Detection, characterization and biological activities of [bisphospho-thr3,9]ODN, an endogenous molecular form of ODN released by astrocytes
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[33–50]) (Ferrero et al., 1986) and the triakontatetraneuropeptide (TTN) (DBI[17–50]) (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.).
References (54)
- et al.
Diazepam binding inhibitor promotes progenitor proliferation in the postnatal SVZ by reducing GABA signaling
Cell Stem Cell
(2012) Electrophysiological characterization of diazepam binding inhibitor (DBI) on GABAA receptors
Neuropharmacology
(1991)- et al.
Endogenous positive allosteric modulation of GABA(A) receptors by diazepam binding inhibitor
Neuron
(2013) - et al.
In vivo action of a new octadecaneuropeptide (ODN) antagonist on gonadotropin-releasing hormone gene expression in the male rat brain
Neuroscience
(2004) - et al.
In vivo action of a new octadecaneuropeptide antagonist on neuropeptide Y and corticotropin-releasing hormone mRNA levels in rat
Mol Brain Res
(2005) - et al.
The dual face of connexin-based astroglial Ca2+ communication: a key player in brain physiology and a prime target in pathology
Biochim Biophys Acta
(2014) - et al.
The complexity of the GABAA receptor shapes unique pharmacological profiles
Drug Discov Today
(2009) - et al.
Involvement of diazepam binding inhibitor and its fragment octadecaneuropeptide in social isolation stress-induced decrease in pentobarbital sleep in mice
Life Sci
(1999) - et al.
A brain octadecaneuropeptide generated by tryptic digestion of DBI (diazepam binding inhibitor) functions as a proconflict ligand of benzodiazepine recognition sites
Neuropharmacology
(1984) - et al.
The stimulatory effect of the octadecaneuropeptide (ODN) on cytosolic calcium in rat astrocytes is not mediated through classical benzodiazepine receptors
Eur J Pharmacol
(1997)
The endozepine ODN stimulates [3H]thymidine incorporation in cultured rat astrocytes
Neuropharmacology
Protein phosphorylation and signal transduction
Pharmacol Ther
On target with a new mechanism for the regulation of protein phosphorylation
Trends Biochem Sci
Extreme stability of Tyr-MIF-1 in CSF
Neurosci Lett
An octadecaneuropeptide (ODN) derived from diazepam binding inhibitor increases aggressive inter-actions in mice
Brain Res
The endogenous benzodiazepine receptor ligand ODN increases cytosolic calcium in cultured rat astrocytes
Brain Res Mol Brain Res
Ontogeny of diazepam-binding inhibitor related peptides (endozepines) in the rat brain
Neuroscience
Effect of diazepam binding inhibitor (DBI) on the fluid intake, preference and the taste reactivity in mice
Behav Brain Res
The role of Ca2+ in the generation of spontaneous astrocytic Ca2+ oscillations
Neuroscience
The endozepine ODN stimulates polyphosphoinositide metabolism in rat astrocytes
FEBS Lett
Temperature and nitric oxide control spontaneous calcium transients in astrocytes
Cell Calcium
Immunocytochemical localization of the endogenous benzodiazepine ligand octadecaneuropeptide (ODN) in the rat brain
Neuropeptides
Endozepines
Activity-dependent expression of acyl-coenzyme A-binding protein in retinal Müller glial cells evoked by optokinetic stimulation
J Neurosci
Effect of intracerebroventricular administration of the octadecaneuropeptide on the expression of pro-opiomelanocortin, neuropeptide Y and corticotropin-releasing hormone mRNAs in rat hypothalamus
J Neuroendocrinol
The octadecaneuropeptide ODN induces anxiety in rodents: possible involvement of a shorter biologically active fragment
Peptides
Reduction of pentylenetetrazol-induced convulsions by the octadecaneuropeptide ODN
Peptides
Cited by (8)
Endozepines and their receptors: Structure, functions and pathophysiological significance
2020, Pharmacology and TherapeuticsCitation Excerpt :This bpODN has a stronger affinity for GABAAR-BZ-binding site than ODN. In agreement, bpODN reduces food consumption with a 10-fold lower efficiency than unphosphorylated ODN (Gach et al., 2015). More recently, Lanfray and colleagues (Lanfray et al., 2013) have shown that intraparenchymal unilateral injection of OP in the ARC of the hypothalamus reduces food intake (Lanfray et al., 2013).
Lactobacillus salivarius and Lactobacillus gasseri supplementation reduces stress-induced sugar craving in mice
2023, European Eating Disorders ReviewNeuroprotective effects of the gliopeptide ODN in an in vivo model of Parkinson’s disease
2018, Cellular and Molecular Life Sciences
- †
These authors contributed equally to this work.