Project Details

Addiction and obesity are national epidemics that are both caused by loss of homeostatic signal transduction mechanisms within the brain’s reward/aversion circuitry. New findings in our lab indicate that substance abuse and diet-induced metabolic disorders share maladaptations in neuronal intracellular signal transduction mechanisms that control mood and are affected by the body’s gastrointestinal, metabolic, and immune states. To understand these comorbidities, we are modeling them in mice and, in collaboration with the Yale/NIDA Neuroproteomics Center, have derived new proteomic and phosphoproteomic signaling libraries. We are using artificial intelligence-based computational analysis to integrate these data with single nuclear RNAseq, ATAC DNAseq, and cell type specific polyribosomal transcriptomics. In this way we are gaining a more comprehensive view of the interacting effects of substance abuse, obesity, and gastrointestinal inflammation on reward/aversion circuitry signaling and gene expression networks. This approach is beginning to reveal previously hidden treatment targets, and we are discovering new neurotropic and GI-specific drugs and repurposing FDA-approved medications that appear to reverse the negative mood that drives the behavior and habits that sustain these chronic comorbidities. These treatments are now being tested preclinically with our first prospective clinical testing now being planned. Our substance abuse disorder research has mainly focused on cocaine and psychomotor stimulant abuse. However, we are now extending our approach to understanding and treating fentanyl and oral opioid substance use disorders. With our Yale-NIDA collaborators, we aim to bring new treatments that disrupt drug abuse and diet-linked metabolic disorder comorbidities to improve the health of our citizens. A new R01 grant application to pursue this question is in preparation.

Overview: Aberrations in neuronal connectivity in addiction-relevant brain regions are correlated with drug seeking and relapse. Trans-synaptic adhesion molecules mediate synapse formation and differentiation, including in brain regions relevant for addiction, and we have previously reported that a mouse model lacking a synaptogenic adhesion protein exhibits altered addiction-relevant behaviors (Giza et al, 2013). This agrees with human genome-wide association studies that support that genetic changes in genes encoding synapse-organizing adhesion proteins can increase the risk for addictive behaviors (Muskiewicz et al., 2018).

These genetic studies need to be complemented by proteomic analysis of synapse-organizing pathways in addiction-relevant brain regions so that functional studies of these molecules can be developed. To aid in these efforts, Dr. Biederer has led during the past funding period proteomic studies of synaptic composition and oversaw a Pilot Project Grant, “Mapping the Proteome of the Synaptic Cleft through Reporter Proteins”. This approach uses a biotin-phenol compound that is turned over by a horse-radish peroxidase (HRP) reporter protein to generate extremely short-lived biotin-phenoxyl radicals and label subcellular proteomes. Labeling specificity arises from the targeting of a HRP fusion protein to subcellular compartments, and we applied this to excitatory synapses (Cijsouw et al, 2018). Our results contributed to the first analyses of the cleft proteome of different synapse types, including synapse-organizing complexes. This progress is now enabling us to determine under this renewal application the synaptic cleft changes that occur in trans-synaptic organizers after psychostimulant exposure.

We will pursue two approaches to attain this goal. First, we will analyze the remodeling of excitatory synapses in striatal slices that occurs after acute cocaine treatment (Javadi-Paydar et al. 2017). This acute effect of cocaine on excitatory synapses agrees with our in vivo finding that a single exposure to cocaine remodels stubby spines in the nucleus accumbens in a manner dependent on the synapse-organizing molecule SynCAM 1 (Giza et al., 2013). We will deliver the proximity labeling reporter HRP-TM (Trans Membrane) that is targeted to postsynaptic sites of excitatory synapses (Cijsouw et al, 2018) into the striatum of mice. We will target medium spiny neurons (MSN), the GABAergic interneurons that make up the vast majority of neurons in this brain region, using the GABA neuron-specific AAV-mDlx (Dimidschstein J et al., 2016) that we already utilize in our group. We will cut slices from the striatum of mice expressing the HRP reporter in MSNs and perform proximity labeling of excitatory inputs to these cells in slices using a permeable biotin-phenol compound. This is followed by affinity purification, on-bead digest, and MS/MS analysis (Cijsouw et al., 2018). We will perform these studies both in wild-type mice and in conditional SynCAM 2 KO mice available in our group. We have selected these KO mice due to genome-wide association studies that linked the gene encoding SynCAM 2, CADM2, to human risk taking and drug abuse (Day et al., 2016; Pasman et al., 2018; Sanchez-Roige et al. 2019). KO mice lacking SynCAM 2 in striatal MSNs will be generated by crossing cKO mice to the Dlx5/6-Cre line (The Jackson Laboratory, line Tg(dlx5a-cre)1Mekk/J). We will analyze how SynCAM 2 loss impacts cocaine-induced remodeling of synapse structure and the molecular makeup of the synaptic cleft, using synaptic labeling (Giza et al, 2013) in conjunction with the proximity labeling approach described above.

As a second approach, we will utilize an in vitro mixed culture system of embryo-derived cortical and MSN neurons that provides for substantially improved MSN differentiation and function compared to MSN monocultures (Penrod et al., 2011). HRP-TM will be expressed in GABAergic neurons, including MSNs that will make up the majority of interneurons in this culture system, using delivery by AAV-mDlx. Neurons will be treated acutely and chronically with increasing amounts of cocaine to establish an in vitro system for psychostimulant-induced synapse remodeling. Molecular changes in synapse organizing molecules of MSNs will be determined using the proximity labeling approach (Cijsouw et al., 2018) and will be correlated with studies of structural changes using immunocytochemistry, as established in our group (Perez de Arce et al., 2015).

Together, the proposed research will provide molecular insights into the synapse-organizing signaling pathways that underlie the changes in neuronal connectivity that are caused by drugs of abuse in the striatum. This can identify permissive and restrictive synapse organizers that serve as novel points of intervention.

References
Cijsouw T, Ramsey AM, Lam TT, Carbone BE, Blanpied TA, Biederer T (2018) Mapping the proteome of the synaptic cleft through proximity labeling reveals new cleft proteins. Proteomes 6, 48. doi:10.3390/proteomes6040048

Day FR, Helgason H, Chasman DI, Rose LM, Loh PR, Scott RA, Helgason A, Kong A, Masson G, Magnusson OT, Gudbjartsson D, Thorsteinsdottir U, Buring JE, Ridker PM, Sulem P, Stefansson K, Ong KK, Perry JRB (2016) Physical and neurobehavioral determinants of reproductive onset and success. Nat Genet 48:617-623.

