone variations due to the estrus cycle on pain sensitivity (Fischer et al., 2008).
The transgenic mouse line C57BL/6 knock-in 5-HT 6 -GFP generated by the Institut Clinique de la Souris (www.ics-mci.fr), was described elsewhere (Deraredj Nadim et al., 2016). The animals were housed under a 12 -h light/dark cycle at 21 ± 2°C and 55 ± 10 % humidity with ad libidum access to food and water in an animal facility free of specified pathogenic organisms.
Experimenters were blinded to the treatment. Treatments were randomized and administered according to the method of blocks in order to assess their effect in the same conditions. Different animals were used in each experiment. At the end of the experiments, the animals were euthanized by progressive carbon dioxide inhalation (10-30 %/min).

Animal models of neuropathic pain
Traumatic neuropathic pain was induced by SNL. The surgery was performed as previously described (Kim and Chung, 1992). Rats (200 ± 20 g) were anesthetized by i.p. injection of xylazine (10 mg/kg) and ketamine (75 mg/kg). Under aseptic conditions, the left L5 spinal nerve was ligated with a nonabsorbable 5−0 braided silk thread. Sham surgery was performed in an identical manner except for the nerve ligation. At the end of the surgery, the rats received a s.c. injection of meloxicam (non-steroidal anti-inflammatory drug) to reduce the postsurgery pain and inflammation.

Behavioral tests
Tactile allodynia was assessed using the von Frey hair test (Chaplan et al., 1994). Animals were habituated to the testing environment 1 h before baseline assessment in a stable temperature and humidity room. Each rat was confined in clear plexiglas boxes placed on an elevated metal mesh floor. Von Frey filaments were applied perpendicularly to the central plantar surface of the ipsilateral hind paw for 5 s in ascending order of force (1.4−26 g). Paw withdrawal or licking was considered as a positive response and the next weaker filament was applied. In case of negative response (no paw withdrawal or licking), we then applied the next stronger filament. This paradigm continued until four measurements have been obtained after an initial change of behavior, or until four consecutive negative responses or five consecutive positive responses. The 50 % response threshold was calculated using the Up-Down method and Dixon's formulae (Dixon, 1980). Thermal allodynia was assessed using the paw immersion test. One day before baseline assessment, rats were habituated to the test in order to get them used to the apparatus and the manual restraint. The ipsilateral hind paw to nerve ligation was immersed in a water bath maintained at 15.0 ± 0.2°C until paw withdrawal or sign of struggle were observed and the paw withdrawal latency was measured (cut-off time, 15 s). A shortened duration of immersion indicated thermal allodynia. The percentage of the maximal possible effect (% MPE) was calculated according to the formulae: % MPE= [(Post-drug latency -Pre-drug latency) / (15 -Pre-drug latency)] x 100.
Mechanical nociception to paw pressure was assessed using a Randall-Selitto analgesimeter (Ugo Basile, Bioseb, France) (Randall and Selitto, 1957) by applying increasing pressure to the left hind paw of the rat until a vocalization or a struggle response occurs, with a cut-off threshold of 750 g to avoid tissue damage.
The social recognition test (ability of an adult rat to recognize a younger conspecific during two 5-min sessions) was performed using a procedure without intersession delay (Loiseau et al., 2008). SNL and sham rats were individually housed for 2 days before testing. On the test day, under dim light conditions (30 lx), a juvenile rat was placed into the home cage for 2 consecutive 5-min sessions. The time spent in active social investigation (i.e. time spent by the adult rat in sniffing, following, biting, jumping, and crawling over or under the juvenile one) during the first (T 1 ) and the second (T 2 ) session was monitored. Social recognition was expressed as the difference between the exploration times during the two sessions (T 1 -T 2 ) (Appendix Fig. 1A).
