2025 Project Descriptions

Ashley Xu, Department of Neuroscience, Indiana University

As legalization of cannabis has spread to more states, there has been a steady rise in its medical and recreational use. The perception of low risk of cannabis product consumption by the general public has led to a surge in usage in all population groups, including pregnant women, especially for cannabis' effect in reducing pregnancy symptoms. Tetrahydrocannabinol (Δ9-THC) is the major psychoactive constituent in cannabis, and due to its lipophilic nature, it easily crosses the placenta and blood-brain barrier to enter the fetal brain. Human longitudinal studies have shown that following perinatal cannabis exposure (PCE) both adolescent and adult offspring have a higher chance of developing affective disorders including anxiety, depression, and schizoaffective disorders. Using a mouse model of PCE with THC, behavioral data from our lab showed that after THC-PCE, fluoxetine was ineffective in enhancing resilience in coping with helplessness-like situations (i.e., reducing immobility time in the forced swim test) for both sexes. Based on human data and our own preclinical studies, we hypothesize that THC-PCE blunts serotonergic system functioning and may lead to hypersensitivity to environmental insults. In the proposed study, we will implement the chronic unpredictable stress (CUS) paradigm to mimic the mild unpredictable stress that humans often experience. The dorsal raphe nucleus (DRN), where most serotonergic neurons reside, plays critical roles in modulating serotonergic CNS signaling. We first aim to interrogate the impacts of CUS on the proteome of the isolated DRN from adult mice with or without THC-PCE. Second, we will attempt to specifically isolate serotonergic neurons within the DRN by using Pet1-Cre::tdTomato mice (reporter mice for serotonergic neurons) and subject dissociated DRN to fluorescence-activated cell sorting (FACS) before proteomic analysis provided by the Yale/NIDA Neuroproteomics Center. Elucidating the proteomic changes in serotonergic neurons between control and CUS from THC-PCE and vehicle-PCE adult progenies will advance our understanding of the underlying cellular mechanisms behind the combined effects of PCE and stress.

Cocaine is the most commonly abused stimulant, yet with no FDA-approved treatments, overdose-related morbidity continues to rise. There is a robust literature demonstrating importance of transcriptional and epigenetic regulation in the nucleus accumbens (NAc) on cocaine induced behavioral changes, but the full scope of effects of cocaine on this molecular machinery is not fully understood. Within the NAc, there are two primary populations of projection neurons, referred to as medium spiny neurons (MSNs), those expressing the D1 type and those expressing the D2 type dopamine receptors. While both populations of neurons are important for regulation of behavior, there is particularly robust evidence for regulation of transcription factors such as FosB and CREB in the D1-expressing neurons in driving cocaine taking and seeking behaviors. Here, we will utilize a transgenic mouse model to isolate and define cell-type specific changes in the nuclear proteome of D1 expressing MSNs in the NAc after cocaine self-administration. In two specific aims, we will utilize unbiased data independent acquisition (DIA) mass spectrometry, to identify differential expression of nuclear proteins in these D1 medium spiny neurons (1) twenty-four hours after conclusion of cocaine self administration, and (2) after a period of prolonged withdrawal that is associated with incubation of cocaine craving. A behavioral approach of cocaine self-administration and saline controls in Drd1Cre x EGFP/Rpl10a mice will be utilized for both aims, allowing the reinforcing effects of the drug to be associated with molecular changes. After self-administering drug or saline for 10 days under a fixed-ratio 1 schedule of reinforcement, mice will be sacrificed at the specified timepoints. At each time point (after self-administration and after withdrawal), tissue from the NAc will be collected, nuclei isolated, and D1+ nuclei purified by fluorescence assisted nuclear sorting. Levels of nuclear proteins from this D1+ population will be quantified at each timepoint using DIA mass spectrometry. Integrated pathway analyses and gene ontology will provide crucial information on the biological processes in which identified proteins are involved. Differentially regulated proteins can be targeted for future studies investigating roles of nuclear proteins in driving addiction like behavior. The complex nature of addiction and underlying biology warrants further understanding of individual components of the cell at distinct behavioral time points of drug use. This project will allow for the identification of cell-type and cell-component specific targets for cocaine use disorder, while enhancing our understanding of the underlying neurobiology of cocaine.

Our research aims to investigate the proteomic changes in the nucleus accumbens (NAc) caused by stress-induced opioid consumption, focusing on differences between sexes. By leveraging the resources of the Yale/NIDA Neuroproteomics Center, we will explore the interaction between chronic social defeat stress (CSDS) and opioid self-administration to understand the molecular mechanisms underpinning addiction and depression comorbidity. The overarching goal is to identify therapeutic targets that can be manipulated to treat the intersection of stress, depression, and opioid use disorder (OUD). Depression and addiction are highly comorbid conditions with significant public health implications. Major depressive disorder (MDD) affects 8% of adults, while over 9 million individuals reported misuse of prescription pain medication in 2021. Chronic stress is a major risk factor for both MDD and OUD, and individuals with one condition are more likely to develop the other. Notably, women are disproportionately affected by the comorbidity of MDD and OUD but females remain underrepresented in preclinical studies. The mesolimbic dopaminergic pathway, particularly the NAc, is crucial in processing rewards and is implicated in both depression and opioid addiction.

