Painful neuropathy is one of the most common complications of diabetes. First‐line therapeutic agents such as tricyclic antidepressants, dual serotonin/noradrenaline reuptake inhibitors, and alpha2‐delta ligands of calcium channels (i.e., gabapentinoids) are poorly effective. New strategies targeting the serotonin type 6 receptor (5‐HT 6 R) and mechanistic Target Of Rapamycin (mTOR) signaling have recently emerged. Until a few years ago, preclinical studies of pain in rodents were more often carried out in males than in females, despite compelling evidence of sex‐specific mechanisms in pain. Here, we investigated the role of 5−HT 6 R/mTOR signaling in neuropathic pain in streptozocin (STZ)‐induced type 1 diabetes (T1D) in female rats. Mechanical hyperalgesia was attenuated in female diabetic (STZ‐D) rats by systemic injection of 5‐HT 6 ‐R inverse agonists. Further, administration of full (PZ‐1386, SB258585) but not partial (PZ‐1179) 5−HT 6 R inverse agonists alleviated cognitive deficits in female STZ‐D rats. Intrathecal administration of the mTOR inhibitor rapamycin or a cell‐penetrating peptide that disrupts the physical interaction between the 5‐HT 6 R and mTOR also reduced pain and cognitive comorbidity in females. Together with previous data obtained in STZ‐D male rats and in spinal nerve ligation (SNL) and oxaliplatin (OXA) models of neuropathic pain, these results suggest that the analgesic and procognitive effects of 5‐HT 6 R inverse agonists are sex‐specific and dependent on the etiology of neuropathic pain, highlighting the importance of personalizing treatment that considers the patient's sex, etiology of neuropathy, and the presence or absence of comorbid cognitive symptoms. image

Painful neuropathy is one of the most common complications of diabetes. First-line therapeutic agents such as tricyclic antidepressants, dual serotonin/noradrenaline reuptake inhibitors, and alpha2-delta ligands of calcium channels (i.e., gabapentinoids) are poorly effective. New strategies targeting the serotonin type 6 receptor (5-HT6R) and mechanistic Target Of Rapamycin (mTOR) signaling have recently emerged. Until a few years ago, preclinical studies of pain in rodents were more often carried out in males than in females, despite compelling evidence of sex-specific mechanisms in pain. Here, we investigated the role of 5-HT6R/mTOR signaling in neuropathic pain in streptozocin (STZ)-induced type 1 diabetes (T1D) in female rats. Mechanical hyperalgesia was attenuated in female diabetic (STZ-D) rats by systemic injection of 5-HT6-R inverse agonists. Further, administration of full (PZ-1386, SB258585) but not partial (PZ-1179) 5-HT6R inverse agonists alleviated cognitive deficits in female STZ-D rats. Intrathecal administration of the mTOR inhibitor rapamycin or a cell-penetrating peptide that disrupts the physical interaction between the 5-HT6R and mTOR also reduced pain and cognitive comorbidity in females. Together with previous data obtained in STZ-D male rats and in spinal nerve ligation (SNL) and oxaliplatin (OXA) models of neuropathic pain, these results suggest that the analgesic and procognitive effects of 5-HT6R inverse agonists are sex-specific and dependent on the etiology of neuropathic pain, highlighting the importance of personalizing treatment that considers the patient's sex, etiology of neuropathy, and the presence or absence of comorbid cognitive symptoms.

Diabetic neuropathy is often associated with chronic pain. Serotonin type 6 (5-HT6) receptor ligands, particularly inverse agonists, have strong analgesic potential and may be new candidates for treating diabetic neuropathic pain and associated co-morbid cognitive deficits. The current study addressed the involvement of 5-HT6 receptor constitutive activity and mTOR signaling in an experimental model of diabetic neuropathic pain induced by streptozocin (STZ) injection in the rat. Here, we show that mechanical hyperalgesia and associated cognitive deficits are suppressed by the administration of 5-HT6 receptor inverse agonists or rapamycin. The 5-HT6 receptor ligands also reduced tactile allodynia in traumatic and toxic neuropathic pain induced by spinal nerve ligation and oxaliplatin injection. Furthermore, both painful and co-morbid cognitive symptoms in diabetic rats are reduced by intrathecal delivery of a cell-penetrating peptide that disrupts 5-HT6 receptor-mTOR physical interaction. These findings demonstrate the deleterious influence of the constitutive activity of spinal 5-HT6 receptors upon painful and cognitive symptoms in diabetic neuropathic pains of different etiologies. They suggest that targeting the constitutive activity of 5-HT6 receptors with inverse agonists or disrupting the 5-HT6 receptor-mTOR interaction might be valuable strategies for the alleviation of diabetic neuropathic pain and cognitive co-morbidities.

