Proteomic Analysis of Cellular Alterations Associated with Drug-Induced Impairments in Decision-Making, Mnemonic Processes, and Relapse Behaviors

Decision-making: The decision-making processes that confer risk for addiction may differ from those that are disrupted by chronic drug use and have important implication for preventing and treating addiction. We have recently identified a computational derived decision-making parameter that predicts subsequent drug-taking behaviors (e.g., + parameter) which, importantly, differs from the decision-making parameter that is altered following drug use (e.g., 0 parameter). These findings suggest that the behavioral processes that mediate an individuals risk for developing an addiction (e.g., ∆+ parameter=”vulnerability”) differ from the behavioral processes that are disrupted by drug use (e.g., ∆0 parameter=”consequence”). Because, these results suggest that the biological mechanisms mediating addiction risk are dissociable they, therefore, can be used to identify novel protein targets for the prevention and treatment of addiction, respectively.

Our approach has used label-free LC/MS-MS to quantify expression in the ventral striatum of rats trained on a flexible decision-making task (probabilistic reversal-learning) that were either drug-naïve or had self-administered methamphetamine. We hypothesized that proteins involved in addiction vulnerability would be: 1) correlated with the ∆+ parameter in both drug-naïve and drug-exposed rats and 2) would not be significantly different in rats that had self-administered methamphetamine compared to drug-naïve rats because the ∆+ parameter was not disrupted following drug use. Of the 2,815 proteins measured in the ventral striatum, three protein targets met this criterion: sorting nexin 1 (Snx1), ryanodine receptor 2 (Ryr2), and ataxin 2-like (Atxn2l). Remarkably, genes encoding these same proteins have been previously linked to addiction in humans. We then sought to identify proteins involved in the addiction consequence phenotype. We hypothesized that proteins responsible for drug-induced changes in decision-making would 1) correlate with the ∆0 parameter in both drug-naïve and drug-exposed rats and 2) would be significantly altered in rats that had self-administered methamphetamine compared to drug-naïve rats since the ∆0 parameter was significantly disrupted following drug self-administration. Only one protein met this criterion: ras-related protein Rab3B. Rab3B is involved in synaptic transmission and vesicle trafficking and is upregulated in animals chronically exposed to sucrose and ethanol suggesting that enhanced expression of Rab3B may be a biological response to chronic exposure to highly reinforcing outcomes.

We have recently performed the same proteomic analysis in orbitofrontal tissue collected from the same rats as used above, and are preparing a manuscript describing these new, exciting results that have identified monoamine oxidase A (MAOA) as an addiction susceptibility protein. Ongoing studies and planned studies for the renewal are examining how viral and pharmacological manipulations of these proteins impacts decision making and drug-taking behaviors in order to provide causal evidence for these alterations in aberant behaviors associated with addiction and to identify novel pharmacotherapies for treating addiction-relevant behaviors. We plan also to extend these behavioral and computational approaches combined with proteomic analyses to other drugs of abuse and to examine sex differences.

Collaborating Center Investigators: These studies were conducted in a collaboration between Drs. Stephanie Groman and Jane Taylor, and the Nairn Lab (Becky Carlyle). Details of these studies that were also part of a Yale/NIDA Neuroproteomics Pilot grant awarded to Dr. Groman. Planned studies will continue this collaboration.

Mnemonic Processes: Our previous work has demonstrated that manipulating cocaine-cue memories by destabilizing them through interfering with the reconsolidation process is one potential therapeutic tool by which to prolong abstinence. We have recently successfully used the naturally-occurring histone acetyltransferase (HAT) inhibitor, garcinol, to block the reconsolidation of a cocaine-cue memory in a manner that is specific to the reactivated memory only, cue-specific, long-lasting and temporally constrained. These data support the hypothesis that garcinol may be a useful novel therapeutic tool by which to interfere with the reconsolidation of cocaine-cue memories. Our recent studies have aimed to examine the neuroproteomic profile of key brain regions involved in addiction and mnemonic processing such as the lateral amygdala (LA) and nucleus accumbens. We collected tissue following cocaine-cue memory retrieval any garcinol administration and in collaboration with Dr. TuKiet Lam (Discovery Proteomics Core) used a label free quantitative approach (LCMS/MS) to examine protein regulation. Our results showed significant regulation of 14 proteins following retrieval and garcinol administration, half of which were identified as regulators of microtubule dynamics. Follow up studies revealed that garcinol is capable of decreasing alpha-tubulin acetylation in both primary neurons and in the human neuroblastoma SH-SY5Y cell line. Garcinol was also seen to decrease the expression of Fez1, a kinesin adaptor protein involved with vesicular transport, in primary neurons. We are working on a manuscript based on these data. We plan to examine differential protein regulation in samples from the nucleus accumbens following cocaine-cue memory retrieval and garcinol, and preliminary analysis shows garcinol may be altering microtubule dynamics in this region as well. Our initial analysis revealed 139 proteins significantly regulated following retrieval alone (no garcinol). One interesting protein we chose to further examine in an aversive memory paradigm (fear conditioning) is Retinol binding protein 1 (RBP1). We found that RBP1 protein expression is associatively regulated in the LA following fear memory retrieval in male, but not female rats. Further, RBP1 mRNA expression in males is enhanced. These studies were initially funded by the Taylor lab and also a previously funded Pilot Project to Dr. Melissa Monsey. Planned studies will examine the effects of dietary and pharmacological retinol manipulations to elucidate what role RBP1, Fez1 and retinol may play during cocaine-cue memory retrieval. These new studies will aim to focus on these targets, and examine how potential sex differences in these signaling molecules are regulated by cocaine memory reconsolidation processes, and can be manipulated therapeutically.

Relapse Behaviors: With the addition of new NIDA funding (Development of Medications to Prevent and Treat Opioid Use Disorders and Overdose” UG3/UH3 DA050322), we are conducting a preclinical and clinical evaluation of the NMDA modulator NYX-783 for Opiate Use Disorders (OUDs). The UG3 component (PIs DiLeone & Taylor) project will use a mouse model of OUD with oxycodone self-administration to study the ability of the NMDA modulator NYX-783 to reduce relapse behavior. NYX-783, a novel small molecule being developed by Aptinyx, has shown evidence of safety/tolerability in Phase 1 studies and is currently in Phase 2 trials for PTSD. Data suggests efficacy in reducing opiate seeking behavior and relapse behaviors measured by cue- and drug-induced reinstatement and the proposed UG3 experiments will evaluate multiple models and dose-ranges while also testing the safety of NYX-783. We have experience with rodent reinstatement models (see Monsey studies above) and we plan to conduct a proteomic analysis of tissue from these oxycodone self-administering animals. We will also determine the effects of NYX-783-induced alterations in signaling proteins with and without oxycodone in animals sacrificed after the completion of our behavioral studies. We will also – where feasible (depending on the experimental design and outcome measures of our other NIDA funded projects) – conduct parallel studies in cocaine and methamphetamine self-administering animals to investigate neuroproteomic correlates of drug-induced signaling changes after behavioral tests of relapse-like behaviors, such as cue and drug induced reinstatement.

Summary: Together these projects are directly related to the Center’s theme of “Proteomics of Altered Signaling in Addiction” because they collectively examine the aspects of addiction vulnerability and consequence from a proteomic basis in brain neurocircuits involved in decision-making, mnemonic and relapse processes.