The novel object recognition (NOR) test provides a measure of episodic memory and was performed as previously described (Ennaceur and Delacour, 1988). Recognition memory is expressed as a recognition index, defined as the ratio of time spent exploring the novel object over the total time spent exploring both familiar and novel objects. One day before the test, SNL and sham rats were singly habituated to the test arena (75 × 75 × 50 cm) for 5 min. On the test day, rats were allowed to freely explore two objects (familiarization trial) under dim light conditions (30 lx) and returned to their home cage. After a 5-min inter-trial interval, rats were reintroduced in the arena with one of the original objects replaced by a novel one (recognition trial). The sequence of presentations and the location of the objects was randomly assigned to each rat. Both trials lasted 5 min. Exploration of each object was recorded separately. Exploratory behavior was defined as looking, licking, or touching the object while sniffing with active vibrissae. Novel object recognition memory was expressed as a recognition index (d2 score), defined as the ratio of time spent exploring the novel object over the total time spent exploring both familiar and novel objects (Appendix Fig. 1B).

Intrathecal injections
Intrathecal (i.t.) injection was performed under isoflurane anesthesia (4% induction, 2% maintenance) according to a previously described procedure (Mestre et al., 1994). The anesthetized rats were held at the pelvic girdle and drug was delivered (10 μl volume) using a 25-gauge × 1-inch needle connected to a 50 μl Hamilton syringe inserted into the subarachnoidal space between lumbar vertebrae L5 and L6, eliciting a tail flick. The syringe was held in position for a few seconds after the injection.

Co-immunoprecipitation experiments
HEK-293 cells were transfected with HA-tagged mTOR and GFPtagged 5-HT 6 constructs. Twenty-four hours after transfection, cells were washed in cold PBS, sonicated and solubilized in lysis buffer (75 mM Tris, 2 mM EDTA, 12 mM MgCl 2 , 10 mM CHAPS, protease inhibitor cocktail EDTA free (Roche cOmplete), pH 7.4) for 5 h at 4°C. Lysates were centrifuged at 12,000 x g for 20 min at 4°C. Immunoprecipitations were performed using an anti-GFP antibody (Invitrogen, 1 μg per condition; overnight 4°C incubation) and A/G plus agarose beads (Santa Cruz). Immunoprecipitated proteins and 50−100 μg of total proteins (lysates) were mixed with Laemmli buffer, heated at 70°C for 10 min and separated by SDS-PAGE. Immunoblots were probed with anti-GFP (Roche) and anti-mTOR antibodies (1:1,000, Cell Signaling) and immunoreactivity was revealed using antirabbit, HRP-conjugated secondary antibodies. All Co-IP experiments were conducted from an identical amount of proteins (starting material); data were normalized over the quantity of immunoprecipitated 5-HT 6 -GFP receptor and quantity of mTOR in the input.

Immunohistochemistry
Immunodetection of 5-HT 6 receptors was performed in allodynic male 5-HT 6 -GFP KI mice. Animals were anesthetized with i.p. injection of 44 mg pentobarbital sodium (Ceva SA) and transcardially perfused with 4% (w/v) paraformaldehyde (Sigma-Aldrich) in 0.1 M phosphate buffered saline (PBS) fixative solution. The brain and the lumbar enlargement of the spinal cord were removed, post-fixed for 48 h in the same solution at 4°C, before being cryoprotected with a 30 % (w/v) sucrose in 0.1 M PBS solution for 3 days at 4°C. All tissues were embedded in tissue freezing medium (TFM, General Data Healthcare) and 40 μm-thick sections were cut at −27°C with a cryotome (Microm KS34 & HM450, Thermo Scientific) and stored at 4°C in 0.1 M PBS containing 0.1 % sodium azide. Sections were permeabilized and saturated with 0.2 % Triton X-100, 20 % goat serum in 0.1 M PBS for 1 h and incubated overnight at room temperature with the primary antibodies: anti-GFP (1:500, Invitrogen) and anti AC3 (1:1,000, Abcam) diluted in the same solution. Sections were then incubated for 1 h with Alexa Fluor 488 or 546-conjugated antibody (1:1,000, Sigma-Aldrich) in PBS containing 20 % goat serum. Immunofluorescence staining was observed with an Eclipse Ni-E microscope (Nikon) equipped with epifluorescence. Images were acquired using the NIS-Elements 4.13 software (Nikon) driving an ORCA Flash 4.0 camera (Hamamatsu).

