Dynamic local mRNA localization and translation occurs during the postnatal molecular maturation of perivascular astrocytic processes.

Astrocytes are highly ramified and send out perivascular processes (PvAPs) that entirely sheathe the brain's blood vessels. PvAPs are equipped with an enriched molecular repertoire that sustains astrocytic regulatory functions at the vascular interface. In the mouse, PvAP development starts after birth and is essentially complete by postnatal day (P) 15. Progressive molecular maturation also occurs over this period, with the acquisition of proteins enriched in PvAPs. The mechanisms controlling the development and molecular maturation of PvAPs have not been extensively characterized. We reported previously that mRNAs are distributed unequally in mature PvAPs and are locally translated. Since dynamic mRNA localization and local translation influence the cell's polarity, we hypothesized that they might sustain the postnatal maturation of PvAPs. Here, we used a combination of molecular biology and imaging approaches to demonstrate that the development of PvAPs is accompanied by the transport of mRNA and polysomal mRNA into PvAPs, the development of a rough endoplasmic reticulum (RER) network and Golgi cisternae, and local translation. By focusing on genes and proteins that are selectively or specifically expressed in astrocytes, we characterized the developmental profile of mRNAs, polysomal mRNAs and proteins in PvAPs from P5 to P60. We found that some polysomal mRNAs polarized progressively towards the PvAPs. Lastly, we found that expression and localization of mRNAs in developing PvAPs is perturbed in a mouse model of megalencephalic leukoencephalopathy with subcortical cysts. Our results indicate that dynamic mRNA localization and local translation influence the postnatal maturation of PvAPs.

Megalencephalic leukoencephalopathy with subcortical cysts is a developmental disorder of the gliovascular unit.

Absence of the astrocyte-specific membrane protein MLC1 is responsible for megalencephalic leukoencephalopathy with subcortical cysts (MLC), a rare type of leukodystrophy characterized by early-onset macrocephaly and progressive white matter vacuolation that lead to ataxia, spasticity, and cognitive decline. During postnatal development (from P5 to P15 in the mouse), MLC1 forms a membrane complex with GlialCAM (another astrocytic transmembrane protein) at the junctions between perivascular astrocytic processes. Perivascular astrocytic processes along with blood vessels form the gliovascular unit. It was not previously known how MLC1 influences the physiology of the gliovascular unit. Here, using the Mlc1 knock-out mouse model of MLC, we demonstrated that MLC1 controls the postnatal development and organization of perivascular astrocytic processes, vascular smooth muscle cell contractility, neurovascular coupling, and intraparenchymal interstitial fluid clearance. Our data suggest that MLC is a developmental disorder of the gliovascular unit, and perivascular astrocytic processes and vascular smooth muscle cell maturation defects are primary events in the pathogenesis of MLC and therapeutic targets for this disease.

A metabolically stable apelin-17 analog decreases AVP-induced antidiuresis and improves hyponatremia.

Apelin and arginine-vasopressin (AVP) are conversely regulated by osmotic stimuli. We therefore hypothesized that activating the apelin receptor (apelin-R) with LIT01-196, a metabolically stable apelin-17 analog, may be beneficial for treating the Syndrome of Inappropriate Antidiuresis, in which AVP hypersecretion leads to hyponatremia. We show that LIT01-196, which behaves as a potent full agonist for the apelin-R, has an in vivo half-life of 156 minutes in the bloodstream after subcutaneous administration in control rats. In collecting ducts, LIT01-196 decreases dDAVP-induced cAMP production and apical cell surface expression of phosphorylated aquaporin 2 via AVP type 2 receptors, leading to an increase in aqueous diuresis. In a rat experimental model of AVP-induced hyponatremia, LIT01-196 subcutaneously administered blocks the antidiuretic effect of AVP and the AVP-induced increase in urinary osmolality and induces a progressive improvement of hyponatremia. Our data suggest that apelin-R activation constitutes an original approach for hyponatremia treatment.

Astrocytes in the regulation of cerebrovascular functions.

Astrocytes are the most numerous type of neuroglia in the brain and have a predominant influence on the cerebrovascular system; they control perivascular homeostasis, the integrity of the blood-brain barrier, the dialogue with the peripheral immune system, the transfer of metabolites from the blood, and blood vessel contractility in response to neuronal activity. These regulatory processes occur in a specialized interface composed of perivascular astrocyte extensions that almost completely cover the cerebral blood vessels. Scientists have only recently started to study how this interface is formed and how it influences cerebrovascular functions. Here, we review the literature on the astrocytes' role in the regulation of the cerebrovascular system. We cover the anatomy and development of the gliovascular interface, the known gliovascular functions, and molecular factors, the latter's implication in certain pathophysiological situations, and recent cutting-edge experimental tools developed to examine the astrocytes' role at the vascular interface. Finally, we highlight some open questions in this field of research.