Dimidschstein J et al. (2016) A viral strategy for targeting and manipulating interneurons across vertebrate species. Nat Neurosci 19:1743-1749.

Giza JI, Jung Y, Jeffrey RA, Neugebauer NM, Picciotto MR, Biederer T (2013) The synaptic adhesion molecule SynCAM 1 contributes to cocaine effects on synapse structure and psychostimulant behavior. Neuropsychopharmacology 38:628-638.

Javadi-Paydar M, Roscoe RF, Jr., Denton AR, Mactutus CF, Booze RM (2017) HIV-1 and cocaine disrupt dopamine reuptake and medium spiny neurons in female rat striatum. PLoS One 12:e0188404.

Muskiewicz DE, Uhl GR, Hall FS (2018) The role of cell adhesion molecule genes regulating neuroplasticity in addiction. Neural Plast 2018:9803764.

Pasman JA et al. (2018) GWAS of lifetime cannabis use reveals new risk loci, genetic overlap with psychiatric traits, and a causal influence of schizophrenia. Nat Neurosci 21:1161-1170.

Penrod RD, Kourrich S, Kearney E, Thomas MJ, Lanier LM (2011) An embryonic culture system for the investigation of striatal medium spiny neuron dendritic spine development and plasticity. J Neurosci Methods 200:1-13.

Perez de Arce K, Schrod N, Metzbower SW, Allgeyer E, Kong GK, Tang AH, Krupp AJ, Stein V, Liu X, Bewersdorf J, Blanpied TA, Lucic V, Biederer T (2015) Topographic mapping of the synaptic cleft into adhesive nanodomains. Neuron 88:1165-1172.

Sanchez-Roige S, Fontanillas P, Elson SL, Gray JC, de Wit H, MacKillop J, Palmer AA (2019) Genome-Wide Association Studies of Impulsive Personality Traits (BIS-11 and UPPS-P) and Drug Experimentation in up to 22,861 Adult Research Participants Identify Loci in the CACNA1I and CADM2 genes. J Neurosci 39:2562-2572.

Overview: The focus of this project is to examine substrates of synaptically localized chaperones and S-acylation machinery and how they impact synaptic structure and function. Spurred by our progress in the last granting period, we have begun examining a) how synaptic chaperones-auxilin and RME-8 impact dopaminergic synaptic function and b) how S-acylation dynamics influences glutamatergic and dopaminergic transmission. We propose to identify RME-8 substrates. We also propose to examine how synaptic protein S-acylation is affected by neuronal activity. To achieve this goal, we will carry out LFQ experiments in synaptic preparations from knockout mice for key components of the S-acylation and chaperone machinery under conditions when neuronal activity is either silenced or stimulated. This topic is directly relevant to the Center’s theme and that of NIDA.

Project background and rationale (related to R01 DA0327089, R01 P50 DA046373, R01 DA061899 (pending)): Epigenetic mechanisms engaged during stimulant or opioid misuse are mechanistically linked to the enduring and prepotent nature of drug-cue associations and relapse vulnerability that characterizes SUD. Specifically, the epigenetic enzyme, histone deacetylase 5 (HDAC5), is reduced in human postmortem NAc tissues of SUD patients, and our past work has shown that HDAC5 functions to limit the strength of cocaine- or heroin- (but not sucrose-) cue/context associations that form during active drug use, thereby reducing future drug seeking (Anderson et al, 2023, PNAS; Taniguchi et al, 2017, Neuron, Wood et al, 2025, Biol. Psychiatry). One HDAC5 target gene that mediates drug-cue memories is the activity-regulated gene and transcription factor, Neuronal Pas Domain protein 4 (Npas4) (Taniguchi et al, 2017, Neuron). Cue-induced drug seeking is gated in the Nucleus Accumbens (NAc) by competing activation of medium spiny neuron (MSN) ensembles that express either D1 dopamine receptors (D1-MSNs), which typically promote, or D2 dopamine receptors (D2-MSNs), which typically oppose, drug seeking. Drug experiences activate a small population of NPAS4-expressing neurons, and they are required for forming cocaine-context/cue associations. We found that NPAS4 is required in the NAc D2-, but not D1-, MSNs, for cocaine Conditioned Place Preference (CPP) and cue-reinstated cocaine seeking, where it functions to block drug-induced strengthening of prefrontal cortex inputs onto NAc D2-MSNs and to maintain the appropriate D1:D2-MSN activation balance (Hughes et al, 2023, Nature Commun.). In addition, we discovered a conserved, long non-coding enhancer RNA (lnc-eRNA) transcribed from an activity-sensitive Npas4 enhancer that forms RNA:DNA hybrid R-loop structures that support 3D chromatin-looping of the enhancer and proximal promoter and stimulus-induced, rapid Npas4 gene induction(Akiki et al, 2024, Science). Npas4 lnc-eRNA, and its associated R-loops, are required in NAc to form drug-cue memories, but the mechanisms by which R-loops function in this context remain unknown.

Core experiment A: Analysis of NAc R-loop complexes that mediate immediate-early gene induction. Following drug or saline conditioning, NAc tissues will be solubilized and processed to purify R-loop-associated complexes using immunoprecipitation and related approaches. LC-MS/MS approaches will be employed to identify constituent proteins.

Core experiment B: Analyze the NPAS4-regulated NAc D2-MSN proteome following cocaine SA. In this experiment, we will take an approach using D2-Cre rodents (male and female) and AAV2-SICO-shNPAS4 or -shControl. Animals will be allowed to acquire cocaine, sucrose, or saline self-administration, followed by home-cage abstinence, and extinction training. We will isolate NAc tissues at 1 day after final extinction training (state of NAc D2-MSNs just prior to the cue test) and perform single-cell isolation using fluorescence-activated cell sorting and gating and collecting mCherry+/GFP- cells and then using LC-MS/MS analysis of differential protein expressions. These data will complement parallel snRNA-seq data to identify candidate target genes that show both protein and mRNA changes.

