2020-2024 Project Descriptions

Mary F. Wang, Department of Chemistry, North Carolina State University

Substance use disorders have become increasingly prevalent in society over the course of time. In particular, from 1990-2016 the use of psychostimulants such as cocaine have increased by 39.7 percent. Cocaine use disorder (CUD) is a relapsing condition without any approved pharmacological treatments or therapies. As this issue has become common in societies, researchers have focused their attention on determining underlying mechanisms of addiction to help guide the development of treatments. Recent findings demonstrate that short-chain fatty acids (SCFAs) found in the gut microbiome are directly correlated with drug-seeking behaviors in cocaine-treated rodent models of addiction. While these findings provide a deeper understanding of the impacts that these metabolites have on the gut microbiome, we seek to further clarify our understanding of the impact of these treatments on SCFAs located within the brain. Preliminary results from Drew Kiraly’s laboratory at Wake Forest University School of Medicine provide the groundwork required for performing effective mass spectrometry imaging (MSI) analyses of treated mouse brain sections. Using the resources of the Yale/NIDA Neuroproteomics Center these experiments will (1) determine the spatial distribution of key SCFAs within the brain and how they are up- and down-regulated in the presence of cocaine- and control-treatment groups, (2) use the advanced data imaging processing algorithms based on human perception developed in our group to derive a list of co-localized metabolites which may be contributing to drug-seeking responses in the mouse model, and (3) perform DIA proteomic MS analysis to enhance the biological coverage of molecular species within the cocaine-treated mouse brain model. The overarching goal is to differentiate the endogenous SCFAs from supplemented ones and provide spatial distributions of these metabolites to reveal where they end up across the brain. Additionally, the resources provided by the Yale/NIDA Neuroproteomics Center will enable DIA proteomic MS analyses of those regions of the brain where SCFAs co-localize. These data will inform research to understand the localization of SCFAs in the brain, in addition to identifying co-localized metabolites and proteins in response to altered signaling in cocaine addiction. These findings can lead to the development of treatment options while elucidating molecular mechanisms of established behavioral effects. In addition to furthering our scientific understanding of cocaine addiction through a multi-OMIC approach, this project will initiate new collaborations between the Kiraly and Muddiman laboratories for future research directed at uncovering underlying mechanisms of addiction.

In just seven years (2010 to 2017) the number of pregnant individuals in the United States with opioid related diagnoses went up by 131%, a number that has continued to grow. An increasing population of children are exposed to opioids in utero. There are marked individual differences in the effects of prenatal opioid exposure on child neurodevelopment and identifying children most likely to be affected is a fundamental challenge. Extracellular vesicles (EVs) isolated from plasma and serum have emerged as clinically relevant biomarkers of several disease states. EVs acquire molecular cargo, including protein, from parent cells providing a window on translational processes within cells of origin. EVs derived from fetal neural tissue can be isolated from maternal plasma, with one study finding that in utero opioid exposure may alter the protein cargo within fetal neuron derived EVs. Existing fetal neuron EV isolation protocols are based on immunoprecipitation targeting Contactin-2 a protein highly expressed in the developing brain. However, our team has shown that existing isolation protocols may lack specificity and require optimization to realize the potential of EV-based biomarkers for understanding fetal and child neurodevelopment. In this project we seek to optimize total EV isolation from cord blood serum and cortical brain organoid media. Using existing cell culture media samples collected from a large collection of brain organoids and accessible cord blood serum, we will compare three alternative exosome isolation techniques used to minimize non-EV related protein contamination (e.g., albumin, APoA/APoB), which may limit the sensitivity of downstream proteomics. Next, we will look at the proteomic cargo within total EVs isolated from cord blood serum of neonates exposed to opioids in utero (versus controls). To identify EVs derived specifically from neuronal tissue, we will use label-free mass spectrometry to characterize the proteins embedded within the surface membrane of brain organoid derived EVs. In doing so we seek to identify candidate proteins that can be used, as an alternative to Contactin-2, that will improve the isolation of fetal neuron derived EVs from cord blood serum. Future work will capitalize on this technical advance to isolate fetal neuron derived EVs from opioid exposed pregnancies as part of a K development award to examine potential EV-based biomarkers of the impact in utero of opioid exposure; a critical first step towards targeted intervention.