The neurotransmitter serotonin (5-hydroxytryptamine, 5-HT) is involved in numerous physiological functions and plays a key role in pain modulation including neuropathic pain. Diabetic neuropathy is a common complication of diabetes mellitus often accompanied by chronic neuropathic pain. Animal models of diabetes offer relevant tools for studying the pathophysiological mechanisms and pharmacological sensitivity of diabetic neuropathic pain and for identifying new therapeutic targets. In this review, we report data from preclinical work published over the last 15 years on the analgesic activity of drugs acting on the serotonergic system, such as serotonin and noradrenaline reuptake inhibitor (SNRI) antidepressants, and on the involvement of certain serotonin receptors-in particular 5-HT1A, 5-HT2A/2c and 5-HT6 receptors-in rodent models of painful diabetic neuropathy.

The endoplasmic reticulum exit of some polytopic plasma membrane proteins (PMPs) is controlled by arginin-based retention motifs. PRAF2, a gatekeeper which recognizes these motifs, was shown to retain the GABAB-receptor GB1 subunit in the ER. We report that PRAF2 can interact on a stoichiometric basis with both wild type and mutant F508del Cystic Fibrosis (CF) Transmembrane Conductance Regulator (CFTR), preventing the access of newly synthesized cargo to ER exit sites. Because of its lower abundance, compared to wild-type CFTR, CFTR-F508del recruitment into COPII vesicles is suppressed by the ER-resident PRAF2. We also demonstrate that some pharmacological chaperones that efficiently rescue CFTR-F508del loss of function in CF patients target CFTR-F508del retention by PRAF2 operating with various mechanisms. Our findings open new therapeutic perspectives for diseases caused by the impaired cell surface trafficking of mutant PMPs, which contain RXR-based retention motifs that might be recognized by PRAF2.

Chronic neuropathic pain is a highly disabling syndrome that is poorly controlled by currently available analgesics. 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-HT6 receptor inverse agonists or by the mTOR inhibitor rapamycin. In contrast, they are not alleviated by the administration of 5-HT6 receptor neutral antagonists. Likewise, activation of mTOR by constitutively active 5-HT6 receptors mediates allodynia in oxaliplatin-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-HT6 receptor/mTOR physical interaction. Collectively, these findings demonstrate a deleterious influence of non-physiological mTOR activation by constitutively active spinal 5-HT6 receptors upon painful and cognitive symptoms in neuropathic pains of different etiologies. They suggest that targeting the constitutive activity of 5-HT6 receptors with inverse agonists or disrupting the 5-HT6 receptor/mTOR interaction might be valuable strategies for the alleviation of neuropathic pain and cognitive co-morbidities.

Cells are sensitive to chemical stimulation which is converted into intracellular biochemical signals by the activation of specific receptors. Mechanical stimulations can also induce biochemical responses via the activation of various mechano-sensors. Although principally appreciated for their chemosensory function, G-protein-coupled receptors (GPCRs) may participate in mechano-transduction. They are indirectly activated by the paracrine release of chemical compounds secreted in response to mechanical stimuli, but they might additionally behave as mechano-sensors that are directly stimulated by mechanical forces. Although several studies are consistent with this latter hypothesis, the molecular mechanisms of a potential direct mechanical activation of GPCRs have remained elusive until recently. In particular, investigating the activation of the catecholamine β2-adrenergic receptor by a pathogen revealed that traction forces directly exerted on the N-terminus of the receptor via N-glycan chains activate specific signaling pathways. These findings open new perspectives in GPCR biology and pharmacology since most GPCRs express N-glycan chains in their N-terminus, which might similarly be involved in the interaction with cell-surface glycan-specific lectins in the context of cell-to-cell mechanical signaling.