1. Anderson EM, Tsvetkov E, Galante A, DeVries D, McCue LM, Wood D, Barry S, Berto S, Lavin A, Taniguchi M, Cowan CW. Epigenetic function during heroin self-administration controls future relapse-associated behavior in a cell type-specific manner. Proc Natl Acad Sci U S A. 2023;120(7):e2210953120. Epub 20230206. doi: 10.1073/pnas.2210953120. PubMed PMID: 36745812; PMCID: PMC9963300.
2. Taniguchi M, Carreira MB, Cooper YA, Bobadilla AC, Heinsbroek JA, Koike N, Larson EB, Balmuth EA, Hughes BW, Penrod RD, Kumar J, Smith LN, Guzman D, Takahashi JS, Kim TK, Kalivas PW, Self DW, Lin Y, Cowan CW. HDAC5 and Its Target Gene, Npas4, Function in the Nucleus Accumbens to Regulate Cocaine-Conditioned Behaviors. Neuron. 2017;96(1):130-44.e6. doi: 10.1016/j.neuron.2017.09.015. PubMed PMID: 28957664; PMCID: PMC5761688.
3. Wood DJ, Tsvetkov E, Comte-Walters S, Welsh CL, Bloyd M, Wood TG, Akiki RM, Anderson EM, Penrod RD, Madan LK, Ball LE, Taniguchi M, Cowan CW. Epigenetic control of an auxiliary subunit of voltage-gated sodium channels regulates the strength of drug-cue associations and relapse-like cocaine seeking. Biol Psychiatry. 2025. Epub 20250207. doi: 10.1016/j.biopsych.2025.01.027. PubMed PMID: 39923817. 4. Akiki RM, Cornbrooks RG, Magami K, Greige A, Snyder KK, Wood DJ, Herrington MC, Mace P, Blidy K, Koike N, Berto S, Cowan CW, Taniguchi M. A long noncoding eRNA forms R-loops to shape emotional experience-induced behavioral adaptation. Science. 2024;386(6727):1282-9. Epub 20241212. doi: 10.1126/science.adp1562. PubMed PMID: 39666799.

Overview: The goal of my laboratory is to elucidate mechanisms that control the dynamics of cell membranes, with special emphasis on their role in neuronal function. My studies on membrane recycling at synapses highlighted the importance of the chemistry of bilayer lipids - phosphoinositides in particular - in the control of membrane traffic. These studies were the starting point for my long-term focus on phosphoinositides in neurons and non-neuronal cells, an area of research that we continue to pursue. These studies, in turn, made us interested in the mechanisms that control membrane lipid homeostasis. Work by our and other labs has shown that vesicle-independent lipid transport at contacts between organelles is far more important in this control than previously appreciated. The endoplasmic reticulum (ER) is the site where most membrane lipids are synthesized. Traffic of lipids from the ER to other membranes, and return of their hydrophobic catabolic products to the ER for metabolic recycling, occurs in part via vesicular carriers along the secretory and endocytic pathways. However, this traffic occurs in parallel with non-vesicular mechanisms mediated by Lipid Transport Proteins (LTPs) that extract lipids from bilayers and deliver them to other membranes while harboring them in hydrophobic cavities. Many of these proteins also function as membrane tethers, holding the ER in proximity of another membrane as they transport lipids. Transfer of lipid via LTPs at membrane contact sites operates in all neuronal compartments, as the ER reaches even the most distal branches of axons and dendrites.

Until a few years ago, we had worked primarily on LTPs that act via a shuttle-like mechanism at membrane contact sites. These proteins typically comprise modules that can accommodate only one or few lipids in their hydrophobic cavities. These modules are connected by linker regions to domains or motifs that tether the two membranes and are thought to shuttle back and forth between them. More recently, we helped identify LTPs that function by a bridge-like mechanism at membrane contacts sites and we have embarked in a systematic functional characterization of some of these proteins. These LTPs, collectively called bridge-like lipid transport proteins (BLTPs), have a rod-like structure and harbor a hydrophobic groove that run along their length in which phospholipids are thought slide from one membrane to another. The founding members of this protein family are the four VPS13 proteins which are of special interest in neuroscience, as mutations in each of them lead to neurodevelopmental or neurodegenerative conditions, including a Huntington-like disease (neuroacanthocytosis, mutations in VPS13A) and Parkinson’s disease (mutations in VPS13C). The special impact of these mutations on brain regions involved in the actions of drugs of addiction (the striatum and dopaminergic neurons) make this topic particularly relevant to the scope of NIDA. Further elucidating the properties, mode of action and function of these proteins are main priorities of our lab for the future.

In addition to studies of lipid dynamics, we are also continuing studies of membrane traffic at synapses with emphasis on two topics: 1) mechanisms in synaptic vesicle biogenesis and role of liquid-liquid phase separation in their clustering at synapses; 2) mechanisms through which mutations of synaptojanin1, a phosphoinositide phosphatase that function in synaptic vesicle recycling, cause dystrophic changes selectively in nerve terminals of dopaminergic neurons in the striatum.

This project seeks to define the actions of nicotine and THC on extracellular vesicle signaling in the brain and periphery. We previously found that nicotine and THC induce the release of extracellular vesicles from cellular subpopulations in the brain and that these vesicles contain various proteins and RNA transcripts. Since these signaling factors are transferred between cells in the brain, extracellular vesicle cargo is thus hypothesized to alter protein expression in target brain regions. Through our collaboration with the Yale/NIDA Neuroproteomics Center, we have determined the specific proteomic changes occurring in the medial habenula during nicotine intake. Given that the habenula has been shown to mediate an aversion signal that controls drug reinforcement, altered habenular neuronal function due to protein modulation may underlie drug intake and/or serve as a novel target for therapeutic development. Thus, we are now conducting follow up studies to further determine the functional significance of these drug-mediated extracellular vesicle signaling changes in vivo. The second aim of our studies has been to determine the specific proteomic changes occurring in circulating extracellular vesicles in the CSF and blood following nicotine and/or THC consumption. In the first completed set of studies, we established differential expression of circulating EV-localized proteins in the CSF based on acute or chronic THC vape exposure in both males and females. Our findings revealed that THC acts in the brain to modulate circulating EV signaling in a sex-specific manner, which has provided a novel understanding for how exogenous factors can regulate intercellular communication in the brain. Our ongoing studies are focused on correlating the findings between the CSF and blood to specifically identify brain-specific EVs that have infiltrated into the blood, which may be detected in the periphery, which will advance our understanding of extracellular signaling mechanisms that could be used as biomarkers underlying the development of an addiction phenotype. In sum, this project has high relevance to the Center’s theme, “Proteomics of Altered Signaling in Addiction”, as it seeks to analyze neuronal signal transduction mechanisms and the adaptive changes in these processes that occur in response to nicotine and THC.

Project Description: Recent evidence has found that teens who use cannabis recreationally are two to four times as likely to develop psychiatric disorders, such as depression and suicidal behavior, than teens who do not use cannabis at all. This is a startling statistic, given these studies were on teens who did not meet the criteria for a substance use disorder. It is believed that cannabis use alters the development of key regions of the brain including the cerebral cortex and ventral striatum and primes the brain for the development of additional psychiatric disorders later in life. These findings are particularly concerning given the popularity of cannabis legalization (both recreationally and for medical use) across the United States. One of the major challenges in the addiction field is determining whether drug use causes additional psychiatric disorders (such as depression or suicide).

