Investigating the Cell Type-Specific Recruitment of the SAGA Complex in Cocaine Use Disorder

Cocaine use disorder (CUD) imposes a large burden on public health, particularly because there are no FDA-approved pharmacotherapies for the disorder. The onset and maintenance of CUD is driven by physiological and molecular changes within the brain that lead to maladaptive behavior associated with cocaine-taking and cocaine-seeking. A key neuronal population in this dysregulation is dopamine 1 receptor expressing medium spiny neurons (D1 MSNs) in the nucleus accumbens (NAc). These cells are activated by acute cocaine, undergo physiological and transcriptional plasticity following repeated cocaine exposure, and are recruited by cocaine-associated cues to drive drug-seeking and self-administration. Although a causal role in drug-induced behavior has been identified, the molecular mechanisms underlying cocaine-induced transcription dysregulation remain poorly understood. Lysine acetyltransferase 2a (KAT2a) is a critical cocaine-induced epigenetic regulator in the NAc, and the Spt-Ada-Gcn5-Acetyltransferase (SAGA) complex is a regulator of stimulus-responsive and cell type-specific gene expression. The goal of this proposal is to characterize KAT2a recruitment and assembly within the SAGA complex in NAc D1 MSNs following cocaine self-administration. We will test the hypothesis that cocaine self-administration recruits KAT2a assembly within the SAGA complex in NAc D1 MSNs.

We present in our preliminary data a detailed series of proteomic and bioinformatic studies through which we identified KAT2a–the acetyltransferase subunit of the SAGA complex–as an upstream regulator of the wide-scale transcriptional dysregulation associated with cocaine exposure in the NAc of both males and females. We also show that a mutation of KAT2a that impairs its function only in D1 MSNs greatly impairs cocaine self-administration. We hypothesize that KAT2a acts as a component of the SAGA complex within NAc D1 MSNs to control the transcriptional plasticity that drives cocaine self-administration. To address this question, we will collaborate with the Yale/NIDA Neuroproteomics Center to conduct Rapid Immunoprecipitation Mass Spectrometry of Endogenous Proteins (RIME) to assess SAGA complex formation following cocaine self-administration in genetic mouse lines that allow for cell-type specific isolation of the SAGA complex from NAc D1 MSNs. The overall goal is to characterize cocaine-induced KAT2a recruitment and SAGA complex formation selectivity within NAc D1 MSNs. In Aim 1, we will express wildtype KAT2a fused to a non-endogenously expressed V5 peptide tag selectively within NAc D1 MSNs and characterize the interacting partners of KAT2a in vivo and the effect that cocaine self-administration exerts on these protein-protein associations compared to saline control. In Aim 2, we will conditionally express a V5-tagged KAT2a mutant that prevents KAT2a recruitment to chromatin by cocaine and assess the effect of this mutation on cocaine-induced recruitment and formation of the SAGA complex. The experimental goals in this pilot grant will provide the technical capability necessary to characterize the proteomic mechanisms underlying the cell type-specific epigenetic and transcriptional mechanisms underlying substance use disorder In addition, the experimental findings will define a cell type-specific neuro epigenetic mechanism of CUD.