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The human neurotensin 1 receptor (hNTS1) is a G protein-coupled receptor involved in many physiological functions, including analgesia, hypothermia, and hypotension. To gain a better understanding of which signaling pathways or combination of pathways are linked to NTS1 activation and function, we investigated the ability of activated hNTS1, which was stably expressed by CHO-K1 cells, to directly engage G proteins, activate second messenger cascades and recruit \b{eta}-arrestins. Using BRET-based biosensors, we found that neurotensin (NT), NT(8-13) and neuromedin N (NN) activated the G{\alpha}q-, G{\alpha}i1-, G{\alpha}oA-, and G{\alpha}13-protein signaling pathways as well as the recruitment of \b{eta}-arrestins 1 and 2. Using pharmacological inhibitors, we further demonstrated that all three ligands stimulated the production of inositol phosphate and modulation of cAMP accumulation along with ERK1/2 activation. Interestingly, despite the functional coupling to G{\alpha}i1 and G{\alpha}oA, NT was found to produce higher levels of cAMP in the presence of pertussis toxin, supporting that hNTS1 activation leads to cAMP accumulation in a G{\alpha}s-dependent manner. Additionally, we demonstrated that the full activation of ERK1/2 required signaling through both a PTX-sensitive Gi/o-c-Src signaling pathway and PLCb-DAG-PKC-Raf-1- dependent pathway downstream of Gq. Finally, the whole-cell integrated signatures monitored by the cell-based surface plasmon resonance and changes in the electrical impedance of a confluent cell monolayer led to identical phenotypic responses between the three ligands. The characterization of the hNTS1-mediated cellular signaling network will be helpful to accelerate the validation of potential NTS1 biased ligands with an improved therapeutic/adverse effect profile., Comment: This is the accepted (postprint) version of the following article: Besserer-Offroy \'E, et al. (2017). Eur J Pharmacol. doi: 10.1016/j.ejphar.2017.03.046, which has been accepted and published in its final form at http://www.sciencedirect.com/science/article/pii/S0014299917302157 V1: Preprint version V2: Accepted version (postprint)

Clinical evidences suggest that an imbalance between descending inhibition and facilitation drives the development of chronic pain. However, potential mechanisms promoting the establishment of a persistent pain state and the increased pain vulnerability remain unknown. This preclinical study was designed to evaluate temporal changes in descending pain modulation at specific experimental endpoints (12, 28, 90 and 168 days) using a novel double-hit model of chronic/tonic pain (first hit: chronic constriction injury (CCI) model; second hit: tonic formalin pain in the contralateral hindpaw). Basal activity of bulbo-spinal monoaminergic systems was further assessed through liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) screening of cerebrospinal fluid (CSF). We found that CCI-operated rats exhibited a reduced nociceptive response profile, peaking on day 28, when subjected to tonic pain. This behavioral response was accompanied by a rapid increase in basal CSF serotonin and norepinephrine levels 12 days after neuropathy, followed by a return to sham levels on day 28. These molecular and behavioral adaptive changes in descending pain inhibition seemed to slowly fade over time. We therefore suggest that chronic neuropathic pain produces a transient hyperactivation of bulbo-spinal monoaminergic drive when previously primed using a tonic pain paradigm (i.e., formalin test), translating into inhibition of subsequent nociceptive behaviors. Altogether, we propose that early hyperactivation of descending pain inhibitory mechanisms, and its potential ensuing exhaustion, could be part of the temporal neurophysiological chain of events favoring chronic neuropathic pain establishment.

Long-term cognitive deficits are observed after treatment of brain tumors or metastases by radiotherapy. Treatment optimization thus requires a better understanding of the effects of radiotherapy on specific brain regions, according to their sensitivity and interconnectivity. In the present study, behavioral tests supported by immunohistology and magnetic resonance imaging provided a consistent picture of the persistent neurocognitive decline and neuroinflammation after the onset of irradiation-induced necrosis in the right primary somatosensory cortex of Fischer rats. Necrosis surrounded by neovascularization was first detected 54 days after irradiation and then spread to 110 days in the primary motor cortex, primary somatosensory region, striatum and right ventricle, resulting in fiber bundle disruption and demyelination in the corpus callosum of the right hemisphere. These structural damages translated into selective behavioral changes including spatial memory loss, disinhibition of anxiety-like behaviors, hyperactivity and pain hypersensitivity, but no significant alteration in motor coordination and grip strength abilities. Concomitantly, activated microglia and reactive astrocytes, accompanied by infiltration of leukocytes (CD45+) and T-cells (CD3+) cooperated to shape the neuroinflammation response. Overall, our study suggests that the slow and gradual onset of cellular damage would allow adaptation in brain regions that are susceptible to neuronal plasticity; while other cerebral structures that do not have this capacity would be more affected. The planning of radiotherapy, adjusted to the sensitivity and adaptability of brain structures, could therefore preserve certain neurocognitive functions; while higher doses of radiation could be delivered to brain areas that can better adapt to this treatment. In addition, strategies to block early post-radiation events need to be explored to prevent the development of long-term cognitive dysfunction.