Arnab Sengupta, Department of Psychiatry, Yale University

Rates of cannabis use are increasing in the USA with over 19 million suffering from cannabis use disorder (CUD) in 2021. CUD is associated with neurocognitive deficits, mood and anxiety disorders, increased risk of psychosis, and decreased quality of life. There has been recent recognition of a withdrawal syndrome associated with cessation of use of cannabis that affects nearly 50% of users and that contributes to relapse. Preclinical studies report synaptic alterations after repeated exposure to delta-9-tetrahydrocannabinol (THC, the main active constituent of cannabis) that return to baseline with cessation of use. But the molecular basis of these changes is yet to be elucidated in clinical populations in part due to the challenges of studying human brain tissue. Peripherally circulating neuronal extracellular vesicles (NDEVs) are a novel tool to study CNS molecular changes and intercellular communication. NDEVs (lipid bi-layered structures containing cargo proteins and RNA) facilitate key functions including synapse assembly and plasticity, can cross the blood brain barrier with intact cargo, and can be isolated from peripheral plasma. A previous Yale/NIDA Neuroproteomics Center pilot research grant found decreased SHANK 1 protein expression in plasma derived NDEVs amongst individuals with CUD relative to healthy controls. SHANK1, which contains an SH3 and multiple ankyrin repeat domains, is involved in integrity of glutamatergic post-synaptic structures in the brain, which plays a role in cognitive impairments. However, this study was not designed to examine clinical or neuroimaging correlates of the NDEV proteome, or the impact of withdrawal or abstinence from cannabis. In a recent positron emission tomography (PET) study, we reported a decrease in synaptic density in CUD assessed using [¹¹C]UCB-J, a PET radioligand with specificity for synaptic vesicle glycoprotein 2A. Reduced synaptic density in CUD positively correlated with reduced verbal memory on the Rey Auditory Verbal Learning Task (RAVLT). CUD is known to be associated with neurocognitive deficits in attention, verbal memory and working memory with mixed data on deficits persisting after prolonged abstinence. Whether the proteomic alterations in CUD25 persist after abstinence and their correlation with clinical and neurocognitive functioning remains unstudied. This proposal aims to leverage existing samples from two ongoing PET studies in our laboratory of individuals with CUD during typical use and after 4 weeks of abstinence to examine the proteomic changes in peripherally isolated NDEVs. Further, correlations between proteomic changes, and cognitive function [measured by verbal memory task (Auditory Verbal Learning Task)], and synaptic density [measured by [¹¹C]UCB-J binding potential (BPND)] also will be examined. Specific aims include, 1: To examine changes in plasma derived NDEV characteristics and proteome in CUD during typical use (day 0) and after abstinence (day 28) relative to matched healthy controls; 2a: To examine correlations between the NDEV proteome and synaptic density in CUD; 2b: To examine correlations between the NDEV proteome and verbal memory function in CUD. Future studies will examine RNA content of NDEV in CUD. Together, these data will provide the basis for a grant application to study peripheral biomarkers of neuropathology in CUD.

Cocaine Use Disorder (CUD) is a pervasive condition with long-lasting effects on brain function, often leading to increased risk for neuropsychiatric conditions such as bipolar disorder (BD). Chronic cocaine use induces profound signaling and epigenome alterations, including histone post-translational modifications (PTMs), which persistently affect gene expression and chromatin structure in key brain regions involved in reward and motivation, particularly the nucleus accumbens (NAc). Understanding these molecular changes is critical for unraveling the mechanisms underlying the comorbidity of CUD and BD. Recent advances in epigenetics have highlighted the central role of histone PTMs and chromatin-interacting proteins in regulating gene responses. Chronic cocaine exposure induces long-lasting changes in histone PTMs and histone variant exchange, playing key roles in transcriptional dysregulation in neurons following prolonged withdrawal. Our prior studies have linked these processes to enhanced genome accessibility and priming of gene programs that control neural plasticity. This proposal aims to elucidate the molecular mechanisms by which cocaine-induced epigenome reprogramming increases BD risk in a translationally relevant mouse model. In Aim 1, we will map histone PTMs in chronic cocaine exposure and assess their role in bipolar disorder susceptibility. We will identify histone PTMs associated with chronic cocaine exposure, chronic unpredictable rhythm disturbance (CURD)—a mouse model for BD—their combination, and a control group with no exposure. Histone PTMs in the NAc will be comprehensively profiled using high-resolution mass spectrometry at the Yale/NIDA Neuroproteomics Center, revealing condition-specific modifications and their roles in gene expression regulation. Integration with mRNA transcriptomics data will help uncover cocaine-induced epigenetic marks that could serve as therapeutic targets for individuals with a history of chronic cocaine use and increased risk of BD onset. In Aim 2, we will study recruitment of protein complexes in chromatin remodeling following chronic cocaine exposure. Our studies indicate that chronic cocaine exposure leads to significant epigenetic changes, including the recruitment of chromatin-interacting proteins KDM1A, SMARCA5, and RBBP7, which are crucial for chromatin remodeling and gene expression. We hypothesize that these enzymes, when recruited to chromatin upon cocaine exposure, result in histone modifications that drive gene expression changes supporting BD pathogenesis. To profile these interactions in mice and the CURD model of BD, we will use rapid immunoprecipitation mass spectrometry of endogenous proteins (RIME) for KDM1A, SMARCA5, and RBBP7 with ChIP-validated antibodies followed by high-resolution mass spectrometry at the Yale/NIDA Neuroproteomics Center. We aim to identify complexes involving these proteins, their interactions with histone modifications, and their influence on gene expression relevant to BD and SUD comorbidity. By addressing the critical areas of cocaine-induced epigenetic changes and their impact on BD risk, this research promises significant insights into the comorbidity of CUD and BD, leading to comprehensive grant proposals for extended funding.

