Biomedical subjects

Patrick Kitabgi

Publications and source records attributed to Patrick Kitabgi.

At least 19 recordsLinked to original sources

PubMed2006

Dendrite-selective redistribution of the chemokine receptor CXCR4 following agonist stimulation.

The chemokine SDF-1 is a secreted protein that plays a critical role in several aspects of neuron development through interaction with its unique receptor CXCR4. A key mechanism that controls neuron responsiveness to extracellular signals during neuronal growth is receptor endocytosis. Since we previously reported that SDF-1 regulates axon development without affecting the other neurites, we asked whether this could correlate with a compartment-selective trafficking of CXCR4. We thus studied CXCR4 behavior upon SDF-1 exposure in rat hippocampus slices and in transfected neuron cultures. A massive agonist-induced redistribution of CXCR4 in endosomes was observed in dendrites whereas no modification was evidenced in axons. Our data suggest that CXCR4 trafficking may play a role in mediating selective effects of SDF-1 on distinct neuronal membrane subdomains.

Animals↗

PubMed2006

Inactivation of neurotensin and neuromedin N by Zn metallopeptidases.

The two related peptides neurotensin (NT) and neuromedin N (NN) are efficiently inactivated by peptidases in vitro. Whereas NT is primarily degraded by a combination of three Zn metallo-endopeptidases, namely endopeptidases 24.11, 24.15 and 24.16, in all systems examined, NN is essentially inactivated by the Zn metallo-exopeptidase aminopeptidase M. In this paper we review the work that has led to the identification of the NT- and NN-degrading enzymes and to the purification and cloning of EP 24.16, a previously unidentified peptidase. We provide a brief description of the three NT-inactivating endopeptidases and of their specific and mixed inhibitors, some of them developed in the course of studying NT degradation. Finally, we review in vivo data obtained with these inhibitors that strongly support a physiological role for EP 24.11, 24.15 and 24.16 in the termination of NT-generated signals and for aminopeptidase in terminating NN action. Knowledge of the NT and NN inactivation mechanisms offers the perspective to develop metabolically stable analogs of these peptides with potential therapeutic value.

Amino Acid Sequence↗

PubMed2006

Functional domains of the subtype 1 neurotensin receptor (NTS1).

The subtype 1 neurotensin receptor (NTS1) belongs to the family of G protein coupled receptors with seven transmembrane domains and mediates most of the known effects of neurotensin. In the past years, mutagenesis studies have allowed to delineate functional regions of the receptor involved in agonist and antagonist binding, G protein coupling, sodium sensitivity of agonist binding, and agonist-induced receptor internalization. These data are reviewed and discussed in the present paper.

Amino Acid Sequence↗

PubMed2006

Differential processing of pro-neurotensin/neuromedin N and relationship to pro-hormone convertases.

Neurotensin (NT) is synthesized as part of a larger precursor that also contains neuromedin N (NN), a six amino acid neurotensin-like peptide. NT and NN are located in the C-terminal region of the precursor (pro-NT/NN) where they are flanked and separated by three Lys-Arg sequences. A fourth dibasic sequence is present in the middle of the precursor. Dibasics are the consensus sites recognized and cleaved by endoproteases that belong to the recently identified family of pro-protein convertases (PCs). In tissues that express pro-NT/NN, the three C-terminal Lys-Arg sites are differentially processed, whereas the middle dibasic is poorly cleaved. Pro-NT/NN processing gives rise mainly to NT and NN in the brain, to NT and a large peptide ending with the NN sequence at its C-terminus (large NN) in the gut and to NT, large NN and a large peptide ending with the NT sequence (large NT) in the adrenals. Recent evidence indicates that PC1, PC2 and PC5-A are the pro-hormone convertases responsible for the processing patterns observed in the gut, brain and adrenals, respectively. As NT, NN, large NT and large NN are all endowed with biological activity, the evidence reviewed here supports the idea that post-translational processing of pro-NT/NN in tissues may generate biological diversity.

Adrenal Glands↗

PubMed2006

The chemokine SDF-1/CXCL12 modulates the firing pattern of vasopressin neurons and counteracts induced vasopressin release through CXCR4.

