Elsevier

Neuroscience

Volume 290, 2 April 2015, Pages 472-484
Neuroscience

Detection, characterization and biological activities of [bisphospho-thr3,9]ODN, an endogenous molecular form of ODN released by astrocytes

https://doi.org/10.1016/j.neuroscience.2015.01.045Get rights and content

Highlights

  • Bisphosphorylated-ODN as well as ODN were released by cultured astrocyte.

  • BpODN and ODN increased Ca2+ event frequency but in a different range of concentration.

  • Our data do not support that ODN phosphorylation is involved in receptor selectivity.

Abstract

Astrocytes synthesize and release endozepines, a family of regulatory neuropeptides, including diazepam-binding inhibitor (DBI) and its processing fragments such as the octadecaneuropeptide (ODN). At the molecular level, ODN interacts with two types of receptors, i.e. it acts as an inverse agonist of the central-type benzodiazepine receptor (CBR), and as an agonist of a G protein-coupled receptor (GPCR). ODN exerts a wide range of biological effects mediated through these two receptors and, in particular, it regulates astrocyte activity through an autocrine/paracrine mechanism involving the metabotropic receptor. More recently, it has been shown that Müller glial cells secrete phosphorylated DBI and that bisphosphorylated ODN ([bisphospho-Thr3,9]ODN, bpODN) has a stronger affinity for CBR than ODN. The aim of the present study was thus to investigate whether bpODN is released by mouse cortical astrocytes and to compare its potency to ODN. Using a radioimmunoassay and mass spectrometry analysis we have shown that bpODN as well as ODN were released in cultured astrocyte supernatants. Both bpODN and ODN increased astrocyte calcium event frequency but in a very different range of concentration. Indeed, ODN stimulatory effect decreased at concentrations over 10−10 M whereas bpODN increased the calcium event frequency at similar doses. In vivo effects of bpODN and ODN were analyzed in two behavioral paradigms involving either the metabotropic receptor (anorexia) or the CBR (anxiety). As previously described, ODN (100 ng, icv) induced a significant reduction of food intake. Similar effect was achieved with bpODN but at a 10 times higher dose (1000 ng, icv). Similarly, and contrasting with our hypothesis, bpODN was also 10 times less potent than ODN to induce anxiety-related behavior in the elevated zero maze test. Thus, the present data do not support that phosphorylation of ODN is involved in receptor selectivity but indicate that it rather weakens ODN activity.

Introduction

The octadecaneuropeptide (ODN) belongs to endozepines, a family of regulatory neuropeptides, initially isolated from rat brain and characterized as the endogenous ligands of benzodiazepine receptors (Guidotti et al., 1983). All endozepines described so far derive from an 86-amino acid polypeptide called diazepam-binding inhibitor (DBI) (Guidotti et al., 1983, Tonon et al., 2013). Proteolytic cleavage of DBI generates several biologically active peptides including ODN (DBI[3350]) (Ferrero et al., 1986) and the triakontatetraneuropeptide (TTN) (DBI[1750]) (Slobodyansky et al., 1989). The mechanism of action of endozepines is still poorly understood but at the molecular level, ODN would interact with two types of receptors. It acts as an inverse agonist of central-type benzodiazepine receptors (CBR) that are intrinsic components of the GABAA receptor-chloride channel complex (Ferrero et al., 1984) and as an agonist of a G protein-coupled receptor (GPCR) (Patte et al., 1995, Gandolfo et al., 1997). Indeed, electrophysiological studies show that DBI and ODN attenuate GABA-induced Cl efflux in neurons and endocrine cells (Bormann, 1991, Louiset et al., 1993, Alfonso et al., 2012), indicating that endozepines act as negative allosteric modulators of the GABAA receptors. In parallel, it has been shown that, in cultured rat astrocytes, ODN activates a GPCR, positively coupled either to phospholipase C (PLC) (Patte et al., 1995, Gandolfo et al., 1997, Leprince et al., 2001) or to adenylyl cyclase (Hamdi et al., 2012). From a functional point of view, ODN stimulates neurosteroid biosynthesis (Do Rego et al., 2001) and glial cell or neuroblast proliferation (Gandolfo et al., 1999, Alfonso et al., 2012) through CBR activation, but increases intracellular calcium concentration ([Ca2+]i) in astrocytes (Gandolfo et al., 1997, Leprince et al., 1998, Leprince et al., 2001) and hypothalamic neuropeptide expressions in neurons (Compère et al., 2003, Compère et al., 2004, Compère et al., 2005) through the ODN–GPCR activation. In vivo, ODN exerts multiple biological effects. Behavioral studies have demonstrated that ODN, acting through CBR, increases in rodent aggressiveness (Kavaliers and Hirst, 1986), induces anxiety and proconflict behavior (De Mateos-Verchère et al., 1998) but reduces pentobarbital-induced sleeping time (Dong et al., 1999), drinking (Manabe et al., 2001) and pentylenetetrazol-evoked convulsions (De Mateos-Verchère et al., 1999). In addition, ODN acting through its metabotropic receptor exerts a potent anorexigenic effect (De Mateos-Verchère et al., 2001, Do Rego et al., 2007) and relays brain glucose sensing (Lanfray et al., 2013).