Determination of cAMP production
NG108−15 cells transiently coexpressing the 5-HT 6 receptor and the CAMYEL probe were washed with PBS containing calcium and magnesium. Coelanterazine H (Molecular Probes) was added at a final concentration of 5 μM, and left at room temperature for 5 min. Cells were then treated with either vehicle or SB258585 or PZ-1388 or CPPQ or IIQ. BRET was measured using a Mithras LB 940 plate reader (Berthold Technologies). BRET values are expressed as percent of the activity measured in cells expressing the 5-HT 6 receptor, and treated with vehicle.

Statistical analyses
Data are expressed as means ± SEMs. The sample size for each experiment is expressed in each Figure legend. Biochemical and behavioral data were analyzed using a two-tailed Student's t-test, a 1-way ANOVA followed by Bonferroni's post-hoc test or a 2-way ANOVA followed by Dunnett's post-hoc test. The statistical significance was set at 5% (P < 0.05). Statistical analyses were run using GraphPad Prism 6.0 software. All statistics are detailed in Table 1. Fig. 2. SB258585, PZ-1388, CPPQ and IIQ induce contrasting effects on receptor constitutive activity and SNL-induced allodynia. A. Impact of SB258585, PZ-1388, CPPQ and IIQ (used at 10 −6 M) on 5-HT 6 receptor constitutive activity (assessed by cAMP production measurement using the CAMYEL sensor) in NG108−15 cells. Data are the mean ± SEM of values obtained in at least three independent experiments performed on different sets of cultured cells. *** P < 0.001 vs. vehicle (ANOVA followed by Dunnett's test). B. NG108−15 cells were exposed to incremental concentrations of either SB258585 or PZ-1388. Data are the mean ± SEM of triplicate determinations. Two independent experiments performed on different sets of cultured cells yielded similar results. Constitutive activity is represented as 100 % of the activity measured in the absence of compound (vehicle). C, D. Intraperitoneal administration of PZ-1388 (1, 5 or 25 μmol/kg) but not vehicle (Veh, water for injections) improved tactile and thermal allodynia in SNL rats (n = 8/group). ### P < 0.001 vs. values measured before SNL; *P < 0.05, **P < 0.01, ***P < 0.001 vs. values measured before the drug/vehicle (water) injection (D14 Post-SNL), 2-way ANOVA. E. Schema illustrating the route of 5-HT 6 receptor ligand administrations in experiments illustrated on panels F-H. F-H. Spinal 5-HT 6 receptor constitutive activity underlies tactile allodynia in SNL rats. Intrathecal administration of IIQ (2 nmol/rat) or CPPQ (2 nmol/rat) but not vehicle (Veh, water for injections) suppressed the anti-allodynic effect of SB258585 (SB, 5 μmol/kg, i.p.) and PZ-1388 (25 μmol/kg, i.p.) in SNL rats (n = 8/group). ### P < 0.001 vs. values measured before SNL; **P < 0.01 ***P < 0.001 vs. values measured before drug/vehicle injection (D14 Post-SNL), 2-way ANOVA.