Here we propose to address this problem by testing the functional impact of cannabis use by perturbing the genotype of Major Depressive Disorder (MDD) risk alleles. We hypothesize that genetic risk and psychoactive drug use contribute to development of MDD by converging on shared downstream epigenetic, transcriptomic, and proteomic changes associated with specific genes and functional pathways in neuronal and non-neuronal cells, which in turn drive the symptoms associated with MDD. To this end, we will employ two complementary approaches: in Part 1, we will use human postmortem brain tissue of cases with cannabis use disorder, major depression (MDD), and neurotypical controls (CON) to identify cell type-specific genomic and proteomic changes for these disorders. We will assess chromatin assemblies of the CUD brain to determine the extent of change at risk loci for MDD. In Part 2, we will use human induced pluripotent stem cell (hiPSC)-derived neurons from donors with a history of CUD but not major depression to identify the function of the CUD-specific epigenetic changes and measure the effects these changes have on regulation of protein expression.

Overview: We are performing experiments to characterize the molecular changes that occur during the process of nicotine upregulation that is tied to nicotine addiction. We recently found that nicotine alters Golgi apparatus morphology causing increases in Golgi satellites, mobile vesicles that derive from the Golgi. In one project, we will use proteomic assays to identity proteins found in purified preparations of the Golgi vesicles using Proteomic Proximity Labeling methods to identify vesicles proteins and their interactions. The nicotine-induced changes in Golgi morphology also causes changes in complex trimming of α4ß2-type nicotinic acetylcholine receptor N-linked glycans, particularly the addition of sialic acid. In the second project, LC MS/MS experiments in collaboration with the Discovery Core are underway to analyze the specific changes in N-linked glycosylation with nicotine exposure. We will be utilizing the Orbitrap Fusion along with the Byonics Software for data collection and site determination, respectively.

Although many people will use a drug of abuse at least once in their lifetime, only a subset of these individuals will develop an addiction. This suggests that some individuals may be more susceptible to developing an addiction compared to others and, importantly, if we can identify the neurobiological mechanisms that mediate this susceptibility, then we may be able to prevent addiction. My laboratory is focused on identifying the protein mechanisms that mediate susceptibility to drug use in rodent models. Our ongoing work – in collaboration with the Yale/NIDA Neuroproteomics Center – uses a reinforcement-learning platform to isolate protein targets that are disrupted by chronic exposure to drugs of abuse from those that influence early-stage drug use. Our goal is to generate mechanistic bridges between proteins and complex behavioral phenotypes. Through a funded NIDA K01 award (DA051598) we are 1) using novel viral approaches to investigate the role of proteins we hypothesize to mediate drug use susceptibility, 2) determining how these proteins and others are altered in a genetic rat model of addiction susceptibility, and 3) identifying the protein mechanisms that are altered in an environmental rat model of addiction susceptibility. This work, collectively, will identify novel biomarkers of addiction susceptibility that can be used to identify at risk humans. A second interest of the laboratory is understanding the neurodevelopmental mechanisms that influence addiction susceptibility. Through pilot funding provided by the Yale/NIDA Neuroproteomics Center we have recently completed a study investigating the protein mechanisms underlying age-related changes in decision-making. Our preliminary data suggests that decision-making improves during adolescence in the rat and that this improvement is associated with changes in the expression of perineuronal net proteins. We hypothesize, therefore, that alterations in the expression of perineuronal net proteins during adolescence could impact susceptibility to drug use in adulthood by altering the formation of neural circuits, and our ongoing work is testing these hypotheses. We plan to integrate our collaborative work with the Yale/NIDA Neuroproteomics Center with magnetic resonance measurements at the University of Chicago in order to identify a non-invasive neuroimaging measure that could be used to assess addiction susceptibility in humans.

While developmental marijuana exposure has been shown to result in life-long vulnerability to reward, motivation and cognitive impairments in the offspring, the cellular and molecular mechanisms that mediate impairments of neuronal development and synaptogenesis remain largely elusive. Recently, epigenetic regulatory mechanisms of gene expression emerged as prime biological candidates to establish and maintain persistent aberrant neuronal processing as a result of developmental drug exposure. Since these mechanisms are highly dynamic and readily influenced by environmental agents including drugs of abuse, the developing brain might be particularly sensitive to epigenetic influences, given the dynamic neuroplasticity characteristic of this period. We have observed that in utero or adolescent exposure to THC leads to epigenetic and transcriptional changes of genes linked to dopamine and glutamate receptor disturbances in the nucleus accumbens that appear to have relevance to specific striatal pathways. Moreover, these patterns persist into adulthood. In this project (DA030359), we will study THC-related changes in epigenetic regulatory mechanisms at affected genes that might underlie the persistent alterations in gene expression. Our preliminary results indicated that post-translational modifications of histone H3 (i.e. H3K4me3) and specific histone modifying enzymes (Kmt2a) are affected by developmental THC exposure. However, histone modifications are known to influence each other and to act in combination. Moreover, several different enzyme complexes might be involved in establishing disease-specific anomalies of these marks. Using a proteomic approach, our goal is to explore the combinatorial histone modification landscape at specific loci, as well as the associated protein complexes that mediate these modifications.

A second important use of the proteomic approach in our research program relates to studying placental specimens from women who used cannabis while pregnant since we have observed significant alterations of the transcriptome (assessed with RNA-sequencing) in which there was a marked alteration of the immune gene expression signature. It is important to determine whether these gene expression changes relate to functional proteins. As such we will use proteomic approaches to determine the protein profile within the human cannabis-exposed placenta as well as placenta from our rat prenatal THC model. This project is a focus of our competitive renewal grant application (DA030359).

Technologies and consultations available from the Yale/NIDA Neuroproteomics Center’s Discovery and Targeted Proteomics Cores will significantly advance our efforts to characterize the complex landscape of post-translational histone modifications regulating exposure to drugs of abuse, as well as our work to explore protein complexes regulating these modifications. In addition, we will work in close collaboration with the Bioinformatics and Biostatistics Core of the Center.

Our laboratory has previously focused on gene expression, thus being part of the Yale/NIDA Neuroprotemics Center will be a unique opportunity for my postdoctoral fellows as well as PhD and MD/PhD students to advance their knowledge of proteomic approaches with respect to neurobiological aspects of addiction. I plan to have a number of my trainees not only learn proteomics, but participate actively in data analysis, which will be critical for their future development. As such my post-doctoral fellows, such as Teesta Naskar PhD, who has already begun to learn about proteomics will receive MS/proteomics training at the Center and will attend the Center’s Research in Progress Meetings.