Meningococcus utilizes β-arrestin selective activation of endothelial cell β2 adrenergic receptor (β2AR) to cause meningitis in humans. Molecular mechanisms of receptor activation by the pathogen and of its species selectivity remained elusive. We report that β2AR activation requires two asparagine-branched glycan chains with terminally exposed N-acetyl-neuraminic acid (sialic acid, Neu5Ac) residues located at a specific distance in its N-terminus, while being independent of surrounding amino-acid residues. Meningococcus triggers receptor signaling by exerting direct and hemodynamic-promoted traction forces on β2AR glycans. Similar activation is recapitulated with beads coated with Neu5Ac-binding lectins, submitted to mechanical stimulation. This previously unknown glycan-dependent mode of allosteric mechanical activation of a G protein-coupled receptor contributes to meningococcal species selectivity, since Neu5Ac is only abundant in humans due to the loss of CMAH, the enzyme converting Neu5Ac into N-glycolyl-neuraminic acid in other mammals. It represents an additional mechanism of evolutionary adaptation of a pathogen to its host.

Serotonin is a neurotransmitter involved in many psychiatric diseases. In humans, a lack of 5HT2B receptors is associated with serotonin-dependent phenotypes, including impulsivity and suicidality. A lack of 5-HT2B receptors in mice eliminates the effects of molecules that directly target serotonergic neurons including amphetamine-derivative serotonin releasers, and selective serotonin reuptake inhibitor antidepressants. In this work, we tested the hypothesis that 5-HT2B receptors directly and positively regulate raphe serotonin neuron activity. By ex-vivo electrophysiological recordings, we report that stimulation by the 5-HT2Breceptor agonist, BW723C86, increased the firing frequency of serotonin Pet1-positive neurons. Viral overexpression of 5-HT2B receptors in these neurons increased their excitability. Furthermore, in-vivo 5-HT2B-receptor stimulation by BW723C86 counteracted 5HT1A autoreceptor-dependent reduction in firing rate and hypothermic response in wildtype mice. By a conditional genetic ablation that eliminates 5-HT2B-receptor expression specifically and exclusively from Pet1-positive serotonin neurons (Htr2b mice), we demonstrated that behavioral and sensitizing effects of MDMA, as well as acute behavioral and chronic neurogenic effects of the antidepressant fluoxetine, require 5-HT2B-receptor expression in serotonergic neurons. In Htr2b mice, dorsal raphe serotonin neurons displayed a lower firing frequency compared to control Htr2b mice as assessed by in-vivo extracellular recordings and a stronger hypothermic effect of 5-HT1A-autoreceptor stimulation was observed. The increase in head twitch response to DOI further confirmed the lower serotonergic tone resulting from the absence of 5-HT2B receptors in serotonin neurons. Together, these observations indicate that the 5-HT2B receptor acts as a direct positive modulator of serotonin Pet1-positive neurons in an opposite way as the known 5-HT1A negative autoreceptor. Introduction Serotonin (5-Hydroxytryptamine, 5-HT) is involved in many psychiatric diseases including depression, addiction, impulsivity or psychosis. The 5-HT neurons that innervate forebrain originate predominantly from the rostral cell group of neurons in the dorsal raphe nucleus (DRN) (Commons, 2016; Okaty et al, 2015). These neurons express the serotonergic markers tryptophan hydroxylase (TPH2), and 5-HT transporter (SERT), and also the negative autoreceptors, 5-HT1A and 5-HT1B receptors, whose expression is restricted to somatodendritic compartments of 5-HT neurons, and to axonal terminals, respectively (Riad et al, 2000). The 5-HT1A autoreceptor activation elicits an outward current carried through G protein-coupled inwardly-rectifying potassium channels (GIRK) of the Kir3 family leading to membrane hyperpolarization and inhibition of 5-HT neuron firing (Aghajanian and Lakoski, 1984). The presence of synaptic vesicles in dendrites of 5-HT neurons led to the suggestion that autoinhibition is mediated via dendritic release of 5-HT, for review see (Andrade et al, 2015). However, activity of 5-HT DRN neurons can also be positively modulated by 5HT2A/2B/2C receptors triggering directly or indirectly inward currents (Boothman et al, 2003; Craven et al, 2001; Kirby et al, 2003; Liu et al, 2000; Quérée et al, 2009). Upon electrical stimulation of leech 5-HT neurons, transmembrane Ca entry through L-type channels first evokes an early dendritic exocytosis; subsequently, the released 5-HT activates dendritic 5HT2 autoreceptors coupled to Gq and phospholipase C, resulting in a positive feedforward loop that maintains sustained exocytosis (Leon-Pinzon et al, 2014). It has thus been proposed that DRN neurons can display responses ranging from inhibition to excitation depending on a balance of functional 5-HT1A and 5-HT2 receptors (Marinelli et al, 2004). However, a direct action of 5-HT2 receptor subtypes at 5-HT neurons has not yet been clearly established. In humans, a loss-of-function polymorphism of 5-HT2B receptors is associated with 5-