Role of the Vasopressin/Apelin Balance and Potential Use of Metabolically Stable Apelin Analogs in Water Metabolism Disorders.

Apelin, a (neuro)vasoactive peptide, plays a prominent role in controlling body fluid homeostasis and cardiovascular functions. In animal models, experimental data demonstrate that intracerebroventricular injection of apelin into lactating rats inhibits the phasic electrical activity of arginine vasopressin (AVP) neurons, reduces plasma AVP levels, and increases aqueous diuresis. In the kidney, apelin increases diuresis by increasing the renal microcirculation and by counteracting the antidiuretic effect of AVP at the tubular level. Moreover, after water deprivation or salt loading, in humans and in rodents, AVP and apelin are conversely regulated to facilitate systemic AVP release and to avoid additional water loss from the kidney. Furthermore, apelin and vasopressin secretion are significantly altered in various water metabolism disorders including hyponatremia and polyuria-polydipsia syndrome. Since the in vivo half-life of apelin is in the minute range, metabolically stable apelin analogs were developed. The efficacy of these lead compounds for decreasing AVP release and increasing both renal blood flow and diuresis, make them promising candidates for the treatment of water retention and/or hyponatremic disorders.

Development of original metabolically stable apelin-17 analogs with diuretic and cardiovascular effects.

Apelin, a (neuro)vasoactive peptide, plays a prominent role in controlling cardiovascular functions and water balance. Because the in vivo apelin half-life is in the minute range, we aimed to identify metabolically stable apelin-17 (K17F) analogs. We generated P92 by classic chemical substitutions and LIT01-196 by original addition of a fluorocarbon chain to the N terminus of K17F. Both analogs were much more stable in plasma (half-life >24 h for LIT01-196) than K17F (4.6 min). Analogs displayed a subnanomolar affinity for the apelin receptor and behaved as full agonists with regard to cAMP production, ERK phosphorylation, and apelin receptor internalization. Ex vivo, these compounds induced vasorelaxation of rat aortas and glomerular arterioles, respectively, precontracted with norepinephrine and angiotensin II, and increased cardiac contractility. In vivo, after intracerebroventricular administration in water-deprived mice, P92 and LIT01-196 were 6 and 160 times, respectively, more efficient at inhibiting systemic vasopressin release than K17F. Administered intravenously (nmol/kg range) in normotensive rats, these analogs potently increased urine output and induced a profound and sustained decrease in arterial blood pressure. In summary, these new compounds, which favor diuresis and improve cardiac contractility while reducing vascular resistances, represent promising candidates for the treatment of heart failure and water retention/hyponatremic disorders.-Gerbier, R., Alvear-Perez, R., Margathe, J.-F., Flahault, A., Couvineau, P., Gao, J., De Mota, N., Dabire, H., Li, B., Ceraudo, E., Hus-Citharel, A., Esteoulle, L., Bisoo, C., Hibert, M., Berdeaux, A., Iturrioz, X., Bonnet, D., Llorens-Cortes, C. Development of original metabolically stable apelin-17 analogs with diuretic and cardiovascular effects.

Directed Molecular Evolution of an Engineered Gammaretroviral Envelope Protein with Dual Receptor Use Shows Stable Maintenance of Both Receptor Specificities.

UNLABELLED: We have previously reported the construction of a murine leukemia virus-based replication-competent gammaretrovirus (SL3-AP) capable of utilizing the human G protein-coupled receptor APJ (hAPJ) as its entry receptor and its natural receptor, the murine Xpr1 receptor, with equal affinities. The apelin receptor has previously been shown to function as a coreceptor for HIV-1, and thus, adaptation of the viral vector to this receptor is of significant interest. Here, we report the molecular evolution of the SL3-AP envelope protein when the virus is cultured in cells harboring either the Xpr1 or the hAPJ receptor. Interestingly, the dual receptor affinity is maintained even after 10 passages in these cells. At the same time, the chimeric viral envelope protein evolves in a distinct pattern in the apelin cassette when passaged on D17 cells expressing hAPJ in three separate molecular evolution studies. This pattern reflects selection for reduced ligand-receptor interaction and is compatible with a model in which SL3-AP has evolved not to activate hAPJ receptor internalization. IMPORTANCE: Few successful examples of engineered retargeting of a retroviral vector exist. The engineered SL3-AP envelope is capable of utilizing either the murine Xpr1 or the human APJ receptor for entry. In addition, SL3-AP is the first example of an engineered retrovirus retaining its dual tropism after several rounds of passaging on cells expressing only one of its receptors. We demonstrate that the virus evolves toward reduced ligand-receptor affinity, which sheds new light on virus adaptation. We provide indirect evidence that such reduced affinity leads to reduced receptor internalization and propose a novel model in which too rapid receptor internalization may decrease virus entry.

