An essential role for LPA signalling in telencephalon development.

Lysophosphatidic acid (LPA) has wide-ranging effects on many different cell types, acting through G-protein-coupled receptors such as LPAR6. We show that Xenopus lpar6 is expressed from late blastulae and is enriched in the mesoderm and dorsal ectoderm of early gastrulae. Expression in gastrulae is an early response to FGF signalling. Transcripts for lpar6 are enriched in the neural plate of Xenopus neurulae and loss of function caused forebrain defects, with reduced expression of telencephalic markers (foxg1, emx1 and nkx2-1). Midbrain (en2) and hindbrain (egr2) markers were unaffected. Foxg1 expression requires LPAR6 within ectoderm and not mesoderm. Head defects caused by LPAR6 loss of function were enhanced by co-inhibiting FGF signalling, with defects extending into the hindbrain (en2 and egr2 expression reduced). This is more severe than expected from simple summation of individual defects, suggesting that LPAR6 and FGF have overlapping or partially redundant functions in the anterior neural plate. We observed similar defects in forebrain development in loss-of-function experiments for ENPP2, an enzyme involved in the synthesis of extracellular LPA. Our study demonstrates a role for LPA in early forebrain development.

Altered Trek-1 Function in Sortilin Deficient Mice Results in Decreased Depressive-Like Behavior.

The background potassium channel TREK-1 has been shown to be a potent target for depression treatment. Indeed, deletion of this channel in mice resulted in a depression resistant phenotype. The association of TREK-1 with the sorting protein sortilin prompted us to investigate the behavior of mice deleted from the gene encoding sortilin (Sort1-/-). To characterize the consequences of sortilin deletion on TREK-1 activity, we combined behavioral, electrophysiological and biochemical approaches performed in vivo and in vitro. Analyses of Sort1-/- mice revealed that they display: (1) a corticosterone-independent anxiety-like behavior, (2) a resistance to depression as demonstrated by several behavioral tests, and (3) an increased activity of dorsal raphe nucleus neurons. All these properties were associated with TREK-1 action deficiency consequently to a decrease of its cell surface expression and to the modification of its electrophysiological activity. An increase of BDNF expression through activation of the furin-dependent constitutive pathway as well as an increase of the activated BDNF receptor TrkB were in agreement with the decrease of depressive-like behavior of Sort1-/- mice. Our results demonstrate that the TREK-1 expression and function are altered in the absence of sortilin confirming the importance of this channel in the regulation on the mood as a crucial target to treat depression.

A novel nucleotide receptor in Xenopus activates the cAMP second messenger pathway.

We describe a Xenopus P2Y receptor that shares only weak homology with members of the mammalian P2Y family, being most similar to human P2Y(11). When activated by nucleotide analogs, it stimulates both calcium and cAMP mobilization pathways, a feature unique, among mammalian P2Y receptors, to P2Y(11). Activity can be blocked by compounds known to act as antagonists of mammalian P2Y(11). Genomic synteny between Xenopus and mammals suggests that the novel gene is a true ortholog of P2Y(11). Xenopus P2Y(11) is transcribed during embryonic development, beginning at gastrulation, and is enriched in the developing nervous system.

Serum sortilin-derived propeptides concentrations are decreased in major depressive disorder patients.

BACKGROUND: Despite intense research on mechanisms underlying the depressive pathophysiology, reliable biomarkers to assess antidepressant treatment response are still lacking. Since the sortilin-derived propeptide (PE) displays potent antidepressant activities and can be measured in the blood of rodents, we wondered whether in human its seric level can vary between patients affected by major depressive disorder (MDD) and healthy controls and after antidepressant treatment. METHODS: By using a specific dosing method, characterized by structure-recognition analysis with various synthesized PE analogues, we conducted a translational study to test whether blood levels of PE are under pathophysiological regulation and could serve as biomarkers of the depression state. RESULTS: The serum concentration of PE, a peptide displaying potent antidepressant activities in rodents, is decreased in patients affected by major depressive disorder (MDD) when compared to healthy non-psychiatric controls cohort (p=0.035). Interestingly, pharmacological antidepressant treatments restore normal PE levels. LIMITATIONS: The limitation of the study concerns the relatively small patient samples that could negatively affect the likelihood that a nominally statistically significant finding actually reflects a true effect. CONCLUSIONS: The longitudinal quantification of the serum PE concentration could assist psychiatrists in the diagnosis of antidepressant response efficacy, and the need to modify the therapeutic strategy.