Overview: Our research is focused on potassium channels, Kv3.4 and Slack (KNa1.1), which emerging evidence suggests play a key role in opiate abuse. In particular, upregulation of Slack channels has been found to be specifically associated with increased drug seeking during extinction in females. Both channels are very highly expressed in the amygdala and the striatum, and in dorsal root ganglia (DRG) and the ventral spinal cord. Reduction of the levels of these channels, either by genetic manipulations or in response to injury, results in enhanced pain sensation. We are testing the hypothesis that changes in these channels, and specifically in the cytoplasmic proteins with which these channel subunits interact, lead to abnormal signaling that is activated with opiate treatment. The MAPK10 (MAP Kinase 10) and the TBK1 (Tank Binding Kinase1)/ ubiquitination/autophagy pathways are both abnormally activated with opioid treatment. We have found that MAPK10 binds directly to the cytoplasmic C-terminal domain of Kv3.4. This channel subunit also forms heteromers with the Kv3.3 subunit. We plan to determine if MAPK10 phosphorylates the channels and map the phosphorylation sites. Most importantly, we plan to determine whether gating of these channels directly alters the activity of this enzyme, a finding that would shed a light on the overactivation of MAPK10 during addiction. Parallel experiments will be carried out with TBK1 and with Protocadherin 9 (PCDH9), a Kv3.4 interacting molecule that, when suppressed, causes long-term social and object recognition deficits. We also plan to further our studies on the interactions of KNa1.1 with two regulators of mRNA translation, CYFIP1 and FMRP. These have been supported by the Yale/NIDA Neuroproteomics Center. This information will be used to test how these interactions are altered by drugs of abuse.

Overview: In recent years there has been growing evidence that neuroimmune and gut-brain interactions have marked effects on brain and behavior, including in models of substance use disorders. While these effects are robust, the underlying molecular pathways are not well characterized. By utilizing the cutting-edge resources available through the Discovery Proteomics Core, we will clarify the molecular mechanism underlying these effects on neuronal and behavioral plasticity in translationally relevant models of substance use disorders.

Our current studies on neuroimmune mechanisms in substance use disorders have focused on cytokine signaling in the brain and found that cytokines interact with cocaine treatment to alter the expression of important synaptic proteins in limbic substructures. Going forward, we will continue to utilize the Discovery Proteomics Core to identify how cytokine signaling alters expression of glutamate and dopamine receptors in isolated synaptic subfractions. Also, we are working with the Core to perform cell type-specific proteomics in key cell populations using non-canonical amino acid tagging and click chemistry. Studies on the gut brain axis have shown that depletion of the host microbiome results in significant dysregulation of the transcriptional response to either opioids or psychostimulants. Working with the Core we will perform discovery proteomics analyses to determine how these transcriptional changes are reflected in protein expression in important limbic structures. This will be coupled with cell sorting of genetically defined cell populations to determine how microbiome shifts are affecting protein expression in distinct populations of neurons and glia. Additionally, given the significant dysregulation of transcription, we will also be using co-immunoprecipitation of nuclear protein complexes to quantitatively determine how alterations in the microbiome affect transcriptional machinery in the brain.

Overview: Many drugs of abuse, such as opioids, act by directly binding to G protein coupled receptors or by indirectly affecting neurotransmitter signaling through such receptors. These receptors activate heterotrimeric G proteins in the brain, and understanding addiction thus requires understanding the mechanism of G protein signaling in neurons. The most abundant heterotrimeric G protein in the brain is the one containing the alpha subunit Go, and this is also the specific G protein activated by important receptors involved in addiction, such as the mu opioid receptor. Remarkably, the mechanism by which Go signals remains poorly defined. Receptors activate Go by causing it to bind GTP and release the G protein beta-gamma subunit complex, and the released beta-gamma complex in turn can then regulate certain ion channels. However, it remains unresolved whether Go-GTP itself also binds to target "effectors" to cause additional signaling effects, analogous to how other G protein alpha subunits are known to work. Genetic studies in C. elegans suggest that Go must indeed signal via regulating effectors, not simply via release of the beta-gamma complex. We are using proteomic analysis to identify neural proteins that bind specifically to the activated form of Go and thus are candidates to serve as the long-sought effectors of Go signaling. We have identified two promising effectors and are investigating the biochemistry of their interactions with Go.

Overview: Proximity labeling is an innovative technique that generates localized reactive intermediates to “tag” nearby proteins in their natural cellular environment, allowing for detailed proteomic analysis. This method has greatly enhanced our understanding of subcellular proteomes, shedding light on the functional and compositional differences in neuronal synaptic clefts, such as those involved in glutamatergic and GABAergic signaling, and revealing new protein-protein interactions within postsynaptic densities. In my lab, we employ both chemical and genetic tools to explore molecular interactions between the peripheral nervous system and other organ systems, focusing on how these interactions change under both normal and pathological conditions. We are also interested in how addictive substances that affect the central nervous system (CNS) can influence the peripheral nervous system and the physiological regulation of organs it innervates. Hence, a major focus of our research is using and improving proximity labeling techniques to map these interactions in living organisms, particularly in mouse models.

While proximity labeling has proven effective in cell culture systems, its application in mice has been limited due to high background interference from biotinylated proteins naturally present in tissues. To address this challenge, we are developing new chemical probes and enzymes specifically designed for in vivo applications. Our long-term goal is to enable studies of neuronal signaling within living mice. This includes creating new chemical probes for existing proximity labeling enzymes like TurboID and engineering novel enzymes with improved pharmacokinetics, bioavailability, and safety profiles. Additionally, we have developed innovative chemical substrates for these enzymes that do not rely on biotin-based enrichment, making them compatible with proximity labeling in both mammalian cell cultures and mouse models. Successful application in both systems will leverage the Yale/NIDA Neuroproteomics Center’s proteomics expertise to apply these new tools for proteomics, and to understand the sites of proximity labeling and improve the proteomic coverage in the MS instrument.

This approach aims to increase the power of proximity labeling, allowing us to study physiological processes, in living organisms that are difficult to replicate in vitro. If successful, these new tools will provide groundbreaking insights into the nervous system and enable the study of physiological and disease processes in vivo. We are also eager to collaborate widely through the center to advance and implement this cutting-edge technology, making it a core tool for future discovery. These objectives align with the Yale/NIDA Neuroproteomics Center’s mission to develop and apply innovative proteomic technologies for studying neuronal signal transduction mechanisms. In addition to support from the Yale/NIDA Neuroproteomics Center, this work is also supported by an ORAU Ralph E. Powe Junior Faculty Enhancement Award.