New structural insights into the apelin receptor: identification of key residues for apelin binding.

Apelin is the endogenous ligand of the orphan 7-transmembrane domain GPCR APJ, now named the apelin receptor (ApelinR). Apelin plays a prominent role in body fluid and cardiovascular homeostasis. To better understand the structural organization of the ApelinR, we built 3 homology 3-dimensional (3D) models of the human ApelinR using the validated cholecystokinin receptor-1 3D model or the X-ray structures of the ß2-adrenergic and CXCR4 receptors as templates. Docking of the pyroglutamyl form of apelin 13 (pE13F) into these models revealed the conservation at the bottom of the binding site of a hydrophobic cavity in which the C-terminal Phe of pE13F was embedded. In contrast, at the top of the binding site, depending on the model, different interactions were visualized between acidic residues of the ApelinR and the basic residues of pE13F. Using site-directed mutagenesis, we showed that Asp 92, Glu 172, and Asp 282 of rat ApelinR are key residues in apelin binding by interacting with Lys 8, Arg 2, and Arg 4 of pE13F, respectively. These residues are only seen in the CXCR4-based ApelinR 3D model, further validating this model. These findings bring new insights into the structural organization of the ApelinR and the mode of apelin binding.

Biased signaling favoring gi over ß-arrestin promoted by an apelin fragment lacking the C-terminal phenylalanine.

Apelin plays a prominent role in body fluid and cardiovascular homeostasis. We previously showed that the C-terminal Phe of apelin 17 (K17F) is crucial for triggering apelin receptor internalization and decreasing blood pressure (BP) but is not required for apelin binding or Gi protein coupling. Based on these findings, we hypothesized that the important role of the C-terminal Phe in BP decrease may be as a Gi-independent but ß-arrestin-dependent signaling pathway that could involve MAPKs. For this purpose, we have used apelin fragments K17F and K16P (K17F with the C-terminal Phe deleted), which exhibit opposite profiles on apelin receptor internalization and BP. Using BRET-based biosensors, we showed that whereas K17F activates Gi and promotes ß-arrestin recruitment to the receptor, K16P had a much reduced ability to promote ß-arrestin recruitment while maintaining its Gi activating property, revealing the biased agonist character of K16P. We further show that both ß-arrestin recruitment and apelin receptor internalization contribute to the K17F-stimulated ERK1/2 activity, whereas the K16P-promoted ERK1/2 activity is entirely Gi-dependent. In addition to providing new insights on the structural basis underlying the functional selectivity of apelin peptides, our study indicates that the ß-arrestin-dependent ERK1/2 activation and not the Gi-dependent signaling may participate in K17F-induced BP decrease.

Structure-activity relationship studies toward the discovery of selective apelin receptor agonists.

Apelin is the endogenous ligand for the previously orphaned G protein-coupled receptor APJ. Apelin and its receptor are widely distributed in the brain, heart, and vasculature, and are emerging as an important regulator of body fluid homeostasis and cardiovascular functions. To further progress in the pharmacology and the physiological role of the apelin receptor, the development of small, bioavailable agonists and antagonists of the apelin receptor, is crucial. In this context, E339-3D6 (1) was described as the first nonpeptidic apelin receptor agonist. We show here that 1 is actually a mixture of polymethylated species, and we describe an alternative and versatile solid-phase approach that allows access to highly pure 27, the major component of 1. This approach was also applied to prepare a series of derivatives in order to identify the crucial structural determinants required for the ligand to maintain its affinity for the apelin receptor as well as its capacity to promote apelin receptor signaling and internalization. The study of the structure-activity relationships led to the identification of ligands 19, 21, and 38, which display an increased affinity compared to that of 27. The latter and 19 behave as full agonists with regard to cAMP production and apelin receptor internalization, whereas 21 is a biased agonist toward cAMP production. Interestingly, the three ligands display a much higher stability in mouse plasma (T1/2 > 10 h) than the endogenous apelin-17 peptide 2 (T1/2 < 4 min).

Ghrelin and obestatin modulate growth hormone-releasing hormone release and synaptic inputs onto growth hormone-releasing hormone neurons.