Xenopus apyrase (xapy), a secreted nucleotidase that is expressed during early development.

We have characterized a cDNA encoding a Xenopus laevis apyrase (XAPY) that is expressed during embryogenesis. XAPY is highly homologous to two recently described mammalian apyrases, human SCAN-1 and rat Ca2+-NDPase, and to a lesser extent the salivary apyrase of the blood-feeding arthropod Cimex lectularis. RT-PCR analysis shows that Xapy is expressed at all the developmental stages tested, from oocytes through to tadpoles. Xapy transcripts are widely distributed in the embryo, but from late neurulae through to late tailbud stages they are highly enriched in the cement gland, an adhesive organ in the epidermis of the head. When expressed in HEK 293 cells, XAPY is largely retained in the endoplasmic reticulum, although some is also secreted. XAPY conditioned media hydrolyses UDP and UTP, confirming that it is a functional apyrase.

Increased Brain Neurotensin and NTSR2 Lead to Weak Nociception in NTSR3/Sortilin Knockout Mice.

The neuropeptide neurotensin (NT) elicits numerous pharmacological effects through three different receptors (NTSR1, NTSR2, and NTSR3 also called sortilin). Pharmacological approaches and generation of NTSR1 and NTSR2-deficient mice allowed to determine the NT-induced antipsychotic like behavior, the inhibitory of weak fear memory and the nociceptive signaling in a rat formalin tonic pain model to NTSR1. Conversely, the effects of NT on thermal and tonic nociceptions were mediated by NTSR2. However, the role of NTSR3/sortilin on the neurotensinergic system was not investigated. Here, by using C57Bl/6J mouse model in which the gene coding for NTSR3/sortilin has been inactivated, we observed a modification of the expression of both NTSR2 and NT itself. Quantitative PCR and protein expression using Western blot analyses and AlphaLisa™ technology resulted in the observation that brain NTSR2 as well as brain and blood NT were 2-fold increased in KO mice leading to a resistance of these mice to thermal and chemical pain. These data confirm that NTSR3/sortilin interacts with other NT receptors (i.e., NTSR2) and that its deletion modifies also the affinity of this receptor to NT.

Potentiation of Calcium Influx and Insulin Secretion in Pancreatic Beta Cell by the Specific TREK-1 Blocker Spadin.

Inhibition of the potassium channels TREK-1 by spadin (SPA) is currently thought to be a promising therapeutic target for the treatment of depression. Since these channels are expressed in pancreatic ß-cells, we investigated their role in the control of insulin secretion and glucose homeostasis. In this study, we confirmed the expression of TREK-1 channels in the insulin secreting MIN6-B1 ß-cell line and in mouse islets. We found that their blockade by SPA potentiated insulin secretion induced by potassium chloride dependent membrane depolarization. Inhibition of TREK-1 by SPA induced a decrease of the resting membrane potential (ΔVm ~ 12 mV) and increased the cytosolic calcium concentration. In mice, administration of SPA enhanced the plasma insulin level stimulated by glucose, confirming its secretagogue effect observed in vitro. Taken together, this work identifies SPA as a novel potential pharmacological agent able to control insulin secretion and glucose homeostasis.

Impairement of HT29 Cancer Cells Cohesion by the Soluble Form of Neurotensin Receptor-3.

