The behavioral adaptations that accompany drug addiction likely result from short and long term regulatory changes in gene expression, protein translation, and protein modification. These three mechanisms underlie stable alterations in brain chemistry and structure of specific neuronal sub-types in specific circuits in the addicted brain. While the effects of drugs of abuse on transcription have been studied in depth little is known about how they regulate protein expression via translational control. Psychostimulant effects are acutely sensitive to protein synthesis inhibitors, yet systemic injection of psychostimulants inhibits protein synthesis in the dorsal and ventral striatum, indicating that regulation of translation of specific mRNAs is complex. Indeed, our preliminary studies suggest a novel role for regulation of protein elongation in striatal neurons in the acute actions of cocaine. While current methods, termed TRAP or Ribo-tag, allow quantification of ribosome bound RNA transcripts in specific neuronal cell types, they do not allow assessment of the dynamics or control of protein translation, i.e. the translatome, at the level of either translaton initiation or elongation. We will extend TRAP using highly complementary biochemical methods for the profiling of ribosome-bound RNA, ribosome footprints, and nascent polypeptides from specific neuronal cell types. These data will be interrogated using a deeply integrated computational analysis infrastructure to enable quantitative assessment of the cell type- specific translatome at isoform-level, rather than gene-level, resolution. In the proposed studies, we will use existing TRAP mice to comprehensively profile the translatome of either D1- or D2-receptor containing medium spiny neurons of the striatum in cohorts of acute cocaine-treated and control animals.
In Aim 1 a: Profiling total-RNA and ribosome footprint RNA from specific neural cell types, we will modify the existing TRAP protocol to enable parallel profiling of the ribosome-associated transcriptome (by RNA-seq) and ribosome occupancy (by ribosome foot printing).
In Aim 1 b: Profiling nascent chain proteins from specific neural cell types, we will analyze nascent chain proteins by puromycin incorporation into purified ribosomes, which we coin protein-TRAP (pTRAP), followed by tandem mass-spectrometry.
In Aim 2 : Integrative statistical analysis, we will integrate the three-level measurements of the translatome provided by Aim 1 using our recently developed expectation-maximization algorithm that probabilistically assigns footprints and/or peptides to specific isoforms based on transcript abundances obtained from RNA-seq. The resulting isoform-level quantitation will enable analysis of translational control at unprecedented resolution and accuracy, in addition to precise characterization of novel mechanisms of translational control such as upstream open reading frames and stalled ribosomes. Through the analysis of cohorts of control and cocaine-treated animals, we will classify sets of isoforms that respond in a variety of ways to different mechanisms of translational control in specific striatal neurons, setting the stage for more in-depth studies in other models of cocaine addiction.
Cocaine addiction, which is a serious medical problem in the USA, is thought to occur via modification of brain chemistry that involves the production of new proteins in specific subsets of neurons in specific brain circuits. However, the mechanisms involved are poorly understood. The proposed research seeks to develop new and highly innovative techniques and methods of data analysis that will allow systematic understanding of the mechanisms of regulation of protein production in these specific neuronal cell types following administration of cocaine.
