Axonal mRNA transport and localized translational regulation of κ-opioid receptor in primary neurons of dorsal root ganglia

Abstract

-opioid receptor (KOR) is detected pre-and postsynaptically, but the subcellular localization, translation, and regulation of kor mRNA in presynaptic compartments of sensory neurons remain elusive. In situ hybridization detected axonal distribution of kor mRNA in primary neurons of dorsal root ganglia (DRG). The MS2-fused GFP tracked kor mRNA transport from DRG neuronal soma to axons, requiring its 5 and 3 UTRs. In Campenot chambers, axonal translation of kor mRNA was demonstrated for DRG neurons, which depended on its 5 UTR and was stimulated by KCl depolarization. KCl depolarization of DRG neurons rendered redistribution of kor mRNA from the postpolysomal fraction to the translationally active polysomal fraction. This study provided evidence for mRNA transport and regulation of presynaptic protein synthesis of nonstructural proteins like KOR in primary sensory neurons and demonstrated a mechanism of KCl depolarizationstimulated axonal mRNA redistribution for localized translational regulation.axonal translation O pioid receptors interact with opioid drugs and endogenous opioid ligands to affect pain sensation, consciousness, and autonomic functions. Three major opioid receptor types, , ␦, and are known (1, 2), each belonging to the super family of G protein-coupled transmembrane receptors (3, 4). Gene knockout studies confirmed the functional roles for opioid receptors in mediating the pharmacological actions of various opioid compounds that are among the most commonly prescribed analgesic agents (5). Furthermore, pharmacological studies revealed that the number of opioid receptors was critical to the manifestation of opioid drugs (6). However, extensive studies of transcriptional regulation of opioid receptor genes (7,8) failed to uncover the mechanisms underlying alteration of receptor number or its protein level in neurons that bear a physiological relevance. Recently, posttranscriptional control for the expression of this gene family has begun to be unraveled, primarily, in studying -opioid receptor (KOR) expression (7, 9, 10).KOR protein is detected both pre-and postsynaptically (11)(12)(13)(14). Like many axonal proteins, presynaptic KOR proteins were thought to be synthesized in the soma and then transported to the axons via the axonal transport mechanism (15, 16). However, using in vitro differentiated neurons (17), we have demonstrated transport of kor mRNA into nerve fibers, suggesting an intriguing possibility that kor mRNA might be transported to the axons of KOR-expressing neurons for local translation and regulation. To this end, the phenomena of mRNA transport/targeting and local protein synthesis in the dendrites of invertebrate and vertebrate neurons have been established, but the source of axonal proteins remained heavily debated (18-21). Evidence for axonal mRNA transport and local protein synthesis was provided primarily for structural proteins, although local synthesis of EphA2 receptor, a protein involved in axonal pathfinding has been demonstrated (22-29). Additionally, it was shown that...

Evidence for Developmentally Regulated Local Translation of Odorant Receptor mRNAs in the Axons of Olfactory Sensory Neurons

“…Even though strong evidence for the presence of functional odorant receptors at growth cone/axon termini surface of olfactory sensory neurons was recently obtained ex vivo and in a hemihead preparation (Maritan et al, 2009), it remains unknown whether these odorant receptors have a local origin. Although the presence of a functional secretory machinery in mature axons is controversial, evidence for the local synthesis of several membrane proteins in developing axons has been reported (Brittis et al, 2002;Bi et al, 2006), and the presence of a functional equivalent of endoplasmic reticulum and Golgi in retinal ganglion cell growth cones has been documented (Merianda et al, 2009). It thus appears very likely that locally synthesized membrane proteins can be inserted into the plasma membrane in growing axons.…”

Identification and quantitative analyses of microRNAs located in the distal axons of sympathetic neurons

“…All of these putative target mRNAs have been identified in the axons of invertebrates (Martin 2004;Crispino et al 2009) and vertebrates (Willis et al 2007;Taylor et al 2009;Vogelaar et al 2009). It is important to note that receptors and transmembrane proteins can be synthesized locally in axons (Spencer et al 2000;Bi et al 2006;Tsai et al 2006) and that functional receptors appear to be incorporated into the axolemma via an ER-and Golgi-dependent mechanism (Merianda et al 2009). Although attention has been focused on miRNAs that are relatively abundant or enriched in the axon in this report (Table 1), it is likely that low abundance miRNAs might also play an important role in the regulation of local translation and axonal function.…”

Copb1-facilitated axonal transport and translation of κ opioid-receptor mRNA

“…Although presynaptic KOR can be derived from proteins that are synthesized in soma and transported by vesicles (26,27), they also may be produced locally from mRNAs located in these areas where endogenous kor mRNAs can be detected and translated (12). Recently, we were able to demonstrate axonal transport and translation of kor mRNA in neurons including dorsal root ganglia (DRG) neurons, a unique example of axonal transport of mRNA encoding a nonstructural component (12). However, it was not yet clear whether this indeed was an active transport process and what machinery or components were required for this process.…”

“…Using the opioid receptor (KOR) as a model, we have begun to uncover several posttranscriptional regulatory events important for the expression of KOR protein. These include alternative mRNA splicing, changing mRNA stability (10), localized translation (11), and mRNA transport (12,13) in neurons.…”