5-HT 6 receptor blockade reduces tactile and thermal allodynia induced by spinal nerve ligation in the rat
As previously reported (Dupuis et al., 2017), 95 % of the SNL rats used in the present study displayed, 14 days after the surgery, tactile allodynia to plantar stimulation with von Frey monofilaments and thermal allodynia in response to a cold stimulus applied on the paw ipsilateral to the side of injury, as shown by the dramatic decrease in 50 % paw withdrawal thresholds (5.62 ± 0.14 vs. 28.30 ± 0.21 g before surgery, one-tailed paired t test P < 0.0001) and paw-withdrawal latencies (6.34 ± 0.29 vs. 13.70 ± 0.29 s before surgery, one-tailed paired t test P < 0.0001). The blockade of 5-HT 6 receptors with systemic administration of SB258585 (5 and 25 μmol/kg) reduced SNLinduced tactile allodynia 60-180 and 30−120 min after injection, respectively (Fig. 1A, Table 1). The maximal effect was similar for both doses (17.43 ± 2.09 g and 17.44 ± 3.12 g at 60 min, corresponding to 72.2 ± 9.5 % and 62.7 ± 20.2 % reversal, respectively). Both doses also produced a similar global anti-allodynic effect, as assessed by the area under the time-course curve (A.U.C.) of 50 % threshold variations (Appendix Fig. 2A, Table 1). Administration of SB258585 likewise increased paw-withdrawal latency to cold bath immersion (60-120 and 30−180 min after systemic injection of SNL rats with 5 and 25 μmol/ kg, respectively (Fig. 1C, Table 1), indicating that 5-HT 6 receptor blockade also alleviates thermal allodynia. The maximal anti-allodynic effect, observed 120 min after injection of both doses, corresponded to 52.9 ± 9.0 % and 58.6 ± 1.5 % of maximal possible effect (MPE), respectively. As observed for tactile allodynia (see Fig. 1A), no gain of effect was obtained with the 25 μmol/kg dose, compared with 5 μmol/ kg. Therefore, the 25 μmol/kg dose was discarded in further behavioral studies. In contrast, systemic administration of SB258585 (5 μmol/kg) failed to increase paw-pressure induced vocalization threshold in healthy rats (Fig. 1D, Table 1), indicating that 5-HT 6 receptor inhibition does not prevent mechanical nociception and specifically alleviates painful symptoms characteristic of neuropathic pain.
In contrast to the anti-allodynic effects of SB258585 and PZ-1388, the administration of the neutral antagonists IIQ or CPPQ did not modify the mechanical threshold to von Frey hair application ( Fig. 2F-H, Table 1). This suggests that SNL-induced allodynia does not result from a tonic activation of spinal 5-HT 6 receptors elicited by endogenously released serotonin, but rather from receptor constitutive activity. To further support the role of constitutive activity of spinal 5-HT 6 receptor in neuropathic pain, we performed joint systemic administration of inverse agonists (SB258585 and PZ-1388) and intrathecal injection of neutral antagonists (Fig. 2E). Intrathecal administration of IIQ (2 nmol/rat, i.t.) totally prevented the anti-allodynic effects of systemic administration of SB258585 (Fig. 2F, Table 1) or PZ-1388 (Fig. 2G, Table 1) in SNL rats. Likewise, spinal injection (2 nmol/ rat) of CPPQ suppressed the anti-allodynic effect induced by systemic administration of PZ-1388 (Fig. 2H, Table 1).

Blocking 5-HT 6 receptor constitutive activity improves co-morbid cognitive symptoms associated with painful neuropathy in SNL rats
In line with the pro-cognitive effects of 5-HT 6 receptor blockade in several rodent models of cognitive impairment (Gravius et al., 2011;Hirst et al., 2006;Loiseau et al., 2008;Vanda et al., 2018;Zajdel et al., 2016), we next examined whether inhibiting 5-HT 6 receptor constitutive activity would also improve cognitive deficits associated with chronic neuropathic pain in SNL rats. We first evaluated the influence of SB258585 and PZ-1388 upon social cognition (social interaction test) and episodic memory (NOR test). SNL-allodynic rats treated with vehicle failed to recognize a younger conspecific rat compared to sham rats, and to discriminate a novel object from the familiar one ( Fig. 4A-G, Table 1). Administration of SB258585 (5 μmol/kg, i.p..) totally restored novelty discrimination in the social recognition (Fig. 4A, Table 1) and NOR (Fig. 4B, Table 1) tests in SNL rats. Injection of PZ-1388 (25 μmol/kg, i.p.) attenuated the social recognition deficit (Fig. 4C) and abolished the episodic memory deficit elicited by SNL (Fig. 4D, Table 1). Importantly, no signs of abnormal behavior were detected in the allodynic SNL rats due to the systemic administration of either SB258585 or PZ-1388. Altogether, these data show pro-cognitive effects of systemic administration of 5-HT 6 receptor inverse agonists to painful neuropathic rats.
