Proteomic Analysis of Brain Regions from Rats Exposed to Cocaine

Abstract

Drugs of abuse are a social problem in many countries. Experimental studies indicate that the dopamine transporter in brain regions including nucleus accumbens has a major role in drug dependence through maintenance of levels of dopamine that reinforce drug-taking behavior. The initial goal of this thesis was to use proteomics including mass spectrometry to characterize the expression and possible post-translational modification of the dopamine transporter in nucleus accumbens of rats exposed to cocaine. There were difficulties, however, in detection of the transporter using one-dimensional gel electrophoresis and Western blotting or using immunoprecipitation and affinity capture with anti-transporter antibodies. Emphasis was therefore given to use of two-dimensional gel electrophoresis to study proteins that showed altered expression after exposure of rats to cocaine. Two-dimensional electrophoresis was carried out using isoelectric focusing of brain extracts on 7 cm immobilized pH gradient strips followed by second dimension separation by SDS polyacrylamide gel electrophoresis. Approximately 400-700 protein spots were detected on each gel using colloidal Coomassie Blue staining. Visual inspection of digital images of the gels showed good repeatability between replicate gels from each of the brain regions studied. There were few detected differences in expression of abundant proteins between frontal cortex, striatum, nucleus accumbens or hippocampus of control rats, but brain stem showed major differences in protein pattern. Proteins that showed differences in expression between brain regions, or following exposure to cocaine, were examined by MALDI mass fingerprinting of tryptic peptides followed by matching to the NCBInr sequence database. Proteins that varied between brain regions included alpha-spectrin 2, clathrin heavy chain, neurofilament 3, heat shock protein 1, protein kinase c inhibitor protein-1, ADP-ribosylation factor 3, protein phosphatase 3, calmodulin 3, IGIF and alpha-synuclein. Alpha-spectrin 2 was expressed most highly in frontal cortex; clathrin heavy chain was expressed most highly in frontal cortex and striatum and least in brainstem. Neurofilament 3 was expressed more highly in frontal cortex, hippocampus and striatum. Heat shock protein 1, calmodulin 3 and IL-18 were expressed least in brainstem. Protein kinase C inhibitor protein-1 was expressed more highly in frontal cortex and brainstem. ADP-ribosylation factor 3 was expressed most highly in hippocampus, followed by nucleus accumbens and striatum, least in frontal cortex, and was not detected in brainstem. Protein phosphatase 3 and alpha-synuclein were not detected in brainstem. Proteins whose expression changed between cocaine dosed (10 mg/kg per day for 8 day) and saline control injected animals included protein kinase C inhibitor protein-1, ADP-ribosylation factor 3, protein phosphatase 3, IL-18, carbonic anhydrase 2 and RAN. Expressions of these proteins were modified in nucleus accumbens. Protein phosphatase 3 showed modified expression in frontal cortex and striatum as well. The current work has used visual inspection of gel images to detect protein change and it is likely that at least two-fold difference in expression was required for detection of a change in abundance. We plan future work using more sensitive methods for detection of protein differences in the brain of rats using a behavioral model of cocaine addiction.