Serotonin is a neurotransmitter involved in many psychiatric diseases. In humans, a lack of 5-HT2B receptors is associated with serotonin-dependent phenotypes, including impulsivity and suicidality. A lack of 5-HT2B receptors in mice eliminates the effects of molecules that directly target serotonergic neurons including amphetamine derivative serotonin releasers, and selective serotonin reuptake inhibitor antidepressants. In this work, we tested the hypothesis that 5-HT2B receptors directly and positively regulate raphe serotonin neuron activity. By ex vivo electrophysiological recordings, we report that stimulation by the 5-HT2B receptor agonist, BW723C86, increased the firing frequency of serotonin Pet1-positive neurons. Viral overexpression of 5-HT2B receptors in these neurons increased their excitability. Furthermore, in vivo 5-HT2B-receptor stimulation by BW723C86 counteracted 5-HT1A autoreceptor-dependent reduction in firing rate and hypothermic response in wild-type mice. By a conditional genetic ablation that eliminates 5-HT2B receptor expression specifically and exclusively from Pet1-positive serotonin neurons (Htr2b5-HTKO mice), we demonstrated that behavioral and sensitizing effects of MDMA (3,4-methylenedioxy-methamphetamine), as well as acute behavioral and chronic neurogenic effects of the antidepressant fluoxetine, require 5-HT2B receptor expression in serotonergic neurons. In Htr2b5-HTKO mice, dorsal raphe serotonin neurons displayed a lower firing frequency compared to control Htr2blox/lox mice as assessed by in vivo extracellular recordings and a stronger hypothermic effect of 5-HT1A-autoreceptor stimulation was observed. The increase in head-twitch response to DOI (2,5-dimethoxy-4-iodoamphetamine) further confirmed the lower serotonergic tone resulting from the absence of 5-HT2B receptors in serotonin neurons. Together, these observations indicate that the 5-HT2B receptor acts as a direct positive modulator of serotonin Pet1-positive neurons in an opposite way as the known 5-HT1A-negative autoreceptor.

