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Proteomic Profiling of Drug-induced Alterations to the Brain Reward System Underlying Addiction

Eric Nestler, Icahn School of Medicine at Mount Sinai

Overview: We are taking several approaches to determine the mechanisms through which cocaine and opioids cause persistent adaptations to intra- and extracellular signaling pathways within interconnected regions of the brain reward system. Currently, we are examining how volitional drug-taking (i.e., drug self-administration) produces long-term changes to the protein makeup of synaptic contacts within the nucleus accumbens (NAc), a brain region critical for drug-seeking.

In a preliminary study of NAc synaptosomes isolated from mice self-administering saline or cocaine analyzed using label-free quantitation (LFQ) with data-independent acquisition (DIA) based targeted protein quantification, we were able to detect 1,818 proteins, many of which were differentially regulated following cocaine exposure. This study confirmed the feasibility and utility of the experimental approach. We are now advancing this work by comparing and contrasting changes to the synaptic proteome induced by cocaine or heroin following self-administration in a large cohort of rats. Once concluded, these studies will reveal many novel synaptic protein targets regulated by drugs of abuse and the rich dataset generated will serve as a reference for other investigators. In parallel, we are refining viral-mediated approaches to enable isolation of specific synaptosomes using fluorescence activated cell-sorting (FACS). Using these approaches we will collaborate with the Yale/NIDA Neuroproteomics Center to investigate drug-induced synaptic remodeling specifically within D1- or D2-receptor expressing NAc neurons, or inputs from other regions of the brain reward system including the ventral hippocampus, prefrontal cortex, and basolateral amygdala. These studies will define cell-type and circuit-specific signaling changes supporting addiction.

In addition to proteomic profiling of synaptosomes, we plan to identify proteins interacting with the promoter region of the FosB gene, products of which play an important role in remodeling neural responses to drugs of abuse. Selective targeting of this gene by synthetic zinc finger proteins or by CRISPR constructs will enable examination of protein constituents at this particular site of the gene, which may help to unravel complexities in the transcriptional regulation of Fos family proteins. We will also identify proteins in complex with full length FosB and ΔFosB at the targeted gene sites, such as Cdk5 and Nfkb. Lastly, we aim to better identify the chromatin-associated proteins that could be responsible for the divergent gene expression patterns between different classes of drugs of abuse (e.g., cocaine vs. opioids). We plan to compare non-nuclear proteins to those specifically bound to chromatin in the NAc following repeated exposure to cocaine, morphine, or saline.