The major goal of our NIDA-funded project (R01 DA053746) entitled “Mechanisms and treatment of adolescent phytocannabinoid impairment of prefrontal cortex function” is to better understand the consequences and mechanisms of cannabis use during adolescence and early adulthood. We model this process in rodents by adolescent administration of ?-9-tetrahydrocannabinol (THC), the primary intoxicating component of cannabis.

Our behavioral studies find enduring deficits in the medial prefrontal cortex (mPFC)- mediated behaviors following adolescent, but not adult, THC exposure. These findings emphasize a specific window of vulnerability of the mPFC to chronic THC exposure.

Our aims include the following: (1) Test the hypothesis that CB1 receptors are required for the detrimental effects of adolescent THC on working memory and evaluate potential therapies to reverse these deficits; (2) Test the hypothesis that adolescent THC exposure reduces the connectivity of the mediodorsal thalamus to mPFC to impair working memory; (3) Test the hypothesis that adolescent THC activates microglia to excessively prune mPFC inputs from the MD thalamus to impair working memory. Recently, our studies using whole-cell electrophysiological recordings with cortical layer V pyramidal neurons located in the prelimbic mPFC region identified significant synaptic functional alterations following adolescent THC exposure. To elucidate the mechanism mediating lasting synaptic and behavioral changes caused by adolescent THC exposure, we would like to conduct unbiased proteomic and post-translational modifications (PTMs) analyses with the support of the Yale/NIDA Neuroproteomics Center. Specifically, we plan to submit two groups of brain tissue punchouts from adult mPFC harvested from both male and female mice with adolescent THC exposure versus mice with vehicle exposure from postnatal day 28-49. The quantitative proteomic data will allow us to generate testable hypotheses on specific signaling cascades. This knowledge will enable us to identify putative pharmacological therapeutic tools and to test their efficacy in reversing the working memory deficits caused by adolescent THC exposure.

We believe our study matches the Yale/NIDA Neuroproteomics Center’s goal of using cutting-edge proteomic technologies to analyze neuronal signal transduction mechanisms and the adaptive changes in these processes that occur in response to drugs of abuse.

Overview: Cannabis is frequently used before, during, and after pregnancy, and during adolescence. Similarly, cannabidiol (CBD) has a large following as a “natural” alternative with promise to treat a number of pregnancy-related maladies. We are interested in how chronic use of THC (the major active component of cannabis) and/or CBD affect the developing brain during bothpernataland adolescent periods.

Nairn laboratory projects carried out with support of the Yale/NIDA Neuroproteomics Center are briefly described below. Some of the recent work supported by the Discovery Proteomics, Targeted Proteomics, and Bioinformatics and Biostatistics Cores is briefly described.

1) ARPP-16 is a striatal-enriched inhibitor of protein phosphatase 2A regulated by MAST3 kinase (Musante et al., 2017). We continue to work on the function of ARPP-16, a small acid-soluble protein highly expressed in medium spiny neurons (MSNs) of striatum. This work continues to be carried out in collaboration with Center Investigator Jane Taylor. Ongoing work, supported by the Discovery and Targeted Proteomics Cores, is aimed at using the BioID proximity biotinylation approach to assess the PP2A interactome and the signaling processes it regulates in striatal MSNs.

2) Striatin-1 is a B subunit of protein phosphatase PP2A that we have found regulates dendritic arborization and spine development in striatal neurons (see Li et al., 2018). As a member of the striatin family of B subunits, striatin-1 is a core component together with PP2A of a multi-protein complex called STRIPAK, the striatin-interacting phosphatase and kinase complex. With the support of the Discovery Core, we used LC-MS/MS to identify proteins from striatum and striatal neurons in culture that include PP2A and the STRIPAK complex. We followed up from these studies by investigating the role of striatin-1 in neuronal maturation. Reduced expression of striatin-1 resulted in increased dendritic complexity and an increased number of dendritic spines, classified as stubby spines. Reduction of striatin-1 did not result in deficits in neuronal connectivity, as we observed no abnormalities in synapse formation or in spontaneous excitatory postsynaptic currents, with the latter work being done in collaboration with Center Investigator, Dr. Marina Picciotto. Together these results suggest that striatin-1 is a regulator of medium spiny neuron development in the striatum.

3) Cell-type specific psychostimulant effects on the neuronal translatome (R21 DA040454; Carlyle et al., 2018). We were funded by NIDA for an R21 grant that proposed to develop biochemical methods for the purification and quantitative profiling of ribosome-affiliated RNA footprints and nascent polypeptides from specific neuronal cell types, combined with integrated methods of data analysis, to characterize all stages of control over the translatome. By integrating mRNA-seq and its ability to reliably quantify isoforms, with ribosome profiling and LC-MS/MS, a more complete understanding of gene regulation at the isoform level can be obtained. As part of the data analysis related to this study, we have been supported by the Bioinformatics Core who have developed an approach termed EMPire to integrate mRNA, ribosomal footprint and proteomic data. Specifically, an expectation maximization algorithm was designed to relate mRNA transcript abundance to protein isoforms from LC-MS/MS, and this was extended to allow analysis of ribosomal footprinting results (see Carlyle et al., 2018). In this approach, we leveraged the principle that most cell types, and even tissues, predominantly express a single principal isoform to set isoform-level mRNA-seq quantifications as priors to guide and improve allocation of footprints or peptides to isoforms. Through tightly integrated mRNAseq, ribosome footprinting and/or LC-MS/MS proteomics we have found that a principal isoform can be identified in over 80% of gene products in homogenous HEK293 cell culture and over 70% of proteins detected in complex human brain tissue. Defining isoforms in experiments with matched RNA-seq and translatomic/proteomic data increases the functional relevance of such datasets and will further broaden our understanding of multi-level control of gene expression.

4) A targeted mass spectrometry-based approach for quantitation of proteins enriched in the postsynaptic density (PSD) (Wilson et al., 2019). The PSD is a structural, electron-dense region of excitatory glutamatergic synapses, which is involved in a variety of cellular and signaling processes in neurons. The PSD is comprised of a large network of proteins, many of which have been implicated in a wide variety of neuropsychiatric disorders. Biochemical fractionation combined with mass spectrometry analyses have enabled an in-depth understanding of the protein composition of the PSD. However, the PSD composition may change rapidly in response to stimuli; and robust and reproducible methods to thoroughly quantify changes in protein abundance are warranted. With the support of the Discovery and Targeted Proteomics Cores, we developed two types of targeted mass spectrometry-based assays for quantitation of PSD-enriched proteins. In total, we quantified 50 PSD proteins in a targeted, parallel reaction monitoring (PRM) assay using heavy-labeled, synthetic internal peptide standards; and were able to identify and quantify over 2,100 proteins through a pre-determined spectral library using a data independent acquisition (DIA) approach in PSD fractions isolated from mouse cortical brain tissue. This work was recently published (Wilson et al., 2019).

