| In the clinical setting, morphine represents the most commonly utilized pharmacological intervention for the management of agonizing or chronic pain. Though best known for its analgesic properties, morphine also exerts a number of additional effects including euphoria, sedation, and constipation. Despite the utility of morphine, its use for these many purposes remains limited by the rapid development of tolerance, dependence, and ultimately addiction that frequently accompanies its use. Substantial evidence has accumulated that implicates alterations in synaptic transmission and neuroplasticity throughout the endogenous reward circuitry of the central nervous system in the etiology of morphine dependence and addiction. However, to date, very few studies have specifically examined morphine-regulated alterations in protein abundance at the postsynaptic density. Fewer still have done so in the striatum, a region of the endogenous reward circuitry known to be integral in the development of dependence and addiction. Thus, the penultimate goals of the present investigations included elucidation of the relationship between morphine dependence and altered protein profiles at the striatal postsynaptic density, as well as the identification of novel protein-protein interactions among proteins that exhibited significant regulation in response to morphine dependence. In the first set of experiments, following the induction of robust morphine tolerance in rats, the striatum was isolated and subjected to subcellular fractionation in order to generate protein fractions significantly enriched in postsynaptic density associated proteins. Using 2-dimensional-liquid chromatography in conjunction with tandem mass spectrometry, the identity and abundance of over 2,600 proteins from the striatal postsynaptic fraction were identified. Among these, 38 proteins exhibited significantly elevated or decreased abundance in response to morphine tolerance. This set of morphine-regulated proteins included many that are involved in G-protein coupled receptor signaling, regulation of transcription and translation, molecular chaperones, glutamatergic neurotransmission and Ca2+ signaling, synaptic transmission, regulation of cytoskeletal dynamics, the ubiquitin-proteasomal system, and several others. The altered expression of several proteins identified by quantitative mass spectrometry was then validated using Western blotting analysis. In the second study, systems biology and bioinformatic approaches were utilized to identify signaling networks and novel interacting proteins that connected the 38 morphine-regulated proteins identified in the previous investigation. Using Genes2FANs, a background database of 15,548 proteins and 64,741 known protein-protein interactions, was queried in order to generate a graph theory inspired protein-protein interaction network. Among the highly significant intermediate nodes predicted by this software tool, three intriguing proteins, of interest as they lacked known localization to or function at the striatal postsynapse, were selected for further characterization and validation. The proteins caspase-3, receptor-interacting serine/threonine protein kinase 3, and the E3-ubiquitin ligase neural precursor cell expressed developmentally downregulated protein 4, previously implicated in apoptosis, necroptosis and ubiquitin-proteasomal degradation respectively, were subsequently revealed to be present in the striatal postsynaptic fraction, and in some cases significantly downregulated in response to morphine tolerance and dependence. Expanding beyond the Genes2FANs analysis, using literature-mining techniques, a larger, more comprehensive protein-protein interaction was generated in an effort to develop an improved contextual understanding of the subnetwork generated using Genes2FANs. Taken together, the results of the present series of investigations have revealed 38 proteins that exhibited significantly regulated expression in response to chronic escalating morphine administration, many of which are involved in the functioning of the ubiquitin-proteasome system. Additional analyses using graph theory inspired protein-protein interaction networks led to the identification of novel proteins involved in protein degradation and cell death, and the ubiquitin system, all of which exhibited significant alterations following morphine treatment. Together, these findings suggest an emerging role for protein degradation and the functioning of the ubiquitin-proteasomal system in the etiology of opiate dependence and addiction. As such, many of these proteins discussed herein may represent potential novel targets for therapeutic intervention in addiction disorders. Further exploration of these protein targets could ultimately contribute to the development of next-generation opiate compounds that retain the analgesic properties of morphine while exhibiting significantly reduced liability for abuse and addiction. |