Ghrelin, a natural ligand of the growth hormone secretagogue receptor (GHS-R), is synthesized in the stomach but may also be expressed in lesser quantity in the hypothalamus where the GHS-R is located on growth hormone-releasing hormone (GHRH) neurons. Obestatin, a peptide derived from the same precursor as ghrelin, is able to antagonize the ghrelin-induced increase of growth hormone (GH) secretion in vivo but not from pituitary explants in vitro. Thus, the blockade of ghrelin-induced GH release by obestatin could be mediated at the hypothalamic level by the neuronal network that controls pituitary GH secretion. Ghrelin increased GHRH and decreased somatostatin (somatotropin-releasing inhibitory factor) release from hypothalamic explants, whereas obestatin only reduced the ghrelin-induced increase of GHRH release, thus indicating that the effect of ghrelin and obestatin is targeted to GHRH neurons. Patch-clamp recordings on mouse GHRH-enhanced green fluorescent protein neurons indicated that ghrelin and obestatin had no significant effects on glutamatergic synaptic transmission. Ghrelin decreased GABAergic synaptic transmission in 44% of the recorded neurons, an effect blocked in the presence of the GHS-R antagonist BIM28163, and stimulated the firing rate of 78% of GHRH neurons. Obestatin blocked the effects of ghrelin by acting on a receptor different from the GHS-R. These data suggest that: (i) ghrelin increases GHRH neuron excitability by increasing their action potential firing rate and decreasing the strength of GABA inhibitory inputs, thereby leading to an enhanced GHRH release; and (ii) obestatin counteracts ghrelin actions. Such interactions on GHRH neurons probably participate in the control of GH secretion.

The RFamide neuropeptide 26RFa and its role in the control of neuroendocrine functions.

Identification of novel neuropeptides and their cognate G protein-coupled receptors is essential for a better understanding of neuroendocrine regulations. The RFamide peptides represent a family of regulatory peptides that all possess the Arg-Phe-NH2 motif at their C-terminus. In mammals, seven RFamide peptides encoded by five distinct genes have been characterized. The present review focuses on 26RFa (or QRFP) which is the latest member identified in this family. 26RFa is present in all vertebrate phyla and its C-terminal domain (KGGFXFRF-NH2), which is responsible for its biological activity, has been fully conserved during evolution. 26RFa is the cognate ligand of the orphan G protein-coupled receptor GPR103 that is also present from fish to human. In all vertebrate species studied so far, 26RFa-expressing neurons show a discrete localization in the hypothalamus, suggesting important neuroendocrine activities for this RFamide peptide. Indeed, 26RFa plays a crucial role in the control of feeding behavior in mammals, birds and fish. In addition, 26RFa up-regulates the gonadotropic axis in mammals and fish. Finally, evidence that the 26RFa/GPR103 system regulates steroidogenesis, bone formation, nociceptive transmission and arterial blood pressure has also been reported. Thus, 26RFa appears to act as a key neuropeptide in vertebrates controlling vital neuroendocrine functions. The pathophysiological implication of the 26RFa/GPR103 system in human is totally unknown and some fields of investigation are proposed.

By interacting with the C-terminal Phe of apelin, Phe255 and Trp259 in helix VI of the apelin receptor are critical for internalization.

Apelin is the endogenous ligand of the orphan seven-transmembrane domain (TM) G protein-coupled receptor APJ. Apelin is involved in the regulation of body fluid homeostasis and cardiovascular functions. We previously showed the importance of the C-terminal Phe of apelin 17 (K17F) in the hypotensive activity of this peptide. Here, we show either by deleting the Phe residue (K16P) or by substituting it by an Ala (K17A), that it plays a crucial role in apelin receptor internalization but not in apelin binding or in Gα(i)-protein coupling. Then we built a homology three-dimensional model of the human apelin receptor using the cholecystokinin receptor-1 model as a template, and we subsequently docked K17F into the binding site. We visualized a hydrophobic cavity at the bottom of the binding pocket in which the C-terminal Phe of K17F was embedded by Trp(152) in TMIV and Trp(259) and Phe(255) in TMVI. Using molecular modeling and site-directed mutagenesis studies, we further showed that Phe(255) and Trp(259) are key residues in triggering receptor internalization without playing a role in apelin binding or in Gα(i)-protein coupling. These findings bring new insights into apelin receptor activation and show that Phe(255) and Trp(259), by interacting with the C-terminal Phe of the pyroglutamyl form of apelin 13 (pE13F) or K17F, are crucial for apelin receptor internalization.