To further explore whether the pro-cognitive effects of SB258585 and PZ-1388 in SNL rats result from neuropathic pain improvement mediated by constitutively active spinal 5-HT 6 receptors, we intrathecally injected the neutral antagonist IIQ concomitantly with the systemic administration of inverse agonists to specifically inhibit the inverse agonist effects in the spinal cord (Appendix Fig. 1A). Intrathecal administration of IIQ (2 nmol/rat, i.t.), which abolished the anti-allodynic effect of SB258585 (5 μmol/kg) and PZ-1388 (25 μmol/kg, see Fig. 2F and G, Table 1), also suppressed the pro-cognitive effect of both inverse agonists ( Fig. 4E and F, Table 1). Furthermore, the intrathecal injection of SB258585 in SNL rats (200 pmol/rat, i.t.) restored mnemonic performance in the social interaction test (Fig. 4G, Table 1), thereby reproducing the effects of a systemic administration.

Agonist-independent 5-HT 6 receptor-operated mTOR signaling underlies neuropathic pain and associated cognitive dysfunction in SNL rats
As 5-HT 6 receptors are known to engage mTOR signaling (Meffre et al., 2012;Teng et al., 2019;Wang et al., 2015) and mTOR has been implicated in neuropathic pain (Duan et al., 2018;Geranton et al., 2009;He et al., 2019;Obara et al., 2011;Wang et al., 2016;Zhang et al., 2013), we examined whether SNL affects mTOR activity in dorsal horn of lumbar spinal cord. Phosphorylation of ribosomal S6 protein at Ser 240/244 , a downstream substrate of mTOR, was significantly increased in the ipsilateral side to the nerve ligation, compared with the contralateral side (Fig. 5A, Table 1). As expected, spinal administration of the mTOR inhibitor rapamycin (10 nmol/rat i.t.) to SNL rats abolished SNL-induced elevation of Ser 240/244 phosphorylation (Fig. 5B, Table 1). We thus reasoned that the enhanced mTOR activation in the ipsilateral spinal cord dorsal horn of SNL rats would result from the constitutive activity of spinal 5-HT 6 receptors. We first demonstrated that the basal level of mTOR phosphorylation (at Ser 2448 , measured as an index of mTOR activation state) in HEK-293 cells expressing 5-HT 6 receptor was significantly reduced by SB258585 or PZ-1388, but not IIQ exposure (Appendix Fig. 4, Table 1). This indicates that 5-HT 6 receptors constitutively activate mTOR signaling and that SB258585 and PZ-1388 behave as inverse agonists while IIQ behaves as neutral antagonist with respect of receptor-operated mTOR activation, corroborating their pharmacological properties on cAMP production (see Fig. 2A and B). Correspondingly, intrathecal injection of SB258585 or PZ-1388 significantly reduced the level of phosphorylated S6 in the dorsal spinal cord of SNL rats (Fig. 5B, Table 1), suggesting that the enhanced mTOR activity measured in these rats results, at least in part, from agonistindependent activation of spinal 5-HT 6 receptors.
We then examined whether mTOR activation underlies tactile and thermal allodynia in SNL rats. Reminiscent of the effects of 5-HT 6 receptor inverse agonists, intrathecal administration of rapamycin (0.3, 3 and 10 nmol/rat) increased mechanical thresholds to von Frey hair application, with a maximal effect measured at 120−240 min post-injection (Fig. 6A, Table 1). The anti-allodynic effect of rapamycin 10 nmol was still significant 24 h after injection but disappeared at 48 h post-injection (Fig. 6A, Table 1). The global anti-allodynic effect of rapamycin, assessed by the A.U.C. of 50 % threshold variations, showed a dose-dependent effect (Appendix Fig. 2D, Table 1). Rapamycin treatment also increased paw-withdrawal latency to cold immersion, with a maximal effect observed 180 min after injection, but this antiallodynic effect persisted no more than 240 min (Fig. 6B, Table 1). In line with its effects upon tactile and thermal allodynia, rapamycin dosedependently reduced SNL-induced social recognition memory deficit (Fig. 6C, Table 1) and restored normal novel object recognition in SNL rats (Fig. 6D, Table 1).