Biotinylation by Antibody Recognition (BAR) is a high-throughput in-situ proximity labeling technique that can identify protein-protein interaction networks. The long-term goal of the proposed research is to apply BAR to measure signaling pathways of addiction. This proposal aims to develop BAR for multiple addiction signaling components in the striatum. The central hypothesis is that BAR can be used to screen the proteome of phenotypically unique populations of cells (e.g., microglia) and known addiction pathways targets (e.g., dopamine transporter). Our rationale for the proposed studies is that by developing BAR for several addiction targets in the striatum, we will provide an unbiased method to map addiction signaling pathways in animal models and human tissues. The central hypothesis will be tested with one aim: 1) Investigate BAR targets of addiction in the striatum of a rodent model of addiction. This pilot proposal is innovative because it applies in situ labeling technology to several models of drug addiction. The proposed research is significant because it will be the first step to developing a high throughput method to map addiction signaling pathways in the striatum. The expected outcome of the proposed research is to validate several BAR targets that can be applied to models of addiction.

Substance use disorder (SUD) has been identified as a major national health emergency with a devastating impact on our society and public health. Substances of abuse, such as psychostimulants and opiates, exert their reinforcing effects by inducing neural alterations in the striatum and many of these changes occur in two main subtypes of medium spiny neurons (MSNs) which are differentiated by enrichment of dopamine receptor 1 (D1) or dopamine receptor 2 (D2) and by their respective projection pathways (striatonigral D1 direct pathway and striatopallidal D2 indirect pathway). Stress is a major risk factor for the development of SUD and addiction relapse, and it significantly contributes to the sex differences in vulnerability to addiction. Stress-induced addiction is thought to be mediated by dysregulated synaptic plasticity in the striatum, resulting in altered synaptic signaling in a cell type-specific manner. Therefore, understanding how stress influences the proteome of the striatum in a sex- and cell type-specific manner is a crucial first step in identifying the protein networks that underlie synaptic alterations that may precipitate addictive behaviors, and that could lead to the development of novel and effective treatment options for SUD. With the state-of-the-art technologies and expertise provided by the Yale/NIDA Neuroproteomics Center, we propose to optimize a laser capture microdissection (LCM)-based proteomics workflow to identify sex- and cell type-specific proteomic changes in D1 and D2 neurons in the striatum of mice subjected to subchronic variable stress (SCVS). In Aim 1, we will refine a method using LCM to isolate D1 and D2 neurons from the striatum of Drd1-EGFP and Drd2-EGFP mice for proteomic analysis. In Aim 2, we will identify proteomic changes in the whole striatum tissue and in striatal D1 and D2 neurons following SCVS in both female and male mice. The proposed study will allow us to optimize an LCM-based proteomics workflow and to generate original data characterizing the sex differences in stress-induced cell type-specific proteomic changes in the striatum. The novel protein targets identified in our discovery-based study may also encourage future hypothesis-driven investigations into sex- and cell type-specific signaling pathways that may precipitate increased vulnerability to substance abuse.

The ongoing opioid crisis has led to increased opioid use in pregnant individuals, resulting in higher rates of fetal drug exposure. Medications for opioid use disorder (MOUD) – such as methadone – are regularly prescribed to pregnant people who are opioid-dependent. While MOUD are highly effective in reducing opioid craving, withdrawal, and relapse, clinical work has reported cognitive deficits in children and adolescents prenatally exposed to MOUD. The prefrontal cortex (PFC) is a late-developing region of the human and rodent brain that acts as a key regulator of cognitive performance. Research suggests that PFC maturation is impacted by prenatal opioid exposure. Moreover, a substantial literature indicates that adolescence is a period of development that is particularly plastic, marking experience-dependent maturation of neurons, glia, and structural components in the PFC. Adolescence consists of distinct stages (early, mid-, and late adolescence) that are affected differentially by adverse experiences early in life in a sex-dependent manner. Therefore, the proposed pilot project aims to characterize how prenatal MOUD – in the form of methadone – alters the PFC proteome throughout adolescence in a sex-dependent manner, focusing on subsets of proteins regulating neuroimmune functioning and extracellular matrix control of neuroplasticity. The proposed project will use osmotic minipumps in rats as a means of drug delivery prior to and during pregnancy. These minipumps are simple to implant and maintain, while removing potential stress-inducing effects of daily injections during pregnancy, which would likely affect immune development in offspring. Following prenatal exposure to methadone or saline, male and female offspring will be sacrificed at postnatal day (PND) 28, 35, and 55 to represent early, mid-, and late adolescent timepoints. This will allow for direct assessment of prenatal MOUD- induced alterations in the trajectory of PFC proteome development. The proposed work will be conducted at Simmons University, a women-centered, primarily undergraduate institution in the heart of Boston. Findings from the proposed project will serve as preliminary data for a federally funded grant application aimed at facilitating research at primarily undergraduate institutions. This pilot grant would therefore lay the foundation for future work researching addiction neurobiology at Simmons, preparing the next generation of addiction researchers by providing opportunities for undergraduates to gain critical lab experience.