5) Collaboration with other Center Investigators (Bertholomey et al., 2018; Miller et al., 2018; Torregrossa et al., 2019). We continue to collaborate on a number of projects with the Taylor, Picciotto and Torregrossa labs.

Literature Cited

Bertholomey, M.L., Stone, K.L., Lam, T.T., Bang, S., Wu, W., Nairn, A.C., Taylor, J.R., Torregrossa, M.M. (2018) Phosphoproteomic analysis of the amygdala response to adolescent glucocorticoid exposure reveals G-protein coupled receptor kinase 2 (GRK2) as a target for reducing motivation for alcohol. Proteomes Special Issue on Neuroproteomics 6(4), 41 (PMCID: PMC6313880, PMID:30322021)

Carlyle, B.C., Kitchen, R.R., Zhang, J., Wilson, R., Lam, T.T., Rozowsky, J.S., Williams, K., Sestan, N., Gerstein, M.B., Nairn, A.C. (2018) Isoform level interpretation of high-throughput proteomic data enabled by deep integration with RNA-seq. J. Proteome Research 17(10), 3431-3444 (PMCID:PMC6392456, PMID: 30125121).

Li, D., Musante, V., Zhou, W., Picciotto, M.R., Nairn, A.C. (2018) Striatin-1 is a B subunit of protein phosphatase PP2A that regulates dendritic arborization and spine development in striatal neurons. J.Biol.Chem. 293(28):11179-11194 (PMCID: PMC6052221, PMID: 29802198).

Miller, M.B., Wilson, R.S., Lam, T.T., Nairn, A.C., Picciotto, M.R., (2018) Evaluation of the phosphoproteome of mouse alpha 4/beta 2-containing nicotinic acetylcholine receptors in vitro and in vivo. Proteomes Special Issue on Neuroproteomics 6(4), 42 (PMCID: PMC6313896, PMID: 30326594).

Musante, V., Li L., Kanyo, J., Lam, T.T., Colangelo, C.M., Cheng, S.K., Brody, H., Greengard, P., Le Novère N., Nairn, A.C. (2017) Reciprocal regulation of ARPP-16 by PKA and MAST3 kinases provides a cAMP-regulated switch in protein phosphatase 2A inhibition. eLife 2017 June 14;6. pii: e24998 (PMCID:PMC5515580, PMID:28613156).

Torregrossa, M., MacDonald, M., Stone, K.L., Lam, T.T., Nairn, A.C., Taylor, J.R. (2019) Phosphoproteomic analysis of cocaine memory extinction and reconsolidation in the nucleus accumbens, Psychopharmacology Epub ahead of print on Nov. 8, 2018 (PMCID:PMC6374162, PMID: 30411139).

Wilson, R. Rauniyar, N., Sakaue, F., Lam, T., Williams, K., Nairn, A. (2019) Development of targeted mass spectrometry-based approaches for quantitation of proteins enriched in the postsynaptic density (PSD), Proteomes Special Issue on Neuroproteomics, 7(2) (PMCID:PMC6630806, PMID:30986977).

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 ?FosB 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.

Overview: Following up on our studies of identification of the high affinity nicotinic acetylcholine receptor (2*-nAChR)-associated proteome and sex differences in the proteome in the mouse ventral tegmental area (VTA), nucleus accumbens (NAc), we are now focusing on the effects of the endogenous neurotransmitter of nAChRs, acetylcholine (nAChR) in stress-induced behaviors. Stress is one of the primary stimuli that induced relapse to drug taking following abstinence, as well as a risk factor for anxiety and depressive-disorders that are strong risk factors for addiction. We have used isobaric labeling with a TMT 10-plex kit (ThermoFisher), then high pH reverse phase peptide fractionation (5 fractions), followed by whole proteome analysis by LC-MS/MS to obtain highly reliable protein identifications that allow sex differences and stress-dependent differences in the proteomes of medial prefrontal cortex (mPFC) synaptosomes to be identified with high reliability and sensitivity. Our proposal going forward will be to establish the baseline and stress-dependent proteome in male and female mice to identify baseline sex differences that differ depending on whether stressors are controllable or uncontrollable.

Our preliminary data show that pathways associated with protein trafficking are regulated in male and female mPFC, and also identify baseline sex differences in mPFC synaptosome proteomes. Interestingly, ‘ACh signaling’ is a significantly regulated pathway across a number of stressful conditions. A number of different pathways are emerging as different between male and female mPFC, including one of the ACh receptor subtypes. These analyses will be carried forward in the next funding period, along with evaluation of the phosphoproteome.

Overview: We are interested in how the anatomical stability versus plasticity of brain wiring is accomplished, and how it is altered by disease. In addiction the rewiring of synaptic connections driven by biochemical changes underlies altered behavior. We study the mechanistic relationship of neuroproteomic patterns to forebrain synaptic turnover and rearrangement during the loss of synapses driven by trauma in spinal cord injury and traumatic brain injury, as well as by misfolded protein aggregation in Alzheimer's Disease and Fronto-Temporal Lobar Degeneration (R35NS097283; U01AG058608; R01AG034924; RF1AG053000; P50AG047270). Our goal is to define the molecular interactions driving synapse loss and the signal transduction mechanisms that link Aßo accumulation to synaptic degeneration. By understanding the basic protein chemistry of synapse stability control in the adult brain across this spectrum of conditions, the basis for disruption in addiction will be clarified.

Our previous investigation of the neurotoxic signaling of amyloid beta oligomers (Aßo) has uncovered critical roles for PrP-C (cellular prion protein), mGluR5 (metabotropic glutamate receptor 5), Fyn (tyrosine-protein kinase Fyn), and Pyk2 (protein-tyrosine kinase 2-beta). Both genetic and pharmacological inhibition of PrP-C, mGluR5, Fyn, and Pyk2 have been demonstrated to rescue synapse loss as well as memory and learning deficits in transgenic mouse models of Alzheimer’s disease. Further, the Fyn inhibitor AZD0530 has been demonstrated safe in Alzheimer’s disease patients and a Phase 2a study is underway (NCT02167256). Previously we have shown that there are early changes in synaptic gene expression in the APPswe/PS1dE9 and are linked to inflammatory changes. To extend our understanding of the neurotoxic signaling induced by Aßo, as well as identify signaling molecules downstream of Fyn and Pyk2, we conducted a proteomic label-free mass spectrometry study of 8 groups of mice with various genetic or pharmacological manipulations: in one (WT vs APPswe/PS1dE9)x(WT vs Pyk2) and in another (WT vs APPswe/PS1dE9)x(Veh vs AZD0530).