Chemokines play a key role in inflammation. They are expressed not only in neuroinflammatory conditions, but also constitutively by different cell types, including neurons in the normal brain, suggesting that they may act as modulators of neuronal functions. Here, we investigated a possible neuroendocrine role of the chemokine stromal cell-derived factor 1 (SDF-1)/CXCL12. We demonstrated the colocalization of SDF-1 and its receptor CXCR4 with arginine vasopressin (AVP) in the magnocellular neurons of the supraoptic nucleus (SON) and the paraventricular hypothalamic nucleus and on AVP projections to the neurohypophysis. Electrophysiological recordings of SON neurons demonstrated that SDF-1 affects the electrical activity of AVP neurons through CXCR4, resulting in changes in AVP release. We observed that SDF-1 can blunt the autoregulation of AVP release in vitro and counteract angiotensin II-induced plasma AVP release in vivo. Furthermore, a short-term physiological increase in AVP release induced by enhanced plasma osmolarity, which was produced by the administration of 1 M NaCl i.p., was similarly blocked by central injection of SDF-1 through CXCR4. A change in water balance by long-term salt loading induced a decrease in both SDF-1 and CXCR4 parallel to that of AVP immunostaining in SON. From these data, we demonstrate that chemokine actions in the brain are not restricted to inflammatory processes. We propose to add to the known autoregulation of AVP on its own neurons, a second autocrine system induced by SDF-1 able to modulate central AVP neuronal activity and release.

Action Potentials↗

PubMed2006

Prohormone convertases differentially process pro-neurotensin/neuromedin N in tissues and cell lines.

Neurotensin (NT) is synthesized as part of a larger precursor that also contains neuromedin N (NN), a six-amino acid neurotensin-like peptide. NT and NN are located in the C-terminal region of the precursor (pro-NT/NN) where they are flanked and separated by three Lys-Arg sequences. A fourth dibasic sequence is present in the middle of the precursor. Dibasics are the consensus sites recognized and cleaved by specialized endoproteases that belong to the family of proprotein convertases (PCs). In tissues that express pro-NT/NN, the three C-terminal Lys-Arg sites are differentially processed, whereas the middle dibasic is poorly cleaved. Processing gives rise mainly to NT and NN in the brain, to NT and a large peptide with a C-terminal NN moiety (large NN) in the gut, and to NT, large NN, and a large peptide with a C-terminal NT moiety (large NT) in the adrenals. Recent evidence indicates that PC1, PC2, and PC5-A are the prohormone convertases responsible for the processing patterns observed in the gut, brain, and adrenals, respectively. As NT, NN, large NT, and large NN are all endowed with biological activity, the evidence reviewed in this paper supports the idea that posttranslational processing of pro-NT/NN in tissues may generate biological diversity of pathophysiological relevance.

Adrenal Glands↗

PubMed2005

Constitutive neuronal expression of CCR2 chemokine receptor and its colocalization with neurotransmitters in normal rat brain: functional effect of MCP-1/CCL2 on calcium mobilization in primary cultured neurons.

Chemokines and their receptors are well described in the immune system, where they promote cell migration and activation. In the central nervous system, chemokine has been implicated in neuroinflammatory processes. However, an increasing number of evidence suggests that they have regulatory functions in the normal nervous system, where they could participate in cell communication. In this work, using a semiquantitative immunohistochemistry approach, we provide the first neuroanatomical mapping of constitutive neuronal CCR2 localization. Neuronal expression of CCR2 was observed in the anterior olfactory nucleus, cerebral cortex, hippocampal formation, caudate putamen, globus pallidus, supraoptic and paraventricular hypothalamic nuclei, amygdala, substantia nigra, ventral tegmental area, and in the brainstem and cerebellum. These data are largely in accordance with results obtained using quantitative autoradiography with [(125)I]MCP-1/CCL2 and RT-PCR CCR2 mRNA analysis. Furthermore, using dual fluorescent immunohistochemistry we studied the chemical phenotype of labeled neurons and demonstrated the coexistence of CCR2 with classical neurotransmitters. Indeed, localization of CCR2 immunostaining is observed in dopaminergic neurons in the substantia nigra pars compacta and in the ventral tegmental area as well as in cholinergic neurons in the substantia innominata and caudate putamen. Finally, we show that the preferential CCR2 ligand, MCP-1/CCL2, elicits Ca(2+) transients in primary cultured neurons from various rat brain regions including the cortex, hippocampus, hypothalamus, and mesencephalon. In conclusion, the constitutive neuronal CCR2 expression in selective brain structures suggests that this receptor could be involved in neuronal communication and possibly associated with cholinergic and dopaminergic neurotransmission and related disorders.

