Opiate agonist-induced re-distribution of Wntless, a mu-opioid receptor interacting protein, in rat striatal neurons☆
Highlights
►Wntless (WLS) mediates Wnt protein secretion, critical for neuronal development. ►Morphine induced a shift of WLS from the cytoplasm to the plasma membrane. ►DAMGO treatment induced a greater distribution of WLS intracellularly. ►Agonist-induced trafficking of WLS in striatum is shown after agonist exposure. ►WLS trafficking may represent a novel target in the treatment of opiate addiction.
Introduction
Wnts belong to a family of secreted glycoproteins essential for cell signaling, development and physiological processes (Fu et al., 2009, Jin et al., 2010a, Jin et al., 2010b, Lie et al., 2005, Logan and Nusse, 2004). As extracellular signaling molecules, Wnt proteins possess neurotrophic properties (Banziger et al., 2006, Ciani and Salinas, 2005). Specifically in neuronal development, Wnt proteins have been shown to stimulate synapse formation and dendritic morphogenesis, in addition to controlling axon pathfinding, remodeling and guidance (Ciani and Salinas, 2005), indicating that Wnt proteins regulate diverse neuronal functions. Biochemical and genetic analyses have shown that perturbations in Wnt signaling have been implicated in various diseases (Grigoryan et al., 2008, Logan and Nusse, 2004). For example, dysregulation of Wnt and its signal transduction cascade may contribute to the development of malignancies in various organs (Clevers, 2006).
A conserved gene that is essential for Wnt secretion was identified in Drosophila named Wntless or Evenness interrupted or Sprinter (Banziger et al., 2006, Bartscherer et al., 2006, Goodman et al., 2006, Jin et al., 2010a, Jin et al., 2010b, Reyes et al., 2010). The Wntless/Evenness interrupted/Sprinter gene encodes a multi-pass transmembrane protein that is conserved from worms to human and is a vital component for Wnt secretion in Drosophila (Banziger et al., 2006, Bartscherer et al., 2006, Franch-Marro et al., 2008). WLS is the mammalian ortholog of Drosophila Wntless/Evenness interrupted/Sprinter. WLS protein is also known as GPR177 (Jin et al., 2010a, Jin et al., 2010b, Reyes et al., 2010). We have recently identified it as a mu-opioid receptor (MOR) interacting protein that may possibly serve as a substrate underlying the alterations in neuronal structure and synaptic organization characteristic of opioid dependence (Jin et al., 2010a).
Upon chronic exposure to a MOR agonist such as morphine or heroin, opiates exert inhibitory effects on axon outgrowth, dendritic arborization, and neurogenesis in brain regions known to be involved in reward processing, as well as learning and memory (Ciani and Salinas, 2005). Using immunoelectron microscopy, we have recently shown that WLS and MOR are co-localized in somata and in dendritic processes in the murine striatum (Jin et al., 2010a, Reyes et al., 2010), a brain region which is also involved in goal oriented behaviors and extrapyramidal motor control (Kehagia et al., 2010, Watson and Stanton, 2009). We demonstrated that 32% of WLS-labeled dendrites contained MOR immunoreactivity while 37% of MOR-labeled profiles contained WLS immunoreactivity (Reyes et al., 2010). It has recently been shown that proteins that interact directly with the MOR influence MOR biosynthesis, trafficking and signaling (Milligan, 2005), suggesting that MOR interacting proteins could regulate multiple mechanisms including signaling and trafficking. The mechanism underlying MOR desensitization or internalization may vary according to agonist exposure (Bailey and Connor, 2005, Bailey et al., 2003, Bailey et al., 2004, Van Bockstaele and Commons, 2001). Thus, in the present study, we examined whether the opiate agonists morphine or [d-Ala2, N-Me-Phe4, Gly-ol5]-enkephalin (DAMGO) cause a redistribution of WLS in striatal neurons using high resolution immunoelectron microscopic analysis. Morphine and DAMGO were selected because morphine causes little MOR internalization (Keith et al., 1996, Van Bockstaele and Commons, 2001), while DAMGO causes significant receptor internalization (Johnson et al., 2006).
Section snippets
Animals
Fifteen adult male Sprague–Dawley rats (Harlan Sprague Dawley Inc., Indianapolis, IN, USA; 250–270 g) housed two to three to a cage (20 °C, 12-h light, 12-h dark cycle lights on 0700) were used in this study. They were allowed ad libitum access to standard chow and water. All procedures were approved by The Institutional Animal Care and Use Committee at Thomas Jefferson University according to the revised Guide for the Care and Use of Laboratory Animals (1996), The Health Research Extension Act
WLS shifts its distribution following opiate agonist treatment
Consistent with our recent reports (Jin et al., 2010a, Reyes et al., 2010), WLS immunoreactivity was distributed within somata and dendritic processes in the rat striatum. Using immunoelectron microscopy, WLS was labeled using immunogold–silver detection where immunolabeling appeared as irregularly shaped black deposits indicative of the antigen of interest (Figs. 1A–H). The localization of WLS, in the present study, supports our recent description provided in a mouse model (Reyes et al., 2010).