The neurotensin (NT) receptor-3 (NTSR3), also called sortilin is a multifunctional protein localized at the intracellular and plasma membrane level. The extracellular domain of NTSR3 (sNTSR3) is released by shedding from several cell lines including colonic cancer cells. This soluble protein acts as an active ligand through its ability to bind, to be internalized in the human adenocarcinoma epithelial HT29 cells and to stimulate the PI3 kinase pathway. The aim of this study was to investigate cellular responses induced by sNTSR3 in HT29 cells. The cellular functions of sNTSR3 were monitored by immunofluocytochemistry, electron microscopy and quantitative PCR in order to characterize the cell shape and the expression of adhesion proteins. We evidenced that sNTSR3 significantly regulates the cellular morphology as well as the cell-cell and the cell-matrix adherens properties by decreasing the expession of several integrins and by modifying the structure of desmosomes. Altogether, these properties lead to an increase of cell detachment upon sNTSR3 treatment on HT29, HCT116 and SW620 cancer cells. Our results indicate that sNTSR3 may induce the first phase of a process which weaken HT29 epithelial properties including desmosome architecture, cell spreading, and initiation of cell separation, all events which could be responsible for cancer metastasis.

The anti-apoptotic role of neurotensin.

The neuropeptide, neurotensin, exerts numerous biological functions, including an efficient anti-apoptotic role, both in the central nervous system and in the periphery. This review summarizes studies that clearly evidenced the protective effect of neurotensin through its three known receptors. The pivotal involvement of the neurotensin receptor-3, also called sortilin, in the molecular mechanisms of the anti-apoptotic action of neurotensin has been analyzed in neuronal cell death, in cancer cell growth and in pancreatic beta cell protection. The relationships between the anti-apoptotic role of neurotensin and important physiological and pathological contexts are discussed in this review.

Focal adhesion kinase dependent activation of the PI3 kinase pathway by the functional soluble form of neurotensin receptor-3 in HT29 cells.

The neurotensin (NT) receptor-3 (NTSR3), also called sortilin, is thought to display several functions including a role as a receptor or a co-receptor, in the sorting to plasma membrane and to lysosomes, and in the regulated secretion. The aim of this study was to investigate the function of the soluble form of NTSR3 (sNTSR3) released from several cell lines including colonic cancer cells. The human adenocarcinoma epithelial cell line HT29 has been used to monitor the release, the binding and internalization of sNTSR3 by radioreceptor assays and confocal microscopy. The modulation of the intracellular signaling pathways by the protein has been investigated by using Fura-2 fluorescence calcium imaging microscopy and Western blots analysis. We demonstrated that sNTSR3 specifically binds and internalizes into HT29 cells. This binding, independent from the transactivation of the epidermal growth factor receptor, leads to the increase of intracellular calcium concentration and to the activation of a FAK/Src-dependent activation of the PI3 kinase pathway. In conclusion, sNTSR3 released from the membrane bound NTSR3 is a functional protein able to activate intracellular pathways involved in cell survival but probably not in cell growth.

Neurotensin and its receptors in the control of glucose homeostasis.

The pharmacological roles of the neuropeptide neurotensin through its three known receptors are various and complex. Neurotensin is involved in several important biological functions including analgesia and hypothermia in the central nervous system and also food intake and glucose homeostasis in the periphery. This review focuses on recent works dealing with molecular mechanisms regulating blood glucose level and insulin secretion upon neurotensin action. Investigations on crucial cellular components involved in the protective effect of the peptide on beta cells are also detailed. The role of xenin, a neurotensin-related peptide, on the regulation of insulin release by glucose-dependent insulinotropic polypeptide is summarized. The last section comments on the future research areas which should be developed to address the function of new effectors of the neurotensinergic system in the endocrine pancreas.

A human TREK-1/HEK cell line: a highly efficient screening tool for drug development in neurological diseases.

TREK-1 potassium channels are involved in a number of physiopathological processes such as neuroprotection, pain and depression. Molecules able to open or to block these channels can be clinically important. Having a cell model for screening such molecules is of particular interest. Here, we describe the development of the first available cell line that constituvely expresses the TREK-1 channel. The TREK-1 channel expressed by the h-TREK-1/HEK cell line has conserved all its modulation properties. It is opened by stretch, pH, polyunsaturated fatty acids and by the neuroprotective molecule, riluzole and it is blocked by spadin or fluoxetine. We also demonstrate that the h-TREK-1/HEK cell line is protected against ischemia by using the oxygen-glucose deprivation model.