D3 PREMRNA PROCESSING IN CHRONIC PSYCHOSIS

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DESCRIPTION (Adapted from applicant's abstract): The D2-class of dopamine receptors are targets for drugs with proven antipsychotic efficacy and have been repeatedly suggested as factors in the pathophysiology of schizophrenia. Although studies have failed to demonstrate a genetic link, studies on brain tissues obtained postmortem from long-term hospitalized patients with chronic psychosis suggest an abnormal posttranscriptional processing of dopamine D3-receptor-encoded pre-mRNA. This results in a decreased expression of D3 mRNA and an increased accumulation of an alternatively spliced, truncated D3-like mRNA, named D3nf. In order to pursue a more detailed investigation of the expression of D3 and D3nf mRNA and protein in postmortem tissues, and in order to begin to examine potential functional consequences of the enhanced D3nf-specific splicing of D3 pre-mRNA in chronic psychosis, it is proposed: (1) To perform RNase protection assays to determine the relative abundance of D3 and D3nf mRNA in defined anatomic regions of brains obtained postmortem from patients with chronic schizophrenia, bipolar affective disorders, and from controls. (2) To perform immunoprecipitation and immunoblot experiments with D3-specific monoclonal antibodies and a D3nf-specific polyclonal antiserum to determine whether (in the same anatomic regions) alterations in D3 and D3nf mRNA expression also result in corresponding changes in the expression of the respective proteins. (3) Furthermore, because co-expression of D3nf in stably D3-expressing rat GH3 cells increases the electrophoretic mobility of D3-immunoreactivity (from 50 kD to 180 kD), and because this co-expression appears to render a significant proportion of D3 receptors inaccessible to ligands, experiments on stably and inducibly D3, D3nf, and D3/D3nf expressing GH3 cells are proposed that aim at determining the protein composition of the D3nf-induced 180 kd D3-immunoreactivity, its posttranslational modifications, the kinetics of its formation, and its stability. In addition, radioligand binding studies are proposed to determine the Bmax of (3H)quinpirole binding of membranes of D3 and D3/D3nf-expressing cells. Finally, it will be tested in transfected polarized epithelial cells (MDCK) whether the membrane targeting of the D3 protein is altered in the presence of D3nf. Results of these studies are expected to provide insight into the extent to which the expression of D3 and D3nf mRNA and protein is altered in schizophrenia, and they will begin to elucidate the influence of D3nf expression of D3 receptor expression and function.