Disrupting 5-HT 6 receptor-mTOR interaction improves neuropathic pain symptoms in SNL rats
We previously demonstrated that mTOR physically associates with the 5-HT 6 receptor C-terminal (Ct) domain and that this interaction is essential for receptor-mediated mTOR activation (Meffre et al., 2012). Expression of several Ct truncation mutants of the 5-HT 6 receptor in HEK-293 cells showed that the 21 Ct residues of the receptor are necessary to the engagement of mTOR signaling upon agonist stimulation of 5-HT 6 receptor (Appendix Fig. 5A). Among these residues, we identified a sequence of 10 amino acids (FFVTDSVEPE) showing homology with the mTOR recognition motif in the S6 protein. We then generated a cell-permeable interfering peptide that encompasses this 10 amino acid sequence fused to the transduction domain (YGRKKRRQ-RRR) of the HIV Tat protein (Tat-VEPE). Treatment of HEK-293 cells with the Tat-VEPE peptide strongly reduced 5-HT 6 receptor-mTOR interaction (Appendix Fig. 5B, Table 1) as well as agonist-dependent (Appendix Fig. 5C, Table 1) and independent (Appendix Fig. 5D, Table 1) mTOR activation.
Intrathecal administration of Tat-VEPE (100 and 300 ng/rat i.t., Fig. 7A) but not a control peptide (Tat-cont, Appendix Fig. 6A), reduced tactile allodynia for 180 min ( Table 1). The maximal anti-allodynic effect occurred at 120 min post-injection, resulting in a 60.4 ± 13.7 % reversal of tactile allodynia. The A.U.C. of threshold variations confirmed the effect of both 100 and 300 ng/rat doses (Appendix Fig. 2E, Table 1). Reminiscent of the pro-cognitive effects of 5-HT 6 receptor inverse agonists and rapamycin, injection of Tat-VEPE also abolished social memory deficit in SNL rats (Fig. 7B, Table 1). The Tat-VEPE peptide also significantly increased paw withdrawal thresholds upon stimulation with von Frey monofilament in OXA rats, resulting in a 49.6 ± 14.9 % of reversal of tactile allodynia at 120 min for the 300 ng/ rat dose ( Fig. 7C and Appendix Fig. 2H, Table 1), as well as the reaction latency to a cold stimulus, the maximal effect resulting in 36.3 ± 6.3 % of reversal of thermal allodynia (Fig. 7D, Table 1). These anti-allodynic effects lasted 120 min i.e. a shorter duration than that observed in SNL rats (180 min). Corroborating the lack of antinociceptive effects of SB258585 and PZ-1388 in healthy rats, intrathecal injection of Tat-VEPE did not change paw pressure-induced vocalization threshold in non-neuropathic rats (Appendix Fig. 6B, Table 1).

