Elsevier

Experimental Neurology

Volume 233, Issue 1, January 2012, Pages 205-213
Experimental Neurology

Opiate agonist-induced re-distribution of Wntless, a mu-opioid receptor interacting protein, in rat striatal neurons

https://doi.org/10.1016/j.expneurol.2011.09.037Get rights and content

Abstract

Wntless (WLS), a mu-opioid receptor (MOR) interacting protein, mediates Wnt protein secretion that is critical for neuronal development. We investigated whether MOR agonists induce re-distribution of WLS within rat striatal neurons. Adult male rats received either saline, morphine or [d-Ala2, N-Me-Phe4, Gly-ol5]-enkephalin (DAMGO) directly into the lateral ventricles. Following thirty minutes, brains were extracted and tissue sections were processed for immunogold silver detection of WLS. In saline-treated rats, WLS was distributed along the plasma membrane and within the cytoplasmic compartment of striatal dendrites as previously described. The ratio of cytoplasmic to total dendritic WLS labeling was 0.70 ± 0.03 in saline-treated striatal tissue. Morphine treatment decreased this ratio to 0.48 ± 0.03 indicating a shift of WLS from the intracellular compartment to the plasma membrane. However, following DAMGO treatment, the ratio was 0.85 ± 0.05 indicating a greater distribution of WLS intracellularly. The difference in the re-distribution of the WLS following different agonist exposure may be related to DAMGO's well known ability to induce internalization of MOR in contrast to morphine, which is less effective in producing receptor internalization. Furthermore, these data are consistent with our hypothesis that MOR agonists promote dimerization of WLS and MOR, thereby preventing WLS from mediating Wnt secretion. In summary, our findings indicate differential agonist-induced trafficking of WLS in striatal neurons following distinct agonist exposure. Adaptations in WLS trafficking may represent a novel pharmacological target in the treatment of opiate addiction and/or pain.

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.

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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.

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