Animals↗

PubMed2005

Constitutive expression of CCR2 chemokine receptor and inhibition by MCP-1/CCL2 of GABA-induced currents in spinal cord neurones.

In the CNS, immune-like competent cells (microglia and astrocytes) were first described as potential sites of chemokine synthesis, but more recent evidence has indicated that neurones might also express chemokines and their receptors. The aim of the present work was to investigate further, both in vivo and in vitro, CC Chemokine Family Receptor 2 (CCR2) expression and functionality in rat spinal cord neurones. First, we demonstrated by RT-PCR and western blot analysis that CCR2 mRNA and protein were present in spinal extracts. Furthermore, we showed by immunolabelling that CCR2 was exclusively expressed by neurones in spinal sections of healthy rat. Finally, to test the functionality of CCR2, we used primary cultures of rat spinal neurones. In this model, similar to what was observed in vivo, CCR2 mRNA and protein were expressed by neurones. Cultured neurones stimulated with Monocyte Chemoattractant Protein-1 (MCP-1)/CCL2, the best characterized CCR2 agonist, showed activation of the Akt pathway. Finally, patch-clamp recording of cultured spinal neurones was used to investigate whether MCP-1/CCL2 could modulate their electrophysiological properties. MCP-1 alone did not affect the electrical properties of spinal neurones, but potently and efficiently inhibited GABA(A)-mediated GABAergic responses in these neurones. These data constitute the first demonstration of a modulatory role of MCP-1 on GABAergic neurotransmission and contribute to our understanding of the roles of CCR2 and MCP-1/CCL2 in spinal cord physiology, in particular with respect to nociceptive transmission, as well as the implication of this chemokine in neuronal adaptation or dysfunction during neuropathy.

Animals↗

PubMed2005

Highly regionalized neuronal expression of monocyte chemoattractant protein-1 (MCP-1/CCL2) in rat brain: evidence for its colocalization with neurotransmitters and neuropeptides.

The monocyte chemoattractant protein-1 (MCP-1/CCL2) and its receptor CCR2 are key modulators of immune functions. In the nervous system, MCP-1/CCL2 is implicated in neuroinflammatory pathologies. However, cerebral functions of MCP-1/CCL2 under normal conditions are still unclear. In this study, using reverse transcriptase-polymerase chain reaction (RT-PCR) and specific rat MCP-1 enzyme-linked immunosorbent assay (ELISA) approaches, we observed that MCP-1/CCL2 mRNA and protein were expressed in different punched regions of the normal rat central nervous system. Immunohistochemical studies further revealed that this chemokine is constitutively expressed not only in astrocytes but also in neurons, in discrete neuroanatomical regions. Neuronal expression of MCP-1/CCL2 is mainly found in the cerebral cortex, globus pallidus, hippocampus, paraventricular and supraoptic hypothalamic nuclei, lateral hypothalamus, substantia nigra, facial nuclei, motor and spinal trigeminal nuclei, and gigantocellular reticular nucleus and in Purkinje cells in the cerebellum. Moreover, we obtained the first evidence that MCP-1/CCL2 is constitutively expressed in cholinergic neurons, notably in the magnocellular preoptic and oculomotor nuclei, and in dopaminergic neurons of the substantia nigra pars compacta. In addition, in the lateral hypothalamic area, MCP-1/CCL2 co-localized with melanin-concentrating hormone-expressing neurons. Interestingly, we demonstrate a co-localization of MCP-1/CCL2 with vasopressin in magnocellular neuronal cell bodies and processes in the supraoptic and paraventricular hypothalamic nuclei, as well as in processes in the internal layer of the median eminence and in the posterior pituitary. Taken together, our data suggest that MCP-1/CCL2 could act as a modulator of neuronal activity and neuroendocrine functions.

Animals↗

PubMed2005

The chemokine SDF-1 differentially regulates axonal elongation and branching in hippocampal neurons.