In the realm of neuroscience, the quest for comprehending the intricacies of the brain at a cell-type- specific and single-cell resolution is steadfast. However, this pursuit is impeded by the challenges posed by achieving accurate tissue isolation and acquiring fresh brain samples. Thus far, cell-type-specific human brain proteomes remain elusive. This poses a substantial knowledge gap in understanding protein expression and RNA-protein correlation at the cell-type-specific and single-cell levels. Moreover, the absence of reference proteomes tailored to distinct cell types within the human brain accentuates this knowledge gap, as these reference proteomic data are essential for emerging fields like subcellular proteomics and single-cell proteomics. In this pilot project, I will establish two parallel isolation and proteome methods to study cell-type-specific proteomics in both fresh and postmortem human brain samples. Firstly, I will employ innovative immunopanning techniques to isolate different cell types in fresh human brains. Subsequently, cell-type specific DDA- and DIA-based LC-MS-MS proteomics will be applied to analyze these samples. To address potential protein loss during tissue isolation, I will utilize a novel antibody-based proximity labeling proteomics pipeline that I previously established to uncover proteins in cell-type-specific compartments, such as cell bodies and processes, in postmortem human brain sections. By comparing the proteome datasets obtained from immunopanning and proximity labeling in fresh and postmortem brain samples, I aim to gain insights into the cell-type-specific proteome in human brains. Ultimately, this work seeks to establish high-quality cell-type-specific reference proteomes in human brains. These reference datasets will be instrumental in understanding the molecular basis of human brains, illuminating RNA-protein correlations, and advancing data analysis in subcellular proteomics and single-cell proteomics in humans. Furthermore, the project will provide valuable tools and resources to enhance our understanding of human brains in both health and diseases, such as drug addiction.

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.

Microglia, the resident immune cells of the brain, respond to environmental cues through morphological, transcriptional, and metabolic signaling adaptations. Throughout their life span microglia dynamically participate in many neural processes from synaptic development to functional and structural plasticity through complex patterns of neuroinflammatory signaling between microglia and other neural cells. Evidence indicates that microglia responses are important in drug-reward behaviors and contribute to addiction to various drugs including alcohol, cocaine, opioid, and nicotine. The conceptual premise of this project is that microglia are drivers of innate immune memory within critical neural circuits that mediate drug-associated behaviors. In support of this, there is evidence that although microglia are widely distributed throughout the adult brain, higher expression has been reported in reward centers such as the substantia nigra, basal ganglia, and hippocampus. In collaboration with ongoing research on sex-specific mechanisms of nicotine addiction and withdrawal at the Yale/NIDA Neuroproteomics Center, this project will identify proteomic changes in microglia of adult mice. Initial work will focus on the hippocampus as an important site for sex-specific neuroinflammatory signaling during nicotine addiction and withdrawal.