Discussion
Active conformations of GPCRs are not only promoted by agonists, but can also occur in their absence, leading to constitutive activity (De Deurwaerdère et al., 2020) Constitutive activity has been established for numerous GPCRs in transfected cells expressing high receptor densities, but often remains to be demonstrated for native receptors. The 5-HT 6 receptor is a GPCR exhibiting a high level of constitutive activity both in recombinant cells and in vivo (Chaumont-Dubel et al., 2019;Deraredj Nadim et al., 2016;Duhr et al., 2014;Kohen et al., 2001). Here, we provide a convergent set of results indicating that 5-HT 6 receptor constitutive activity is involved in painful symptoms in two preclinical models of traumatic (SNL) and toxic (OXA administration) neuropathy of high translational value. In both models, systemic administration of the 5-HT 6 receptor inverse agonists SB258585 and PZ-1388 (one of the leads from the newly designed arylpyrrole carboxamide series), attenuated tactile and thermal allodynia associated with traumatic nerve injury and chemotherapy, while 5-HT 6 receptor ligands that behave as neutral antagonists were ineffective. The antiallodynic effects of 5-HT 6 receptor inverse agonists were of similar magnitude in both models, emphasizing the relevance of blocking 5-HT 6 receptor constitutive activity for the management of neuropathic pains of different etiologies. Interestingly, these anti-allodynic effects were long-lasting, as usually observed with gabapentin, one of the reference treatments in neuropathic pain (Hunter et al., 1997;Pastrana-Quintos et al., 2020). Moreover, they were observed at much lower doses than the gabapentin doses (100 mg/kg p.os or 300 mg/kg, i.p.) necessary to produce a similar anti-allodynic effect (50 % reversal of pain) (Hunter et al., 1997;Pastrana-Quintos et al., 2020), minimizing the risk of off-target adverse effects.
The anti-allodynic effects of SB258585 in neuropathic rats corroborates previous studies indicating that this compound improves chemical hyperalgesia (Castaneda-Corral et al., 2009;Godinez-Chaparro et al., 2012), tactile allodynia in SNL rats (Pineda-Farias et al., 2017) and thermal hyperalgesia in STZ-induced diabetic mice (Sari et al., 2019). The present study suggests that these previously described effects result from SB258585 inverse agonist activity at 5-HT 6 receptors. Further supporting that SNL-induced allodynia involves 5-HT 6 receptor constitutive activity rather than tonic activation of spinal 5-HT 6 receptors elicited by endogenously released serotonin, intrathecal administration of a neutral antagonist prevented the antiallodynic effects of SB258585 and PZ-1388. These findings together with the antiallodynic effect induced by intrathecal injection of SB258585 demonstrate a crucial role of constitutively active 5-HT 6 receptors located in the spinal cord.
Corroborating these observations and previous findings that revealed the presence of 5-HT 6 receptor mRNA in the dorsal horn spinal cord (Ward and Dorsa, 1996), we provide direct evidence of 5-HT 6 receptor expression in dorsal spinal cord neurons of 5-HT 6 -GFP KI mice. The present results are also consistent with recent observations made in BAC-CreER mouse line where the 5-HT 6 receptor was found in excitatory interneurons of the dorsal spinal cord in a region below lamina II inner to lamina IV outer called "low-threshold mechanoreceptor-recipient zone" and involved in tactile perception processing (Abraira et al., 2017). Furthermore, we show that 5-HT 6 receptors are mainly localized in the primary cilium of dorsal spinal cord neurons, corroborating previous observations in striatal neurons where 5-HT 6 receptors were found to finely regulate cilia length and signaling as well as dendrite outgrowth and neuronal morphology (Brodsky et al., 2017;Lesiak et al., 2018). The functional relevance of the specific location of 5-HT 6 receptors in primary cilium of spinal cord neurons remains to be established.
Beyond the demonstration that spinal 5-HT 6 receptors are constitutively active in experimental models of neuropathic pain and that their blockade with inverse agonists alleviates allodynic symptoms, we show for the first time that 5-HT 6 receptors constitutively activate a non-canonical signaling pathway (mTOR signaling), as assessed by measuring the phosphorylation of mTOR (at Ser 2448 ) and its downstream substrate S6 (at Ser 240/244 ). To our knowledge, these findings provide the first demonstration of agonist-independent activation of mTOR signaling by a GPCR. Constitutive activation of mTOR was prevented by an interfering peptide disrupting 5-HT 6 receptor/mTOR physical interaction, suggesting that this interaction might stabilize the receptor in an active conformation able to stimulate mTOR kinase in an agonist-independent manner. Importantly, we established constitutive activity of 5-HT 6 receptor at mTOR signaling not only in HEK-293 cells transiently expressing recombinant receptors, but also in the spinal cord dorsal horn of SNL rats, specifically in the ipsilateral side of the lesion.