Recent data have shown that the chemokine SDF-1 plays a critical role in several aspects of brain development such as cell migration and axon pathfinding. However, its potential function in the generation of axons and dendrites is poorly characterized. In order to better understand the role of SDF-1 in the development of central neurons, we studied the cellular distribution of the SDF-1 receptor CXCR4 by immunocytochemistry of developing hippocampal neurons and tested the effect of SDF-1 in process patterning at the early stages of neuronal development. We found that CXCR4 immunoreactivity undergoes a striking redistribution during development. At the early stages, from day 2 to day 4 in culture, CXCR4 is particularly concentrated at the leading edge of growing neurites. As the cells mature, staining declines at the tip of the processes and becomes more broadly distributed along axons and, to a lesser extent, dendrites. SDF-1 stimulation of neurons at day 1-2 in culture triggers several effects on neuronal morphogenesis. SDF-1 reduces growth cone number and axonal outgrowth but stimulates axonal branching. These latter two effects are not observed in other neurites. This study unravels a new role for SDF-1/CXCR4 in specifying hippocampal neuron morphology by regulating axonal patterning at an early stage of neuronal development.

Animals↗

PubMed2005

Chemokines and brain functions.

Chemokines are small secreted proteins that chemoattract and activate immune and non-immune cells both in vivo and in vitro. Besides their well-established role in the immune system, several recent reports have suggested that chemokines and their receptors may also play a role in the central nervous system (CNS). The best-known central action is their ability to act as immuno-inflammatory mediators. Indeed, these proteins regulate the leukocyte infiltration in the brain during inflammatory and infectious diseases. However, recent studies clearly demonstrate that chemokines and their receptors are constitutively expressed by glial and neuronal cells in the CNS, where they are involved in intercellular communication. The goal of this review is to summarize recent information concerning the role of chemokines in brain functions. The first part will focus on the expression of chemokines and their receptors in the CNS with the main spotlight on the neuronal expression. In the second part, we will discuss the role of chemokines and their receptors in normal brain physiology. Because several chemokines are involved in neuroinflammatory and neurodegenerative disorders, the role of chemokines and their receptors in these diseases is reviewed further in this section. In conclusion, the implication of chemokines in cellular communication could allow: i) to identify a new pathway for neuron-neuron and/or glia-glia and/or neuron-glia communications that are relevant to both normal brain function and neuroinflammatory and neurodegenerative diseases; ii) to develop new therapeutic approaches for still untreatable diseases further.

Animals↗

PubMed2003

Highly regionalized distribution of stromal cell-derived factor-1/CXCL12 in adult rat brain: constitutive expression in cholinergic, dopaminergic and vasopressinergic neurons.

The stromal cell-derived factor-1 (SDF-1)/CXCL12 and its receptor CXCR4 are key modulators of immune functions. In the nervous system, SDF-1/CXCL12 is crucial for neuronal guidance in developing brain, intercellular communication and the neuropathogenesis of acquired immunodeficiency syndrome. However, cerebral functions of SDF-1/CXCL12 in adult brain are poorly understood. The understanding of its role in the adult brain needs a detailed neuroanatomical mapping of SDF-1/CXCL12. By dual immunohistochemistry we demonstrate that this chemokine is constitutively expressed not only in astrocytes and microglia but also in neurons, in discrete neuroanatomical regions. Indeed, neuronal expression of SDF-1/CXCL12 is mainly found in cerebral cortex, substantia innominata, globus pallidus, hippocampus, paraventricular and supraoptic hypothalamic nuclei, lateral hypothalamus, substantia nigra and oculomotor nuclei. Moreover, we provide the first evidence that SDF-1/CXCL12 is constitutively expressed in cholinergic neurons in the medial septum and substantia innominata and in dopaminergic neurons in substantia nigra pars compacta and the ventral tegmental area. Interestingly we also show, for the first time, a selective co-localization of SDF-1/CXCL12 with vasopressin-expressing neurons in the supraoptic and paraventricular hypothalamic nuclei. In addition, in the lateral hypothalamic area, SDF-1/CXCL12 was found to be located on melanin concentrating hormone-expressing neurons. Altogether, these original data suggest that SDF-1/CXCL12 could be a modulatory neuropeptide regulating both central cholinergic and dopaminergic systems. In addition, a key role for SDF-1/CXCL12 in neuroendocrine regulation of vasopressin-expressing neurons represents an exciting new field of research.