Endozepines are widely distributed in the central nervous system (CNS) and peripheral tissues (for review Tonon et al., 2013). In the mammalian brain, endozepines are exclusively synthesized by glial cells. Within the brain, the highest level of DBI-like immunoreactivity has been reported in astroglial cells of the cerebral cortex (Tonon et al., 1990), ependymocytes bordering the third ventricle (Tonon et al., 1990, Malagon et al., 1993, Do Rego et al., 2001), tanycytes of the median eminence (Tonon et al., 1990, Malagon et al., 1993) and Bergmann cells of the cerebellum (Tonon et al., 1990, Vidnyánszky et al., 1994, Yanase et al., 2002 In the retina, DBI is expressed and released by radially oriented Müller glial cells and by stellate astrocytes (Holländer et al., 1991, Barmack et al., 2004, Qian et al., 2008). Moreover, it has been shown that retinal Müller glial cells also secrete a multiphosphorylated form of DBI (Qian et al., 2008). Indeed, four phosphorylation sites were identified in position Ser2, Thr36, Thr42 and Thr65 that fit a protein kinase C and/or a casein kinase II phosphorylation pattern. Interestingly, Thr36 and Thr42 residues of DBI are located in the sequence of ODN (Thr3 and Thr9 residues, respectively). The functional consequence of this post-translational modification is still unclear but it has been reported that phosphorylation of DBI increases its affinity for the GABAA receptor (Qian et al., 2008). Similarly, threonine-phosphorylated ODN, [bisphospho-Thr3,9]ODN (bpODN), has a higher affinity for the GABAA receptor than unphosphorylated ODN (Qian et al., 2008).

It is well established that cultured rat astrocytes contain and release DBI-related peptides, including ODN (Lamacz et al., 1996). There is now evidence that ODN acts as both an autocrine factor regulating glial cell activity and a gliotransmitter modulating neurotransmission (Hamdi et al., 2011, Lanfray et al., 2013). However, little is known regarding a possible selectivity of ODN toward the CBR or metabotropic receptors. The aim of the present study was thus to investigate whether bpODN is released by mouse astrocytes, affects their calcium mobilizing dynamics, and exerts different in vivo effects than unphosphorylated ODN.

Section snippets

Reagents

All Fmoc-amino-acid residues, O-benzotriazol-1-yl-N,N,N′,N′-tetramethyluronium tetrafluoroborate (TBTU), and 1-hydroxybenzotriazole (HOBt) were purchased from PolyPeptide (Strasbourg, France), Novabiochem Merck Chemicals (Nottingham, UK) or Christof Senn Laboratories (Dielsdorf, Switzerland). Preloaded 4-hydroxymethyl-phenoxymethyl-copolystyrene-1%-divinylbenzene resin (Fmoc-Lys(Boc)-HMP) was from Life Technologies (Villebon sur Yvette, France). N,N-Diisopropylethylamine (DIEA), piperidine,

Detection and characterization of endozepines released from cultured mouse astrocytes

HPLC analysis of supernatant from secondary cultured cortical mouse astrocytes revealed the presence of spontaneously released bpODN-LI compounds with retention times similar to bpODN, ODN and OP (Fig. 1). To further characterize the molecular forms of endozepines released, mass spectrometry analysis of the highest immunoreactive fraction was used. The total ion current chromatogram showed two major signals in this pre-purified fraction (Fig. 2A). Extraction ion current revealed the presence of

Discussion

Protein phosphorylation is one of the most prevalent intracellular protein modifications that plays a pivotal role to regulate various cellular processes including cell proliferation, differentiation, and apoptosis (Pawson and Scott, 1997, Graves and Krebs, 1999, Shumyantseva et al., 2014). It is estimated that 30% of all proteins in a cell are phosphorylated at any given time. Phosphorylation of serines and threonines is one of the most widespread post-translational modifications in nature,

Conclusion

In the present study, we have shown that cortical astrocytes, like Müller glial cells release bpODN and also two other active peptides, [pGlu1]ODN and OP. Our results showed that phosphorylation of ODN resulted in a remarkable decrease in metabotropic receptor functionality and did not support that this post-translational modification is involved in receptor selectivity. The weak effect on GABAA receptor may be associated with its lower receptor affinity or may be related to its interaction

Acknowledgments

This work was supported by grants from Inserm, European Regional Development Fund (ERDF) PeReNE, and Polish Ministry of Science ‘Mobilnosc Plus 625/MOB/743/2011/0’ (K.G.).

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