Compounds acting via the GPCR neurotensin receptor type 2 (NTS2) display analgesia in relevant preclinical models. The amide bond in nonpeptide NTS1 antagonists plays a central role in receptor recognition and molecular conformation. Using NTS2 FLIPR and binding assays, we found that it is also a key molecular structure for binding and calcium mobilization at NTS2. We found that reversed amides display a shift from agonist to antagonist activity and provided examples of the first competitive nonpeptide antagonists observed in the NTS2 FLIPR assay. These compounds will be valuable tools for determining the role of calcium signaling in vitro to NTS2 mediated analgesia.

Compounds acting via the GPCR neurotensin receptor type 2 (NTS2) display analgesic effects in relevant animal models. Using a pharmacophore model based on known NT receptor nonpeptide compounds, we screened commercial databases to identify compounds that might possess activity at NTS2 receptor sites. Modification of our screening hit to include structural features known to be recognized by NTS1 and NTS2, led to the identification of the novel NTS2 selective nonpeptide, N-{[6-chloro-4-(2,6-dimethoxyphenyl)quinazolin-2-yl]carbonyl}-l-leucine (9). This compound is a potent partial agonist in the FLIPR assay with a profile of activity similar to that of the reference NTS2 analgesic nonpeptide levocabastine (5).

Processing of polypeptide precursors by proprotein convertases (PCs) such as furin typically occurs within the trans-Golgi network. Here, we show in a variety of cell types that the propeptide of ADAMTS9 is not excised intracellularly. Pulse-chase analysis in HEK293F cells indicated that the intact zymogen was secreted to the cell surface and was subsequently processed there before release into the medium. The processing occurred via a furin-dependent mechanism as shown using PC inhibitors, lack of processing in furin-deficient cells, and rescue by furin in these cells. Moreover, down-regulation of furin by small interference RNA reduced ADAMTS9 processing in HEK293F cells. PC5A could also process pro-ADAMTS9, but similarly to furin, processed forms were absent intracellularly. Cell-surface, furin-dependent processing of pro-ADAMTS9 creates a precedent for extracellular maturation of endogenously produced secreted proproteins. It also indicates the existence of a variety of mechanisms for processing of ADAMTS proteases.

We have characterized ADAMTS7B, the authentic full-length protein product of the ADAMTS7 gene. ADAMTS7B has a domain organization similar to that of ADAMTS12, with a total of eight thrombospondin type 1 repeats in its ancillary domain. Of these, seven are arranged in two distinct clusters that are separated by a mucin domain. Unique to the ADAMTS family, ADAMTS7B is modified by attachment of the glycosaminoglycan chondroitin sulfate within the mucin domain, thus rendering it a proteoglycan. Glycosaminoglycan addition has potentially important implications for ADAMTS7B cellular localization and for substrate recognition. Although not an integral membrane protein, ADAMTS7B is retained near the cell surface of HEK293F cells via interactions involving both the ancillary domain and the prodomain. ADAMTS7B undergoes removal of the prodomain by a multistep furin-dependent mechanism. At least part of the final processing event, i.e. cleavage following Arg(220) (mouse sequence annotation), occurs at the cell surface. ADAMTS7B is an active metalloproteinase as shown by its ability to cleave alpha(2)-macroglobulin, but it does not cleave specific peptide bonds in versican and aggrecan attacked by ADAMTS proteases. Together with ADAMTS12, whose primary structure also predicts a mucin domain, ADAMTS7B constitutes a unique subgroup of the ADAMTS family.

The metalloprotease ADAMTS-1 (a disintegrin and metalloprotease with thrombospondin type I motif), similarly to other members of the ADAMTS family, is initially synthesized as a zymogen, proADAMTS-1, that undergoes proteolytic processing at the prodomain/catalytic domain junction by serine proteinases of the furin-like family of proprotein convertases. The goals of this study were to identify residues of the prodomain that play an essential role in ADAMTS-1 processing and to determine the identity of the convertase required for zymogen processing. To gain insight into the putative roles of specific prodomain residues in ADAMTS-1 biosynthesis, we performed biosynthetic labeling experiments in transiently transfected human embryonic kidney 293 cells expressing wild-type and prodomain mutants of proADAMTS-1. Cells expressing wild-type ADAMTS-1 initially produced a 110-kDa zymogen form that was later converted to an 87-kDa form, which was also detected in the media. Although convertases such as PACE4 and PC6B processed proADAMTS-1, we found that furin was the most efficient enzyme at producing the mature ADAMTS-1 87-kDa moiety. Site-directed mutagenesis of the two putative furin recognition sequences found within the ADAMTS-1 prodomain (RRNR173 and RKKR235) revealed that Arg235 was the sole processing site. Use of the Golgi disturbing agent, Brefeldin A, and monensin suggests that the cleavage of proADAMTS-1 takes place in the Golgi apparatus prior to its secretion. Conserved residues within the prodomain of other ADAMTS members hinted that they might act as maturation determinants. Replacement with alanine of selected residues Cys106, Tyr108, Gly110, Cys125, and Cys181 and residues encompassing the 137-144 sequence significantly affected the biosynthetic profile of the enzyme. Our results suggest that conserved residues other than the furin cleavage site in the prodomain of ADAMTS-1 are involved in its biosynthesis.