Furthermore, mimicking the effects of 5-HT 6 receptor inverse agonists, intrathecal delivery of the Tat-VEPE peptide reduced tactile allodynia with the same magnitude in SNL and OXA rats. These results indicate that the activation of mTOR, under the control of constitutively active spinal 5-HT 6 receptors, might contribute to tactile and thermal allodynia induced by traumatic nerve injury and chemotherapy. Similarly, intrathecal administration of rapamycin reversed SNL-induced neuropathic pain. The global effect of rapamycin, measured by the A.U.C. was similar to that of SB258585, PZ-1388 and Tat-VEPE. This antiallodynic effect was long-lasting (between 4 and 24 h) and more prolonged than that of inverse agonists, suggesting the involvement of additional (5-HT 6 receptor-independent) mechanisms, such as neurotrophin-induced activation of Trk receptors and mTOR signaling (Pezet, 2014). These results are also consistent with recent findings showing that i) mTOR signaling plays a central role in the sensitization of DRG and dorsal horn spinal cord neurons (Uttam et al., 2018) contributing to the development of chronic pain, and ii) intrathecal or systemic administration of rapalogs alleviates painful symptoms in various rodent models of neuropathic pain of different etiologies (Asante et al., 2010;Duan et al., 2018;He et al., 2019;Obara et al., 2011;Wang et al., 2016;Zhang et al., 2013).
The present study suggests that 5-HT 6 receptor inverse agonists currently in clinical development as symptomatic treatment of cognitive impairment in dementia and psychoses, might also be proposed for alleviating neuropathic pains. 5-HT 6 receptor inverse agonists are generally well tolerated (Atri et al., 2018) and thus certainly more relevant than mTOR inhibitors which exhibit strong adverse effects linked to their immunosuppressive activity. Disrupting the 5-HT 6 receptor/ mTOR interaction might also be considered, as strategies based on interfering peptides to disrupt membrane receptor interaction with intracellular proteins (DOP receptor/Cdk5 (Beaudry et al., 2015); 5-HT 2A receptor/PSD-95 (Pichon et al., 2010); NMDA receptor/Src (Liu et al., 2008)) have already proved their efficacy in animal models of chronic pain.
Notably, blocking 5-HT 6 receptor-mediated mTOR signaling not only improved painful symptoms but also co-morbid cognitive deficits in neuropathic rats. Again, only the 5-HT 6 receptor inverse agonists SB258585 and PZ-1388 restored normal social and episodic memory in SNL rats, whereas a neutral antagonist (IIQ) failed to reverse cognitive deficits, underscoring the need for developing 5-HT 6 receptor inverse agonists rather than neutral antagonists for the treatment of neuropathic pain and co-morbid symptoms. The pro-cognitive effects of 5-HT 6 receptor antagonists (or inverse agonists) in various models of cognitive impairment such as scopolamine or PCP administration are mediated by the blockade of striatal, hippocampal and prefrontal 5-HT 6 receptors and rely on their abilities to restore normal cholinergic, GABAergic and glutamatergic transmissions in these brain regions (Codony et al., 2011). In contrast, the present results suggest that the beneficial effects of 5-HT 6 receptor inverse agonists upon cognitive impairment caused by neuropathic pain are independent of a supraspinal (cortico-limbic) mechanism but rely on the spinal analgesic effect of the compounds.

Conclusions
The present findings identify the activation of mTOR signaling by constitutively active spinal 5-HT 6 receptors as a key mechanism underlying tactile and thermal allodynia elicited by SNL or OXA administration in the rat. They provide the first demonstration of a pathological influence of agonist-independent activation of non-canonical signaling by a GPCR. They suggest that targeting constitutively active 5-HT 6 receptors or the 5-HT 6 receptor/mTOR complex might be efficient strategies to alleviate painful symptoms and associated cognitive comorbidities in neuropathic pain.