Animals↗

PubMed2003

The Arg617-Arg618 cleavage site in the C-terminal domain of PC1 plays a major role in the processing and targeting of the enzyme within the regulated secretory pathway.

The C-terminal domain of the prohormone convertase PC1 is involved in targeting of the enzyme to secretory granules in neuroendocrine cells and is subsequently processed in this compartment at an Arg617-Arg618 site. Three other dibasics are found in the C-terminal domain of mouse PC1. Here, we examined the role of the four dibasics in targeting PC1 to secretory granules. All 15 possible combinations of dibasic mutations were performed. Wild-type (WT) and mutant PC1 were stably expressed in neuroendocrine PC12 cells that lacked endogenous PC1. Processing, secretion and intracellular localization of PC1 and its mutants were analyzed. Leaving intact Arg617-Arg618 and mutating any combination of the three other dibasics yielded proteins that were stored and processed in secretory granules, similarly to WT PC1. Mutating Arg617-Arg618 alone or with any one of the three remaining dibasics generated proteins that were efficiently stored in secretory granules but were not processed further. Mutating Arg617-Arg618 with more than one of the remaining dibasics produced proteins that reached the TGN but were not stored in secretory granules and exited the cells through the constitutive secretory pathway. These data demonstrate that the Arg617-Arg618 plays a prominent role in targeting PC1 to secretory granules.

Amino Acid Substitution↗

PubMed2002

Production of recombinant large proneurotensin/neuromedin N-derived peptides and characterization of their binding and biological activity.

Proneurotensin/neuromedin N (pro-NT/NN) is the common precursor of two biologically active related peptides, neuromedin N (NN) and neurotensin (NT). It undergoes a tissue-specific processing leading to the formation in some tissues and cancer cell lines of large peptides ending with the NT (large NT) or NN (large NN) sequence. In this study, we prepared and purified high amounts of recombinant large NT and large NN using the Drosophila S2 cell expression system. The binding and pharmacological properties of recombinant large peptides were characterized and compared to those of NT and NN using either COS cells transfected with the human subtype-1 NT receptor (hNTS1) or the human colon adenocarcinoma HT29 cell line that endogenously expresses hNTS1. Furthermore, the metabolic stability of the large peptides, when exposed to HT29 cells, was compared to that of NT and NN. Both large NT and large NN were able to bind to and activate hNTS1 with potencies that were approximately 10 times lower than that of their small counterpart. In addition, the large forms proved to be far less sensitive to degradation than the small peptides. Taken together, these data suggest that the large forms might represent endogenous, long-lasting activators of hNTS1 in a number of physiopathological situations.

Animals↗

PubMed2002

Insertion of dibasic residues directs a constitutive protein to the regulated secretory pathway.

The mechanisms for sorting proteins to the regulated secretory pathway (RSP) remains poorly understood. We recently reported that dibasic sequences that are cleaved by pro-protein convertases (PCs) in pro-neurotensin also acted as sorting signal for the precursor. Here we addressed two questions regarding the role of dibasics as sorting signal: (i) Are dibasics sufficient to direct proteins to the RSP? (ii) Do they sort proteins by virtue of their interaction with PCs? The first question was studied by inserting dibasics in beta-lactamase, a constitutively secreted protein and comparing the regulated secretion of beta-lactamase to that of its mutant in transfected endocrine cells. The second question was investigated by comparing the regulated release of pro-neurotensin in PC12 cells that are devoid of PCs to that in PC1- and PC2-transfected PC12 cells. The data show that the mutant beta-lactamase was indeed targeted in part to the RSP and that pro-neurotensin was sorted to the RSP without the assistance of the PCs, thus indicating that dibasics can act as sorting signal by themselves independently of their interaction with PCs.

Amino Acid Sequence↗

PubMed2002

Constitutive activation of the neurotensin receptor 1 by mutation of Phe(358) in Helix seven.

