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Abstract
Despite its efficacy, the use of morphine for the treatment of chronic pain remains limited because of the rapid development of tolerance, dependence and ultimately addiction. These undesired effects are thought to be because of alterations in synaptic transmission and neuroplasticity within the reward circuitry including the striatum. In this study we used subcellular fractionation and quantitative proteomics combined with computational approaches to investigate the morphine-induced protein profile changes at the striatal postsynaptic density. Over 2,600 proteins were identified by mass spectrometry analysis of subcellular fractions enriched in postsynaptic density associated proteins from saline or morphine-treated striata. Among these, the levels of 34 proteins were differentially altered in response to morphine. These include proteins involved in G-protein coupled receptor signaling, regulation of transcription and translation, chaperones, and protein degradation pathways. The altered expression levels of several of these proteins was validated by Western blotting analysis. Using Genes2Fans software suite we connected the differentially expressed proteins with proteins identified within the known background protein-protein interaction network. This led to the generation of a network consisting of 116 proteins with 40 significant intermediates. To validate this, we confirmed the presence of three proteins predicted to be significant intermediates: caspase-3, receptor-interacting serine/threonine protein kinase 3 and NEDD4 (an E3-ubiquitin ligase identified as a neural precursor cell expressed developmentally down-regulated protein 4). Because this morphine-regulated network predicted alterations in proteasomal degradation, we examined the global ubiquitination state of postsynaptic density proteins and found it to be substantially altered. Together, these findings suggest a role for protein degradation and for the ubiquitin/proteasomal system in the etiology of opiate dependence and addiction.
Highlights
From the ‡Department of Pharmacology and Systems Therapeutics, §Department of Neuroscience and Friedman Brain Institute, Icahn School of Medicine at Mount Sinai, New York, New York, 10029; ¶Center for Advanced Proteomic Research and Department of Biochemistry and Molecular Biology, New Jersey Medical School Cancer Center, Rutgers University, Newark, New Jersey, 07103; ʈDepartment of Anesthesiology, Columbia University Medical Center, New York, New York, 10027
The present study seeks to elucidate alterations in striatal postsynaptic density (PSD) protein expression in response to prolonged exposure to morphine using a combination of proteomics and network biology
In the case of synaptophysin we detect a strong signal in homogenate fractions from both saline and morphine treated animals but no detectable signal was observed in the PSD fractions of these animals (Fig. 1B)
Summary
Materials—Rabbit-anti-PSD-95 (Catalog# 2507), rabbit-anti-synaptophysin (Catalog# 5467S), rabbit-anti-G␣o (Catalog# 3975S), rabbit-anti-HSP70 (Catalog# 4872), rabbit-anti-GAP43 (D9C8) (Catalog# 8945), rabbit-anti-USP8 (Catalog# 8728), mouse-anti-Ubiquitin (P4D1) (Catalog# 3936), mouse-anti-Caspase-3 (3G2) (Catalog# 9668), rabbit-anti-NEDD4 (C5F5) (Catalog# 3607) were from Cell Signaling Technology, Danvers, MA. Identification and Quantification of Proteins—The MS/MS spectra were searched against UniRef 100 rat database (51,862 entries) using both Mascot (v.2.3) and Sequest search engines via the Proteome Discoverer platform (v 1.3; Thermo Scientific) In conducting this search, the following search parameters were used: [1] fixed modifications included iTRAQ 8plex (K), iTRAQ 8plex (N-terminal), and methylthio (C); [2] variable modifications included iTRAQ 8plex (Y) and oxidation (M); [3] trypsin was selected as the digestive enzyme; [4] a maximum of one missed cleavage site was allowed; [5] the peptide precursor mass tolerance was 10 ppm; and (vi) MS/MS mass tolerance was 0.1 Da. Scaffold (v.3.6.3, Proteome Software Inc., Portland, OR) was used to validate MS/MS based peptide and protein identification.
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