Discussion
This study provides the first in vivo evidence of opiate agonist-induced trafficking of WLS that is consistent with data obtained using in vitro systems (Jin et al., 2010a). Here, we show that following morphine treatment, WLS is more frequently distributed along the plasma membrane when compared to saline-treated rats. Interestingly, following DAMGO treatment, WLS was more often distributed within the cytoplasm. These data suggest that opiate agonists induce differential trafficking of WLS in
Acknowledgments
This project was supported by the National Institutes of Health grants , to W.H.B. and DA 09082 to E.V.B.
References (60)
- et al.
Opioids: cellular mechanisms of tolerance and physical dependence
Curr. Opin. Pharmacol.
(2005) - et al.
Wntless, a conserved membrane protein dedicated to the secretion of Wnt proteins from signaling cells
Cell
(2006) - et al.
Secretion of Wnt ligands requires Evi, a conserved transmembrane protein
Cell
(2006) - et al.
Optimization of differential immunogold–silver and peroxidase labeling with maintenance of ultrastructure in brain sections before plastic embedding
J. Neurosci. Methods
(1990) Wnt/beta-catenin signaling in development and disease
Cell
(2006)- et al.
The basal ganglia
- et al.
The frequency and distribution of medium-sized neurons with indented nuclei in the primate and rodent neostriatum
Brain Res.
(1985) - et al.
Learning and cognitive flexibility: frontostriatal function and monoaminergic modulation
Curr. Opin. Neurobiol.
(2010) - et al.
Morphine activates opioid receptors without causing their rapid internalization
J. Biol. Chem.
(1996) - et al.
Loss of surface N-methyl-d-aspartate receptor proteins in mouse cortical neurones during anaesthesia induced by chloral hydrate in vivo
Br. J. Anaesth.
(2009)
Distinct effects of individual opioids on the morphology of spines depend upon the internalization of mu opioid receptors
Mol. Cell. Neurosci.
Ultrastructural relationship between the mu opioid receptor and its interacting protein, GPR177, in striatal neurons
Brain Res.
Amygdalar peptidergic circuits regulating noradrenergic locus coeruleus neurons: linking limbic and arousal centers
Exp. Neurol.
Impaired neuropsychological performance in chronic nonmalignant pain patients receiving long-term oral opioid therapy
J. Pain Symptom Manage.
Wnts: up-and-coming at the synapse
Trends Neurosci.
Membrane properties and synaptic connectivity of fast-spiking interneurons in rat ventral striatum
Brain Res.
Wnt signaling in neuroprotection and stem cell differentiation
Prog. Neurobiol.
Internalization of mu-opioid receptors produced by etorphine in the rat locus coeruleus
Neuroscience
Functional dissociation of mu opioid receptor signaling and endocytosis: implications for the biology of opiate tolerance and addiction
Neuron
Differential activation and trafficking of micro-opioid receptors in brain slices
Mol. Pharmacol.
Mu-opioid receptor desensitization in mature rat neurons: lack of interaction between DAMGO and morphine
J. Neurosci.
Protein kinase C activation enhances morphine-induced rapid desensitization of mu-opioid receptors in mature rat locus ceruleus neurons
Mol. Pharmacol.
Isolation and characterization of postsynaptic densities from various brain regions: enrichment of different types of postsynaptic densities
J. Cell Biol.
Function of a calmodulin in postsynaptic densities. III. Calmodulin-binding proteins of the postsynaptic density
J. Cell Biol.
WNTs in the vertebrate nervous system: from patterning to neuronal connectivity
Nat. Rev. Neurosci.
Opiates inhibit neurogenesis in the adult rat hippocampus
Proc. Natl. Acad. Sci. U. S. A.
Wingless secretion requires endosome-to-Golgi retrieval of Wntless/Evi/Sprinter by the retromer complex
Nat. Cell Biol.
Reciprocal regulation of Wnt and Gpr177/mouse Wntless is required for embryonic axis formation
Proc. Natl. Acad. Sci. U. S. A.
Sprinter: a novel transmembrane protein required for Wg secretion and signaling
Development
Axosomatic and axo-dendritic synapses of the cerebral cortex: an electron microscopic study
J. Anat.
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Grant information: This project was supported by the National Institutes of Health grant P20 DA #025995 and DA #05186 to W.H.B. and DA #09082 to E.V.B.