Of the 50 million+ adults in the United States suffering from chronic pain, 19.6 million suffer from high-impact chronic pain that interferes with life or work activities. Though cannabinoid-based therapies offer a needed alternative to opioids for the treatment of chronic pain, tolerance to their therapeutic effects can rapidly develop, limiting their efficacy while also facilitating escalating consumption and dependence. Consequently, identification of the mechanism(s) of tolerance to the analgesic effects of cannabinoids may serve to prolong their clinical utility. Desensitization (uncoupling of a receptor from its G proteins) and internalization (loss of receptor from the cell surface) are cell-based correlates of tolerance that involve G protein-coupled receptor kinase phosphorylation. For cannabinoid type 1 receptors (CB1R), desensitization and internalization appear to be mediated by distinct receptor domains. Previous work in vitro suggests that desensitization of CB1R is mediated by residues S426 and S430 while six different carboxy terminal serines and threonines (T461, S463, S465, T466, T468, and S469) are critical for CB1R internalization. We subsequently evaluated the contribution of these 8 different phosphorylation sites in vivo through the creation first of a desensitization-resistant (S426A/S430A) and then an internalization-resistant six-point mutant (6PM) mouse by mutating each of the phosphorylation sites to a non-phosphorylated alanine. The central hypothesis is that internalization of CB1R counteracts tolerance to strongly internalizing cannabinoids, such as CP55,940, and that tolerance to CP55,940 would be more profoundly impacted in 6PM mice relative to ?9-THC, a weakly internalizing, partial agonist. In contrast, desensitization of CB1R would have a greater effect on tolerance development to ?9-THC compared to CP55,940. Behaviorally, we have repeatedly shown that desensitization-resistant S426A/S430A mice display enhanced sensitivity and delayed tolerance to ?9-THC-mediated antinociception. Preliminary data collected from our recently generated six-point mutants (T461A/S463A/S465A/T466A/T468A/S469A; 6PM) has revealed decreased sensitivity and faster tolerance to CP55,940-induced antinociception. This proposal seeks to identify molecular neuroadaptations associated with cannabinoid tolerance that will complement our behavioral data, to be utilized in a larger proposal for NIDA funding on cannabinoid tolerance. Specifically, this pilot project will utilize the cutting-edge use of phosphoproteomics to identify agonist-specific downstream targets in commercially available HEK293 cells stably expressing HA-CB1R that are treated with either vehicle, ?9-THC or CP55,940 at time points (5 and 60 minutes) sufficient to induce desensitization and/or internalization. Our hypothesis is that treatment with CP55,940, a strongly internalizing cannabinoid agonist, will result in robust phosphorylation of the six internalization residues while treatment with ?9-THC, a weakly internalizing agonist, will result in phosphorylation at the residues involved in desensitization, S426 and S430, with limited phosphorylation at the six internalization residues. The results of this project will be utilized in a larger grant to devise the precise nature of diverse downstream signaling pathways dictated through CB1R agonist-specific binding with the eventual aim of devising appropriate therapeutic strategies to combat cannabis tolerance and dependence in managing chronic pain.

Despite strides toward understanding circuit and molecular mechanisms of substance use disorders (SUDs), treatment options remain largely unchanged. This impasse is due, in part, to limitations in the construct and predictive validity of animal models of drug self-administration and relapse, which rarely incorporate social factors. In both humans and laboratory animals, adverse social interactions and social isolation promote drug self-administration and relapse, while positive social interaction tends to be protective. We recently developed an operant rat model of choice between drugs and social interaction and showed the profound protective effects of the latter on drug self-administration and relapse. Our research revealed two major findings. First, rats strongly prefer operant social interaction over heroin, methamphetamine (Meth), and cocaine. Additionally, social choice-induced abstinence (voluntary abstinence) decreases incubation (the progressive increase in drug seeking during abstinence) of heroin craving and prevents incubation of Meth craving. This protective effect was associated with activation of protein kinase-Cd (PKCd) in the central amygdala lateral part (CeL). In contrast, after forced abstinence, the reliable expression of incubation of Meth craving was associated with activation of CeL-somatostatin (SOM). The cellular and molecular mechanisms mediating the protective effect of social reward on heroin craving after voluntary abstinence remain unknown. Based on our preliminary data, we hypothesize that molecular changes within CeL PKCd-expressing neurons in CeL are selective for the social-based buffering of incubation of heroin craving. In contrast, molecular changes within SOM-expressing neurons are selective for the forced abstinence-promoting expression of incubation of heroin craving. The overarching aim of this proposal is to elucidate changes in the neuronal proteome of CeL PKCd/SOM- expressing neurons in heroin craving in order to understand the functional molecular pathways driving either the protective or promoting effects of voluntary versus forced abstinence procedures, respectively. By combining viral-mediated immunolabeling of CeL PKCd-expressing neurons with laser capture microdissection (LCM) followed by isobaric tags for relative and absolute quantitation (iTRAQ), we predict differential protein expression as well as differential post translational modifications (PTMs) within the CeL PKCd/SOM-expressing neurons during heroin craving after either voluntary or forced abstinence. Our proposal will provide new insights into the molecular mechanisms mediating the protective effect of social reward on addiction related measures merging cutting-edge tools for the investigation of neural circuits with an original behavioral model. This direction will provide collaborative potential and introduce new ideas and conceptual frameworks to the Yale/NIDA Neuroproteomics Center’s repertoire with potential direct translational applicability.

Addiction is a chronic relapsing disease with devastating consequences to the health and well-being of patients and their families. Addiction is frequently not restricted to one drug: cannabis and alcohol are amongst the two most concomitantly used and abused drugs. Addiction vulnerability has been associated with altered prefrontal cortex (PFC) activity, leading to a more impulsive executive function. Repeated drug exposure increases glutamate release by PFC glutamatergic neurons projecting to NAc, increasing the sensitivity of the reward circuit to prior drug use-associated environmental cues. Previous in vivo studies identified important signaling networks involved in drug-seeking behaviors, but focused on VTA and NAc rather than PFC, and did not clarify whether the observed changes map directly onto changes in the protein machinery of the neurons. Moreover, addiction has a strong inheritable component, but animal models cannot reproduce the genetic environment of individuals with addiction or that are vulnerable to develop substance abuse. Using human iPSC-derived glutamatergic neurons as a model system of human cortical excitatory neurons is a suitable strategy to overcome the limitations of animal models.