The serotonin receptor subtypes 2 comprises 5HT2A, 5-HT2B and 5-HT2C that are Gαq-coupled receptors and display distinct pharmacological properties. Although co-expressed in some brain regions and involved in various neurological disorders, their functional interactions have not been studied yet. We report that 5-HT2 receptors can form homo and heterodimers when expressed alone or co-expressed in transfected cells. Coimmunoprecipitation and bioluminescence resonance energy transfer studies confirmed that 5-HT2C receptors interact with either 5-HT2A or 5-HT2B receptors. Although heterodimerization with 5-HT2C receptors does not alter 5-HT2C Gαq-dependent inositol-phosphate signaling, 5HT2Aor 5-HT2B-receptor-mediated signaling was totally blunted. This feature can be explained by a dominance of 5-HT2C on 5-HT2A and 5-HT2B receptor binding: in 5-HT2Ccontaining heterodimers, ligands bind and activate exclusively the 5-HT2C protomer. This dominant effect on the associated protomer was also observed in neurons, supporting a physiological relevance of 5-HT2 receptors heterodimerization in-vivo. Accordingly, exogenous expression of an inactive form of the 5-HT2C receptor in the Locus ceruleus is associated with decreased 5-HT2A-dependent noradrenergic transmission. These data demonstrate that 5-HT2 receptors can form functionally asymmetric heterodimers in-vitro and in-vivo that must be considered when analyzing the physiological or pathophysiological roles of serotonin in tissues where 5-HT2 receptors are co-expressed. Many members of the G-protein-coupled receptor (GPCR) family have the capacity to form homoor hetero-oligomers with biochemical and functional characteristics, including receptor pharmacology, signaling and regulation, which are unique to these oligomeric conformations. These GPCR oligomers have been found not only to occur within a type of GPCR but also across different families and subtypes (1,2). Metabotropic serotonin (5hydroxytryptamine, 5-HT) subtype 2 receptors (5-HT2), which belong to the class A-1 GPCR family, display a widespread expression in the nervous system and are involved in an important array of physiological and pathological processes. The 5-HT2 subfamily consists in three Gαq/Gα11-coupled receptors, 5-HT2A, 5-HT2B, and 5-HT2C, which mediate excitatory neurotransmission (3). Interestingly, 5-HT2 subtypes coexist in multiple areas of the brain http://www.jbc.org/cgi/doi/10.1074/jbc.M117.779041 The latest version is at JBC Papers in Press. Published on March 3, 2017 as Manuscript M117.779041 Copyright 2017 by The American Society for Biochemistry and Molecular Biology, Inc. by gest on M arch 8, 2017 hp://w w w .jb.org/ D ow nladed from Dimerization among 5-HT2 receptor subtypes 2 (4,5). For example, 5-HT2A and 5-HT2C receptors are co-expressed in GABAergic interneurons and in a subpopulation of pyramidal neurons of the prefrontal cortex (PFC) (6-8), in dopaminergic neurons of the ventral tegmental area (VTA) (9,10) and 5-HT2C and 5-HT2B receptors are expressed in pro-opiomelanocortin (POMC) neurons of the hypothalamic arcuate nucleus (11). Although 5-HT2 receptors are similar in structure, there are differences in their pharmacology and signaling outputs (12). It has been reported that 5-HT2A and 5-HT2C receptors can function as stable homodimers (13-16) whereas the existence of 5-HT2B homodimers has not been documented yet. Dimers have also been reported for other 5-HT receptors, including 5-HT1A, 5-HT1B, 5-HT1D, 5-HT4, and 5-HT7 receptor subtypes in heterologous expression systems (13,17-20). In addition, there are observations suggesting that the 5-HT2 receptors subfamily can form heterodimeric complexes with other types of GPCRs. For example, the formation of heterodimers has been reported for the 5-HT2A with mGluR2, D2dopamine and CB1 receptors (21-23), for 5-HT2C with ghrelin receptors (GHS-R1a) (24) and MT2 receptor (25), for 5-HT1A with μ-opioid (26) and adenosine A2A receptors (27), and for 5-HT2B with angiotensin AT1 receptors (28). As mentioned above, oligomerization can occur between receptors of different GPCR families (i.e. 5-HT and dopamine for example) but also within the same family. A seminal study reported the identification of the first heterodimer between the 5-HT1B and 5-HT1D receptor subtypes (18). However, no significant pharmacological differences were reported between homo and heterodimers for these closely related 5-HT receptor subtypes. Recently, a study identified other 5-HT receptor heterodimers with functional implication: heterodimers between 5-HT1A and 5-HT7 receptors have been reported to regulate GIRK channel activity in heterologous systems and in hippocampal neurons (29). Heterodimerization was found to inhibit 5-HT1A-mediated activation of Gαi and GIRK channel activity, without affecting 5-HT7-receptor-mediated signaling, indicating a unidirectional dominant effect of the 5-HT7 protomer. Of note, cross talks between 5HT receptors have been reported