Identification and pharmacological properties of E339-3D6, the first nonpeptidic apelin receptor agonist.

Apelin plays a prominent role in body fluid and cardiovascular homeostasis. To explore further upstream the role played by this peptide, nonpeptidic agonists and antagonists of the apelin receptor are required. To identify such compounds that do not exist to date, we used an original fluorescence resonance energy transfer-based assay to screen a G-protein-coupled receptor-focused library of fluorescent compounds on the human EGFP-tagged apelin receptor. This led to isolated E339-3D6 that displayed a 90 nM affinity and behaved as a partial agonist with regard to cAMP production and as a full agonist with regard to apelin receptor internalization. Finally, E339-3D6 induced vasorelaxation of rat aorta precontracted with noradrenaline and potently inhibited systemic vasopressin release in water-deprived mice when intracerebroventricularly injected. This compound represents the first nonpeptidic agonist of the apelin receptor, the optimization of which will allow development of a new generation of vasodilator and aquaretic agents.

Cellular localization of apelin and its receptor in the anterior pituitary: evidence for a direct stimulatory action of apelin on ACTH release.

Apelin is a bioactive peptide recently identified as the endogenous ligand of the human orphan G protein-coupled receptor APJ. The presence of apelin-immunoreactive nerve fibers, together with the detection of apelin receptor mRNA in the parvocellular part of the paraventricular nucleus and the stimulatory action of apelin on corticotropin-releasing hormone release, indicate that apelin modulates adrenocorticotropin (ACTH) release via an indirect action on the hypothalamus. However, a direct action of apelin in the anterior pituitary cannot be excluded. Here, we provided evidence for the existence of an apelinergic system within the adult male rat pituitary gland. Double immunofluorescence staining indicated that apelin is highly coexpressed in the anterior pituitary, mainly in corticotrophs (96.5 +/- 0.3%) and to a much lower extent in somatotropes (3.2 +/- 0.2%). Using in situ hybridization combined with immunohistochemistry, a high expression of apelin receptor mRNA was also found in corticotrophs, suggesting a local interaction between apelin and ACTH. In an ex vivo perifusion system of anterior pituitaries, apelin 17 (K17F, 10(-6) M) significantly increased basal ACTH release by 41%, whereas apelin 10 (R10F, 10(-6) M), an inactive apelin fragment, was ineffective. In addition, K17F but not R10F induced a dose-dependent increase in K(+)-evoked ACTH release, with maximal increase being observed for a 10(-6) M concentration. Taken together, these data outline the potential role of apelin as an autocrine/paracrine-acting peptide on ACTH release and provide morphological and neuroendocrine basis for further studies that explore the physiological role of apelin in the regulation of anterior pituitary functions.

Adenosine does not bind to the growth hormone secretagogue receptor type-1a (GHS-R1a).

Ghrelin regulates GH secretion and energy homeostasis through the GH secretagogue receptor type-1a (GHS-R1a). This G-protein coupled receptor shows the peculiarity to transduce information provided not just by ghrelin as well as by adenosine through a supposed binding site different from the characterized ghrelin-binding pocket. Indeed, adenosine triggers intracellular calcium rise through a distinct signaling pathway to the one described for ghrelin, although it fails to stimulate GH secretion. Despite multiple active conformations of GHS-R1a, suggested as an explanation for a ligand-dependent activation of the downstream signaling, the concept of adenosine as agonist for GHS-R1a has been re-evaluated. The results revealed that calcium rise of both ghrelin and adenosine appears to be mediated by receptors that did not show the same sensitivity to protein kinase C (PKC) activity in GHS-R1a-transfected HEK 293 cells (HEK-GHS-R1a cells). The binding analyses showed the same number of adenosine-binding sites in both HEK 293 (B(max) = 2.01 +/- 0.15 fmol/cell) and HEK-GHS-R1a cells (B(max) = 1.90 +/- 0.11 fmol/cell). This binding was unaltered by different GHS-R1a antagonists. Western blot analysis showed a similar endogenous expression of endogenous adenosine receptor type-2b and -3 in both cell lines. The K(d) values for adenosine were 1.78 microM in HEK 293 cells and 6.30 microM in HEK-GHS-R1a cells, pointing to a modification of agonist affinity induced by overexpression of the GHS-R1a. Additionally, adenosine failed to induce the GHS-R1a endocytosis, although it attenuates the ghrelin-induced GHS-R1a endocytosis. In conclusion, adenosine is not an agonist of the GHS-R1a and its action is mediated by the endogenous adenosine receptor type-2b and -3, which is able to partially use the intracellular signaling machinery of HEK-GHS-R1a cells.