Combining GWAS summary statistics and addiction related traits, our collaborators from the Collaborative Studies on Genetics of Alcoholism (COGA) have calculated polygenic risk scores (PRS) for a cohort of healthy individuals, as well as for cannabis and alcohol addicted individuals, generating an addiction-PRS distribution. hiPSCs from these individuals will be differentiated into excitatory neurons using the NGN2-induction protocol and chronically exposed to THC, ethanol, a combination of both drugs, or vehicle. The goal of the present study is to identify protein changes in the synaptic machinery of the neurons due to chronic THC and/or ethanol exposure using cutting-edge iTRAQ proteomics, and compare this changes between individuals with high and low addiction-PRS. The findings of this study will be further integrated with transcriptomic and functional datasets that have been generated in this cohort of addiction-PRS individuals, to converge on drug-induced disruptions in glutamatergic signaling in human hiPSC-derived excitatory neurons. Elucidating the whole synaptosomal proteome alterations due to chronic THC and/or ethanol exposure in these hiPSC-derived glutamatergic neurons and evaluating the potential differences in these drug-associated changes between individuals with high and low PRS for addiction will markedly advance our understanding of the molecular underpinnings behind genetic predisposition to addiction.

Opioid overdoses continue to increase, despite newly available treatments and enhanced legal regulations. New therapeutic targets are urgently needed. Opioid use disorder (OUD) is twice as likely to develop in PTSD patients, and OUD patients with PTSD have a greater risk of increased OUD severity. This suggests predisposing risk factors exist for the development and severity of OUD in patients with PTSD and perhaps other psychiatric disorders. The amygdala is a significant brain region of convergence between OUD and PTSD, regulating positive and negative emotional states for both conditions. Additionally, altered amygdala structure and volume have been reported in both conditions. We previously conducted transcriptomic profiles in human postmortem amygdala and identified differentially expressed transcripts for both OUD and PTSD. In collaboration with Yale/NIDA Neuroproteomic Center’s Discovery Proteomics Core, we propose a pilot study to identify differentially expressed proteins and causal genes in OUD+, PTSD+, OUD+PTSD, and normal control (NC) postmortem amygdala. In Aim 1, we will generate comprehensive proteomic profiles for each condition. We will use our uniquely developed, state-of-the-art bioinformatic pipeline to integrate transcriptomic and proteomic profiles, and identify sex-specific differences. In addition, we will conduct proteomic co-expression analysis to identify cell type-specific differences in OUD. We will extend our studies to identify single cell type proteomic changes in Aim 2 by using laser capture microdissection on frozen sections of OUD and PTSD postmortem tissue. We will then conduct proteomic profiling and pathway analysis to identify dysregulated expression of neuronal and non-neuronal cells. These preliminary findings will identify biological factors that can be mechanistically interrogated in animal studies to potentially advance diagnostics and therapeutics of OUD.

Unbiased, proteomic approaches for defining the molecular components of a spatially defined region of interest often rely on long extensive purification schemes to isolate the region of interest. Proximity labeling based proteomic approaches have revolutionized this workflow for molecular discovery because they circumvent the need for classical forms of biochemical fractionation, allowing access to many previously unpurifiable subcellular structures. Most iterations of proximity labeling rely on the expression of an enzyme in the region of interest to generate a reactive intermediate that tags neighboring proteins with a biotin handle, for subsequent streptavidin enrichment and identification by proteomics. Although applied widely and successfully in vitro and in cell culture systems, proximity labeling suffers from several limitations when applied in vivo. The major limitation when applied in vivo arises from additional sources of background in tissue, particularly from endogenous biotinylated proteins that similarly compete for the same binding site during streptavidin enrichment. This limitation is particularly debilitating when the region of interest is a small subpopulation of cells within the tissue, such as a small subpopulation of neurons within the brain. This challenge could, however, be overcome by using non-biotin-streptavidin based approaches for chemical enrichment of labeled proteins. As a step towards expanding the in vivo applications of proximity labeling, this proposal will identify or engineer new enzyme scaffolds, capable of generating reactive intermediates like those generated by the proximity labeling enzyme TurboID, that have been functionalized for chemical enrichment instead of biotin-streptavidin enrichment.

Psychedelic drugs, which act on the serotonin-2a receptor, have recently generated interest as potential novel treatments for substance use and mood disorders. A single dose of psilocybin confers antidepressant effects lasting for several months in humans and rodent studies have demonstrated that psilocybin induces structural plasticity in the prefrontal cortex on a timescale which appears to correlate with its antidepressant effects. Causal manipulations of dendritic spine formation can occlude the rapidly acting antidepressant effect for other novel antidepressants like ketamine, suggesting its necessity. Despite great interest in this area, there continue to be fundamental questions about how these spines may integrate into circuits to produce therapeutic effects. Understanding how dendritic spines form and persist in response to rapidly acting antidepressants may facilitate the utilization of these compounds as therapeutics for substance use disorders and could lead to development of pharmacological strategies that prolong their therapeutic effects.