without obligatory physical interaction. For instance, coexpression of 5-HT1B and 5-HT2B receptors influences the internalization pathways and kinetics of both receptors without heterodimerization (i.e. lack of FRET signal) (30). To date, neither the basic pharmacological profiles of putative 5-HT2 heterodimers nor their signaling properties have been characterized. We specifically addressed this issue here, by studying the interactions between the three members of the 5-HT2 subfamily and their functional consequences invitro and in-vivo. Using co-immunoprecipitation and Bioluminescence Resonance Energy Transfer (BRET) approaches, we found that 5HT2A, 5-HT2B and 5-HT2C form heterodimers when co-expressed in heterologous expression systems. Although 5-HT2C-containing heterodimers did not show alterations in coupling properties, the signaling of associated 5-HT2A or 5-HT2B protomers was blunted, while 5-HT2C protomer maintained its signaling properties. Moreover, no blunting occurred in 5HT2A/2B heterodimers. We next showed that this asymmetry in Gαq-protein activation was related to a dominant effect of the 5-HT2C protomer on ligand binding to the other partner. Using AAVmediated exogenous expression of a 5-HT2C receptor truncated C-tail mutant (5-HT2CΔCter) in brain regions expressing endogenous 5-HT2A receptor, we also observed a blunting effect of this inactive 5-HT2C receptor leading to a complete binding inhibition of 5-HT2A selective ligand. Accordingly, this lack of ligand binding was associated with impaired 5-HT2A-induced excitatory neurotransmission in neurons expressing the 5-HT2C inactive protomer. Results Interactions between 5-HT2 receptors The putative formation of heterodimers between 5-HT2 receptor subtypes was investigated using BRET and co-immunoprecipitation experiments (Fig. 1). The coding region of Renilla luciferase (Rluc, BRET donor) or the yellow variant of the green fluorescent protein (YFP, BRET acceptor), were fused in phase downstream of the coding region of 5-HT2A, 5-HT2B and 5-HT2C receptors. Saturation BRET experiments were conducted in HEK293 cells co-transfected with constant amounts of BRET donor plasmids and increasing amounts of BRET acceptor plasmids. In case of a close proximity between the investigated partners, hyperbolic saturation of the BRET signal is expected (see methods). Hyperbolic curves were indeed obtained when 5-HT2A or 5HT2C receptors were tested for self-association (5-HT2A/2A and 5-HT2C/2C, respectively, Fig. 1a), by gest on M arch 8, 2017 hp://w w w .jb.org/ D ow nladed from Dimerization among 5-HT2 receptor subtypes 3 confirming previous studies showing that these receptors are able to homodimerize in transfected cells (13,14). Noteworthy, the same experiment with 5-HT2B BRET pairs led to a linear plot, consistent with a bystander (non-specific) BRET and thus with the absence of self-association. Hyperbolic curves were also obtained with 5HT2A and 5-HT2C BRET pairs (5-HT2A/2C), 5HT2B and 5-HT2C BRET pairs (5-HT2B/2C) and 5HT2A and 5-HT2B (5-HT2A/2B) BRET pairs (Fig. 1b), suggesting that 5-HT2 receptors can form heterodimers in intact cells. BRET50 values (values of YFP/Rluc for half-maximal BRET) reflect the propensity of association between the investigated proteins. Interestingly, BRET50 values for heterodimeric association are significantly lower than those measured for homodimeric association of 5-HT2 subtypes, suggesting that in native cells expressing more than one 5-HT2 receptor subtype, heterodimerization is favored over homodimerization (Fig. 1b). The physical interaction between these receptor subtypes was confirmed by coimmunoprecipitation studies in the same cells, using epitope (FLAG)or GFP-tagged proteins (Fig. 1c). Consistent with BRET data, FLAG-5HT2C receptor co-immunoprecipitated with 5HT2A-GFP and 5-HT2C-GFP receptors. In complementary experiments FLAG-5-HT2B receptor co-immunoprecipitated with 5-HT2AGFP and 5-HT2C-GFP receptors. Impact of dimerization of 5-HT2 receptors on signaling We next examined the consequence of 5-HT2 receptors heterodimerization on agonist-inducedGαq activation. The 5-HT2 receptors consistently activate the PLC-β pathway in native tissues and heterologous cells (3,12). We first determined whether 5-HT2 receptor signaling was altered when expressed in the presence of other 5-HT2 receptors (Fig. 2). Dose-response curves of 5HT2-mediated production of IP in response to 5HT or the partial agonist DOI in cells expressing 5-HT2A, 5-HT2B or 5-HT2C receptors and combination were analyzed using the operational model (31) to determine the Gαq coupling efficiency of single receptors and heterodimers (Fig. 2). No significant difference in Gαq coupling efficiency as determined by the transduction coefficient (τ/KA) (32) was found among the groups, supporting a lack of major difference in coupling efficiency between individual 5-HT2 receptors to Gαq activation and no modification of this coupling efficiency by heterodi