1. The neurotensin receptor 1, NTS1, is a G protein-coupled receptor with seven transmembrane domains (TM) that mediates most of the known effects of the neuropeptide. Our previous studies have pointed to extracellular loop 3 and adjacent TM7 as being potentially involved in agonist-induced activation of the NTS1. 2. Here we investigated residues in these domains that might be involved in transconformational activation of the rat NTS1. Single amino acid mutated receptors were expressed in COS cells and inositol phosphate (IP) and cyclic AMP productions were studied. 3. The F358A mutation in TM7 resulted in a time- and receptor concentration-dependent increase in spontaneous IP production. At expression levels of 12 pmol mg(-1), agonist-independent IP production was increased 10 fold over basal for the F358A mutant receptor whereas the wild type NTS1 exhibited virtually no spontaneous activity at expression levels of 7.5 pmol mg(-1). 4. Neurotensin remained agonist on the F358A mutant receptor with a maximal effect that amounted to greater than twice basal IP levels. SR 48692 was inverse agonist at the mutant receptor, reversing IP production almost back to the levels measured in wild type NTS1-transfected cells. 5. Cyclic AMP production was not constitutively activated with the F358A mutant receptor but was stimulated by neurotensin with the same concentration dependence as that observed with the wild type NTS1. 6. This is the first report, to our knowledge, of a constitutively active mutant of the NTS1. The data are consistent with TM7 being involved in the transconformational changes that lead to agonist-induced coupling of the NTS1 to Gq.

Animals↗

PubMed2002

Neuroanatomical distribution of CXCR4 in adult rat brain and its localization in cholinergic and dopaminergic neurons.

Accumulating evidence supports a role of chemokines and their receptors in brain function. Up to now scarce evidence has been given of the neuroanatomical distribution of chemokine receptors. Although it is widely accepted that chemokine receptors are present on glial cells, especially in pathological conditions, it remains unclear whether they are constitutively present in normal rat brain and whether neurons have the potential to express such chemokine receptors. CXCR4, a G protein-coupled receptor for the chemokine stromal cell-derived factor-1 (SDF-1/CXCL12) was reported to have possible implications in brain development and AIDS-related dementia. By dual immunohistochemistry on brain sections, we clearly demonstrate that CXCR4 is constitutively expressed in adult rat brain, in glial cells (astrocytes, microglia but not oligodendrocytes) as well as in neurons. Neuronal expression of CXCR4 is mainly found in cerebral cortex, caudate putamen, globus pallidus, substantia innominata, supraoptic and paraventricular hypothalamic nuclei, ventromedial thalamic nucleus and substantia nigra. Using confocal microscopy, a differential distribution of CXCR4 in neuronal perikarya and dendrites can be observed according to the brain structure. Furthermore, this work demonstrates for the first time the coexistence of a chemokine receptor with classical neurotransmitters. A localization of CXCR4 is thus observed in neuronal cell bodies expressing choline acetyltransferase-immunoreactivity in the caudate putamen and substantia innominata, as well as in tyrosine hydroxylase-positive neurons in the substantia nigra pars compacta. In conclusion, the constitutive neuronal CXCR4 expression suggests that SDF-1/CXCL12 could be involved in neuronal communication and possibly linked up with cholinergic and dopaminergic neurotransmission and related disorders.

Adrenergic Fibers↗

PubMed2002

Altered processing of the neurotensin/neuromedin N precursor in PC2 knock down mice: a biochemical and immunohistochemical study.

Neurotensin (NT) and neuromedin N (NN) are generated by endoproteolytic cleavage of a common precursor molecule, pro-NT/NN. To gain insight into the role of prohormone convertases PC1, PC2, and PC7 in this process, we investigated the maturation of pro-NT/NN in the brain of PC7 (PC7-/-), PC2 (PC2-/-), and/or PC1 (PC1+/- and PC2-/-; PC1+/-) knock down mice. Inactivation of the PC7 gene was without effect, suggesting that this convertase is not involved in the processing of pro-NT/NN. By contrast, there was a 15% decrease in NT and a 50% decrease in NN levels, as measured by radioimmunoassay, in whole brain extracts from PC2 null as compared with wild type mice. Using immunohistochemistry, we found that this decrease in pro-NT/NN maturation products was uneven and that it was most pronounced in the medial preoptic area, lateral hypothalamus, and paraventricular hypothalamic nuclei. These results suggest that PC2 plays a critical role in the processing of pro-NT/NN in mouse brain and that its deficiency may be compensated to a regionally variable extent by other convertases. Previous data have suggested that PC1 might be subserving this role. However, there was no change in the maturation of pro-NT/NN in the brain of mice in which the PC1 gene had been partially inactivated, implying that complete PC1 knock down may be required for loss of function.

Animals↗