Interestingly, the duration of drug effects on plasticity may not correlate with duration of psychedelic effects. We have demonstrated that even a drug which elicits very brief psychedelic effects can lead to long-term structural plasticity. Using, in vivo two-photon dendritic spine imaging, we have shown that the psychedelic compound 5-methoxy-DMT (5-MeO-DMT) enhances spinogenesis for >1 month after a single dose. In contrast, prior studies have demonstrated that the rapidly acting antidepressant ketamine only enhances spinogenesis for up to 1 week, which correlates with the duration of antidepressant effects. The goal of this study is to investigate the synaptic proteome of newly generated and persistent spines after administration of psychedelics with differing temporal effects on plasticity. We propose to do so using a novel approach via proximity labeling of synaptic proteins in collaboration with Dr. Angus Nairn and the NIDA Proteomics Center. This pilot study will enable us to identify targets for future functional studies testing the necessity of these identified proteins in spinogenesis. Future studies will couple these proximity labeling approaches and findings with in vivo two photon imaging of neural structure and second messenger systems in order to identify spatiotemporal events in therapeutic spinogenesis.

Major depression is highly comorbid with tobacco smoking, the leading cause of preventable death in the United States. Nicotinic acetylcholine receptors are a key subgroup of receptors involved in ACh signaling and are the primary target of nicotine, the addictive component of tobacco smoke. Importantly, increased ACh signaling is implicated in the etiology of depression and chronic nicotine use has been shown to desensitize nicotinic receptors, suggesting that cigarette used by people with depression is a form of self-medication. Increases in ACh are also important in learning, memory, and attentional processes, suggesting that optimal levels of ACh are beneficial while excessive increases are detrimental to affective health. In this scenario, excessively increased ACh during stressful events would lead to a negative encoding bias, in which stronger encoding occurs, leading to increased depressive symptoms and given the co-morbidity with smoking, increased tobacco use. To test this theory, I used the GRAB ACh4.3 sensor to record ACh transients in mice subjected to inescapable shocks as part of learned helplessness testing and found that mice classified as helpless in a later active avoidance test had increased ACh signaling in the medial prefrontal cortex relative to resilient mice. Furthermore, although the number of male and female mice classified as helpless was similar, the increased ACh signaling was more robust in male mice, a potentially important sex difference. Because synaptic dysfunctions in the mPFC are a hallmark of stress response and depression, I plan to use synaptosomal proteomics and phospho-proteomics to discover the differences between helpless and resilient mice responsible for alterations in ACh signaling. The findings from the study, along with previous work detailing the nicotinic ACh receptor-associated proteome will more clearly elucidate protein-dependent mechanisms contributing to the comorbidity of tobacco smoking and depression.

G protein-gated inwardly rectifying potassium (GIRK) channels are widely expressed in the brain and mediate GPCR-dependent slow inhibition. Malfunctioning and dysregulation of GIRK channels can result in various neurological and psychiatric disorders. Drugs of abuse can cause sustained alterations in GIRK function and trafficking in different brain regions, including the medial prefrontal cortex (mPFC), hippocampus and ventral tegmental area (VTA), important nodes in the brain reward pathway. In order to identify new therapeutic targets and develop more effective treatments for drug addiction, it is critical that we understand the molecular mechanisms behind such drug-induced plasticity. Our goal is to use the novel in vivo iBioID technique to define the GIRK channel interaction proteome (i.e., proteins in close proximity to GIRK channels in vivo) to discover novel regulators of GIRK channel function and trafficking. Furthermore, we will investigate how psychostimulant drugs such as methamphetamine alter the GIRK interaction proteome in a regional and cell type-specific manner. We expect to identify a few candidate proteins whose interaction with GIRK channels is modulated by repeated methamphetamine exposure, and we plan to investigate their functional significance with a new R01 grant in the future.