Transient reduced food intake (hypophagia) following high stress could have beneficial effects on longevity, but paradoxically, hypophagia can persist and become anorexia-like behavior. The neural underpinnings of stress-induced hypophagia and the mechanisms by which the brain prevents the transition from transient to persistent hypophagia remain undetermined. In this study, we report the involvement of a network governing goal-directed behavior (decision). This network consists of the ascending serotonergic inputs from the dorsal raphe nucleus (DR) to the medial prefrontal cortex (mPFC). Specifically, adult restoration of serotonin 4 receptor (5-HT4R) expression in the mPFC rescues hypophagia and specific molecular changes related to depression resistance in the DR (5-HT release elevation, 5-HT1A receptor, and 5-HT transporter reductions) of stressed 5-HT4R knockout mice. The adult mPFC-5HT(4)R knockdown mimics the null phenotypes. When mPFC-5-HT(4)Rs are overexpressed and DR-5HT(1A)Rs are blocked in the DR, hypophagia following stress persists, suggesting an antidepressant action of early anorexia.

The serotonin receptor subtypes 2 comprise 5-HT2A, 5-HT2B, and 5-HT2C, which are Gαq-coupled receptors and display distinct pharmacological properties. Although co-expressed in some brain regions and involved in various neurological disorders, their functional interactions have not yet been studied. We report that 5-HT2 receptors can form homo- and heterodimers when expressed alone or co-expressed in transfected cells. Co-immunoprecipitation and bioluminescence resonance energy transfer studies confirmed that 5-HT2C receptors interact with either 5-HT2A or 5-HT2B receptors. Although heterodimerization with 5-HT2C receptors does not alter 5-HT2C Gαq-dependent inositol phosphate signaling, 5-HT2A or 5-HT2B receptor-mediated signaling was totally blunted. This feature can be explained by a dominance of 5-HT2C on 5-HT2A and 5-HT2B receptor binding; in 5-HT2C-containing heterodimers, ligands bind and activate the 5-HT2C protomer exclusively. This dominant effect on the associated protomer was also observed in neurons, supporting the physiological relevance of 5-HT2 receptor heterodimerization in vivo. Accordingly, exogenous expression of an inactive form of the 5-HT2C receptor in the locus ceruleus is associated with decreased 5-HT2A-dependent noradrenergic transmission. These data demonstrate that 5-HT2 receptors can form functionally asymmetric heterodimers in vitro and in vivo that must be considered when analyzing the physiological or pathophysiological roles of serotonin in tissues where 5-HT2 receptors are co-expressed.