We are studying the entire proteome with a focus on synaptic proteins, as well as samples enriched for phospho-proteins. Recent data have revealed increased JNK3 levels and phospho-JNK3 in APPswe/PS1?E9 mice. In WT mice, loss of Pyk2 does not alter these levels, but the elevation in AD mice is suppressed to WT levels by deletion of Pyk2 (Cox et al, 2019). These unbiased data fit with previous evidence for JNK activation in AD mouse models and with Pyk2 activation of JNK in non-neuronal cells. We have now utilized these data to guide histological and immunoblot studies in APPswe/PS1dE9 mice. We find that p-Jun levels are increased in the hippocampus and cerebral cortex of these mice. Because JNK activation has been linked to neuronal apoptosis, this expands the role of the Aß/PrP/mGluR5/Fyn/Pyk2 signaling pathway from synaptic dysfunction to cell loss.

Due to the essential but ill-defined role of Pyk2 in AD signal transduction we extracted Pyk2 interacting proteins from brain by affinity chromatography. Isolated proteins were analyzed by mass spectrometry, and Graf1 a rhoA regulating protein was identified. We verified that Graf1 mediates Aßo driven, Pyk2-mediated effects on dendritic spine anatomical loss (Lee et al, 2019).

Literature Cited
Cox, T. O. et al. (2019) Anti-PrP(C) antibody rescues cognition and synapses in transgenic alzheimer mice. Ann Clin Transl Neurol 6, 554-574.

Lee, S., Salazar, S. V., Cox, T. O. & Strittmatter, S. M. (2019) Pyk2 Signaling through Graf1 and RhoA GTPase Is Required for Amyloid-beta Oligomer-Triggered Synapse Loss. J Neurosci 39, 1910-1929.

Overview: Our projects collectively examine aspects of addiction vulnerability and consequence from a proteomic basis in brain neurocircuits involved in decision-making, mnemonic processes and relapse behavior in rodent models. Several ongoing and new projects involve collaborative studies with center investigators.

Decision- making: The decision-making processes that confer risk for addiction may differ from those that are disrupted by chronic drug use. Our work focuses on mechanisms mediating addiction risk vs. consequence to identify novel protein targets for the prevention and treatment of addiction, respectively. Ongoing studies examine how viral and pharmacological manipulations of select proteins impacts value-based decision-making strategies and drug-taking behaviors in order to provide causal evidence for these alterations in behaviors associated with addiction vulnerabilities and drug exposures, and to identify novel pharmacotherapies for treating addiction-relevant behaviors. We plan also to extend our behavioral and computational approaches combined with proteomic analyses to other drugs of abuse and to examine sex differences. These studies are in collaboration with Drs. Stephanie Groman and the Nairn Lab. New studies also will examine the relationship between individual differences in social-affective decision-making processes and addiction risk vs. consequence, and conduct proteomic analyses of key brain regions involved in social behaviors.

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. 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. Planned studies will focus on targets (e.g., RBP1, Fez1 and retinol), in cocaine cue-memory destabilization and restabilization processes, and to examine how potential sex differences in signaling molecules are regulated by cocaine cue-memory reconsolidation processes, and those that can be manipulated therapeutically.

Relapse Behaviors: With Dr. Ralph DiLeone we have investigated the ability of the NMDA modulator NYX-783 to reduce opioid-relapse behavior. NYX-783, a novel small molecule that has shown evidence of safety/tolerability for PTSD, and our data suggests efficacy in reducing opiate seeking behavior and relapse behaviors measured by cue- and drug-induced reinstatement. We are conducting 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, and also will conduct parallel studies in cocaine and methamphetamine self-administering animals to investigate proteomic correlates of drug-induced signaling changes after behavioral tests of relapse-like behaviors, such as cue- and drug-induced reinstatement.

Overview: We have identified TARPg-8 as a critical CaMKII substrate for LTP (Park et al., 2016 Neuron). Since then, we have been working on the stoichiometry of TARPg-8 phosphorylation. We identified ten phosphorylation sites in TARPg-8. Among them, CaMKII phosphorylates two sites, and several sites are phosphorylated by PKC, while unidentified kinases phosphorylate remaining four sites. Since combinations of these phosphorylation sites are enormous, we focused on identifying functional phosphorylation sites by measuring synaptic activity in neurons expressing TARPg-8 mutants and identified minimum sets of phosphorylation sites for potentiating synaptic activity. Since TARP phosphorylation plays a crucial role in synaptic plasticity, which is also implicated in addiction, this study provides valuable insights into how addiction may arise through synaptic plasticity mediated by multi-site phosphorylation of TARPγ-8. Additionally, we have expanded our research to investigate cannabinoid-induced changes in synaptic proteins and their roles in synaptic transmission.

Overview: My laboratory continues to work with the Yale/NIDA Neuroproteomics Center to identify changes in protein phosphorylation that regulate many aspects of the addiction cycle: 1) Vulnerability -- we have determined the effects of adolescent stress on the adult amygdala phosphoproteome, and have plans to perform similar studies that directly investigate the effects of adolescent sleep disruption and circadian misalignment on protein expression and phosphorylation in corticolimbic circuits. This latter project is part of a NIDA P50 Center grant (PI Colleen McClung). 2) Acute intoxication – we were funded by NIAAA to perform phosphoproteomic analysis of what proteins are regulated by low dose alcohol exposure. We examined effects in the nucleus accumbens after acute administration of varying doses of alcohol to identify what molecules may be involved in the acute intoxicating/rewarding effects of alcohol. These results can be compared to other drugs of abuse in future studies. 3) Craving and relapse – we have performed several studies to investigate dynamic changes in protein phosphorylation after a cocaine-associated memory is retrieved, which is thought to trigger a craving-like response and compared these results to effects after that cue memory has been extinguished. We have examined two brain regions and validated several identified targets. 4) Polysubstance use -- We have recently received an R01 to perform cell type specific proteomics of dopamine neurons using APEX-mediated biotinylation in a model of nicotine-THC polysubstance use.