Roughly one third of Americans suffer from chronic pain, yet little progress has been made to develop new pain remedies beyond non-steroidal anti-inflammatory agents and opioids. While opioids can be effective analgesic agents, these drugs harbor poor side effects and the risk of addiction because opioid receptors are expressed in neurons and brain regions outside the pain producing somatosensory neural network. An attractive therapeutic approach to develop new analgesic and anti-inflammatory agents, which will not harbor these poor side effects, is to identify receptors predominantly expressed in peripheral nociceptor neurons that initiate pain signals. One such receptor is the wasabi receptor, TRPA1, which is activated directly by a diverse panel of environmental and endogenous chemical irritants, as well as indirectly by pro-inflammatory mediators downstream of several G-protein coupled receptors, to originate painful sensations. Pain is not a single entity, but instead has many causes, types, and symptoms, including mechanical pain, temperature hypersensitivity, and pain from chronic inflammation or some chemotherapeutic agents. Despite these diversities, TRPA1 plays a central role in each etiology to generate pain signals. Indeed, two identified gain-of-function human TRPA1 natural variants, characterized by pronounced pain phenotypes in patients, provide strong evidence for the critical role of TRPA1 in human pain physiology. Accordingly, TRPA1 has been deemed a bona fide therapeutic target for analgesic and anti-inflammatory agent development, however, all known TRPA1 antagonists have failed in clinical trials due to poor pharmacokinetics. Despite its importance to human pain, little is known about how TRPA1 is regulated. A detailed understanding of the diverse mechanisms by which TRPA1 activity is modulated could uncover novel avenues for therapeutic agent development that may fare better in clinical trials.

Plasma membrane localization of TRPA1 is increased after direct channel activation or indirect activation by pro-inflammatory mediators, which could contribute to neuronal sensitization and, if unchecked, to the development of chronic pain. Nonetheless, little is known about how TRPA1 trafficking is regulated. Here, we propose to use BioID2-fused TRPA1 constructs in combination with proteomics analysis to identify interacting and proximity protein partners that govern proper channel trafficking and functionality, as our preliminary data suggests these processes are decoupled in TRPA1. In our first aim, we intend to identify protein partners necessary to mediate TRPA1 plasma membrane trafficking by using a nonfunctional TRPA1 natural variant when it is expressed alone or co-expressed with wild type protein. We recently discovered this nonfunctional natural variant exhibits aberrant trafficking alone and confers gain-of-function by assembling with wild type TRPA1 subunits to form hyperactive channels that are functional at the plasma membrane, though their trafficking route is unclear. In our second aim, we want to identify interacting and proximity protein partners necessary to confer channel functionality using wild type, functional yet N-linked glycosylation incompetent, or nonfunctional yet properly trafficking TRPA1 variants. The machinery involved in both of these processes are predicted to interact with the channel on its cytoplasmic face, which could uncover novel druggable sites that are distinct from the transmembrane regions targeted by current TRPA1 antagonists.

Background: The prevalence of cannabis use is highest amongst adolescents and young adults, who are also more vulnerable to the development of Cannabis Use Disorder (CUD) and its neuropsychiatric and cognitive consequences. This is supported by preclinical data demonstrating neuronal/synaptic pathology and immunomodulatory changes with repeated cannabinoid exposure especially in adolescents. The underlying neuropathology in humans, however, remains unclear, in part due to difficulty in obtaining brain tissues from patients with CUD. Exosomes are vesicles, that are secreted from various cell types in the brain including neurons, and which can be isolated from blood. Importantly, exosomes cross the blood brain barrier with their contents intact, making them particularly attractive vehicles for potential signatures of neuropathology in the periphery. Proteomic analyses of exosome protein cargo may help identify patterns of altered protein signaling in the brain associated with recurrent cannabis exposure. Advances in Mass Spectrometry (MS) approaches present an unprecedented opportunity for profiling proteomes in body-fluids or within organelles of interest (e.g. exosomes). MS approaches have been used to evaluate the exosomal proteome of disease states but this promising approach has not been examined in CUD. We propose to examine the proteomic changes related to recurrent cannabis exposure in plasma neuron derived exosomes (NDE) using a label-free proteomics approach to identify broad proteomic signatures of altered signaling in CUD.

Specific Aim 1: To collect plasma samples from 20 adolescents and young adults with CUD and those without any exposure to cannabis (n = 10, 5 females in each group), from the ongoing studies. Extraction of NDE from the total pool of plasma exosomes (TPE) will be done under an MTA with NeuroDex (https://www.neurodex.co/) a company with proprietary technology, ExoSORT for capturing NDE.

Specific Aim 2: To examine the broad differential proteomic profile of TPE and NDE in CUD compared to matched HCs in a label-free, discovery proteomics approach using MS. To examine the enrichment of the differentially expressed proteins to cellular components, biological processes, and molecular functions with in silico bioinformatics approaches with a focus on brain specific proteins. To specifically examine the enrichment of proteins of relevance to synaptic function using SynGo database. To validate selected proteins with western blot.

Exploratory Aim: To examine the impact of acute exposure to cannabinoids (delta-9-tetrahydrocannabinol and cannabidiol) on plasma composition and size distributions of TPEs and NDEs.

Future directions: The data collected in this pilot project will be used to support larger grant applications to NIH for examining proteomic signatures in CUD. The results of this analysis will provide valuable preliminary confirmation regarding the feasibility of deriving CNS relevant signals from the periphery in developmental neuropsychiatric disorders. This would also allow us to explore the biophysical properties of TPE and NDE in further detail in future studies. Replication of the results of this study in larger studies will have numerous translational implications for research and clinical care of persons with substance use disorders.