Addiction is a maladaptive pattern of behavior following repeated use of reinforcing drugs in predisposed individuals, leading to lifelong changes. Common among these changes are alterations of neurons releasing dopamine in the ventral and dorsal territories of the striatum. The serotonin 5-HT2B receptor has been involved in various behaviors, including impulsivity, response to antidepressants, and response to psychostimulants, pointing toward putative interactions with the dopamine system. Despite these findings, it remains unknown whether 5-HT2B receptors directly modulate dopaminergic activity and the possible mechanisms involved. To answer these questions, we investigated the contribution of 5-HT2B receptors to cocaine-dependent behavioral responses. Male mice permanently lacking 5-HT2B receptors, even restricted to dopamine neurons, developed heightened cocaine-induced locomotor responses. Retrograde tracing combined with single-cell mRNA amplification indicated that 5-HT2B receptors are expressed by mesolimbic dopamine neurons. In vivo and ex vivo electrophysiological recordings showed that 5-HT2B-receptor inactivation in dopamine neurons affects their neuronal activity and increases AMPA-mediated over NMDA-mediated excitatory synaptic currents. These changes are associated with lower ventral striatum dopamine activity and blunted cocaine self-administration. These data identify the 5-HT2B receptor as a pharmacological intermediate and provide mechanistic insight into attenuated dopamine tone following exposure to drugs of abuse. SIGNIFICANCE STATEMENT Here we report that mice lacking 5-HT2B receptors totally or exclusively in dopamine neurons exhibit heightened cocaine-induced locomotor responses. Despite the sensitized state of these mice, we found that associated changes include lower ventral striatum dopamine activity and lower cocaine operant self-administration. We described the selective expression of 5-HT2B receptors in a subpopulation of dopamine neurons sending axons to the ventral striatum. Increased bursting in vivo properties of these dopamine neurons and a concomitant increase in AMPA synaptic transmission to ex vivo dopamine neurons were found in mice lacking 5-HT2B receptors. These data support the idea that the chronic 5-HT2B-receptor inhibition makes mice behave like animals already exposed to cocaine with higher cocaine-induced locomotion associated with changes in dopamine neuron reactivity.

Prenylated Rab acceptor family, member 2 (PRAF2) is a four transmembrane domain protein of 19 kDa that is highly expressed in particular areas of mammalian brains. PRAF2 is mostly found in the endoplasmic reticulum (ER) of neurons where it plays the role of gatekeeper for the GB1 subunit of the GABA(B) receptor, preventing its progression in the biosynthetic pathway in the absence of hetero-dimerization with the GB2 subunit. However, PRAF2 can interact with several receptors and immunofluorescence studies indicate that PRAF2 distribution is larger than the ER, suggesting additional biological functions. Here, we conducted an immuno-cytochemical study of PRAF2 distribution in mouse central nervous system (CNS) at anatomical, cellular and ultra-structural levels. PRAF2 appears widely expressed in various regions of mature CNS, such as the olfactory bulbs, cerebral cortex, amygdala, hippocampus, ventral tegmental area and spinal cord. Consistent with its regulatory role of GABA(B) receptors, PRAF2 was particularly abundant in brain regions known to express GB1 subunits. However, other brain areas where GB1 is expressed, such as basal ganglia, thalamus and hypothalamus, contain little or no PRAF2. In these areas, GB1 subunits might reach the cell surface of neurons independently of GB2 to exert biological functions distinct from those of GABA(B) receptors, or be regulated by other gatekeepers. Electron microscopy studies confirmed the localization of PRAF2 in the ER, but identified previously unappreciated localizations, in mitochondria, primary cilia and sub-synaptic region. These data indicate additional modes of GABA(B) regulation in specific brain areas and new biological functions of PRAF2.

Depressive disorders are among the most prevalent neuropsychiatric dysfunctions worldwide, with high rates of resistance to antidepressant treatment. Genetic factors clearly contribute to the manifestation of depression as well as to the response to antidepressants. Transgenic mouse models appear as seminal tools to disentangle this complex disorder. Here, we analyzed new key aspects of the phenotype of knock-out mice for the gene encoding the serotonin 2B receptor (Htr2B), including basal phenotype, ability to develop a depressive-like phenotype upon chronic isolation, and effect of chronic exposure to fluoxetine on chronically stressed Htr2B mice. We find, here, that Htr2B mice display an antidepressant-like phenotype, which includes reduced latency to feed in the Novelty Suppressed Feeding test, basal increase in hippocampal BDNF levels, no change in TrkB and p75 protein levels, and an increased preference for sucrose consumption compared to wild type (Htr2B) mice. Nevertheless, we show that these mice can develop depressive-like behaviors when socially isolated during four weeks. Selective serotonin reuptake inhibitors (SSRI) have been previously shown to be ineffective in non-stressed Htr2B mice. We evaluated, here, the effects of the SSRI fluoxetine in chronically stressed Htr2B mice and, similarly, no behavioral or plastic effect was induced by this antidepressant. All together, these results highlight the suitability to study resistance to SSRI antidepressants of this mouse model, displaying panoply of conditions among which, behavioral, neurotrophic and plastic causative factors can be analyzed.