Our approach is innovative in several ways:

1. Combined Study of Stress and Drug Abuse: We will not study stress or opioid use in isolation but will explore their comorbidity.
2. Use of Oral Oxycodone Self-Administration: This method allows continuous drug access in a familiar context, providing a more accurate model of human opioid use.
3. Inclusion of Both Sexes: By including male and female mice, we aim to uncover sex-specific proteomic changes, addressing a significant gap in current research.
4. Proteomic Analysis: Bilateral punches of the NAc will be analyzed using LC-MS mass spectrometry. Data-dependent Acquisition (DDA) will be used to help identify stress and drug-specific proteomic changes across sexes.

This pilot study will be the first to provide a detailed analysis of proteomic changes caused by stress-induced opioid consumption in both male and female mice. Our findings will pave the way for preclinical studies aimed at identifying molecular mechanisms involved in the comorbidity of MDD and OUD.

By integrating our results with existing transcriptomic and proteomic datasets, we hope to identify therapeutic targets for genetic manipulation and better understand the molecular adaptations caused by stress-induced opioid abuse. Our collaboration with the Yale/NIDA Neuroproteomics Center will ensure robust analysis and validation of our findings. This research holds the potential to significantly advance our understanding of depression and opioid use disorder comorbidity, ultimately guiding the development of new therapeutic interventions.

With the advent of legalization, cannabis (Cannabis Sativa) is rapidly growing in popularity with ~22% of Americans reporting use. Legalization efforts have had multiple positive outcomes, including reduced arrests and incarcerations for possession and use. However, easing laws have also been associated with changing attitudes towards the drug with many individuals believing that cannabis is not addictive, including adolescents. Many teens and emerging adults are now regularly using cannabis by vaping or consuming edibles (popularized due to high concealability) and research shows that adolescent use increases the risk of developing cannabis use disorder (CUD), especially when use is initiated before age 16, and that CUD symptoms at age 18 predicts CUD severity in adulthood. These effects are observed at higher rates in males, indicating important sex specific mechanisms underlying vulnerability to CUD. As cannabis becomes more broadly available, it is essential that we determine mechanisms contributing to CUD risk that emerge after adolescence use to help in the development of novel therapeutics. Seminal animal studies including our own have established that exposure to delta-9-tetrahydrocannabinol (THC) during adolescence has enduring impact into adulthood associated with increased vulnerability to addiction-like behaviors and related psychiatric phenotypes. Our recent data shows that THC exposure in male rats increases risky decision-making and impulsivity in adulthood, two phenotypes associated with the onset and maintenance of CUD. What was unclear from these data was whether these cognitive phenotypes predicted adult drug intake. Our novel experimental data using a translational model of edible consumption shows that volitional consumption of THC during adolescence significantly increases risky decision-making which predicts escalation of consumption of THC in adulthood. Surprisingly, this effect was only observed in males, recapitulating results from clinical data and reveal that important sex differences contribute to cognitive outcomes that impact CUD-like behavior. Critical neurobiological knowledge is needed to understand how adolescent THC consumption impacts the maturation of mechanisms relevant to cognition and reward and the impact of biological sex on these outcomes.

Understanding protein expression profiles within brain regions is essential to uncovering cellular function and is key to exploring disease pathology. To further deepen the brain proteome atlas in the spatial context of brain regions, there is a need for unbiased, quantitative technology capable of mapping proteomes within brain regions with high accuracy and precision.

In this technology pilot project, I propose to use the newly released 11K cell and tissue lysate (CTL) SomaScan kit to carry out comparative tissue proteome analyses on anatomically different human and mouse brain regions including cortex, striatum, and cerebellum. Two of these brain regions (i.e., cortex and striatum) showed the largest inter-regional differences in gene expression by RNA sequencing in the BrainSpan project, while the cerebellum and cortex exhibit wide differences in patterns of protein expression in various human and rodent proteomic studies. The SomaScan method employs affinity-based platforms to bind target proteins for identification using single-strand DNA aptamer reagents with slow off-rate kinetics. In order to expand the understanding of the mouse and human brain proteomes, I will carry out proteomic analysis on human and mouse brain region extracts using the recently released 11K CTL kit. The SomaScan assay is compatible with both human and mouse samples.

Direct analyses to address the relative strengths and weaknesses of the 11K CTL platform, and how it compares with mass spectrometry (MS) approaches, are lacking. For a detailed comparison of the quantitative protein expression data obtained from the SomaScan versus MS platforms, I will also analyze samples using Data Independent Acquisition (DIA) based MS together with the Discovery Core. The SomaScan assay should allow us to identify and quantify ~6000 proteins and DIA based MS approach should allow us to identify and quantify ~3000-4000 proteins in brain tissues. Working together with the Biostatistical and Bioinformatics Core, the comparative SomaScan versus DIA mass spectrometry analyses on the brain proteome will assess the data quality and correlation between the two platforms. The identification of common and exclusive proteins measured by the two different platforms should improve the depth of profiling of region-specific brain proteomes beyond that afforded by any one platform due to their inherent methodologies and limitations. If successful, the project may offer an alternative approach to the Center’s 27 investigators at 10 institutions who are all carrying out research projects directed at understanding the proteomics of altered signaling in addiction.