2015 - 2019 Project Details

Despite more than fifty years of research, no cures for psychiatric disease exist and the standard of treatment remains unsatisfactory. Genomic studies have identified hundreds of genetic variants that confer increased risk for psychiatric disease, including both highly penetrant rare variants and common variants of small effect, some of which have been linked to alternative splicing. Neurexin genes are amongst the most highly alternatively spliced genes in the human genome; non-recurrent heterozygous mutations of neurexin-1 (NRXN1) have been repeatedly associated with schizophrenia (SZ), autism spectrum disorder (ASD), and other psychiatric disorders, while changes in NRXN3 have been linked to addiction, reward behavior and obesity. Our preliminary data developed new bioinformatic strategies to quantify the NRXN1 alternative splice repertoire, comparing control fetal and adult cortical tissue to human induced pluripotent stem cell (hiPSC)-derived neurons from NRXN1+/- cases and controls. Here, we propose to establish methods with which to evaluate the effect on synaptic composition of experimental manipulation of the NRXN1 isoform repertoire in both control and patient-derived neurons. Once established, we anticipate that these innovative methodologies will be easily applied to NRXN3 and other alternatively spliced synaptic genes linked to psychiatric disease. Our overall objective is to resolve how changes in alternative splicing impact synaptic function - across donors, cell-types and drug exposures. Ultimately, we hope to directly correlate genomic and functional deficits across increasingly refined populations of patient-derived neurons.

Neuronal subcellular compartments, such as dendritic spines, growth cones and synaptosomes, are critical for neuronal function in health and diseases. Glial subcellular constituents, such as myelin and astrocytic endfeet, support neuronal functions and maintain brain homeostasis. However, the molecular mechanisms and signaling pathways present in these subcellular compartments are not fully understood. Limited neuronal and glial subcellular proteomics data have been reported and the current protocols are mainly restricted to fresh samples, which pose barriers to study postmortem human and mouse pathologies with fixed tissue.

In this grant, we aim to implement and perfect methods for subcellular fraction isolation and proteomics methodologies with the goal of studying neuronal and glial subcellular compartments in both fresh and fixed mouse/human brain samples. Ultimately, our goal is to understand molecular mechanisms and signaling pathways governing subcellular compartments in diseases and health. Firstly, we innovatively apply a recently developed method called proximity labeling by using an in situ antibody recognition approach in fixed mice and human brain sections. With this approach, we can label and isolate endogenous subcellular proteins specifically and efficiently in a wide range of neuronal and glial subcellular compartments for subsequent label free LC-MS/MS proteomic analysis. Secondly, in order to compare proteome datasets generated by different isolation approaches, we will establish a Percoll gradient-FACS approach to isolate axonal subcellular compartments in fresh mouse brains, followed by label free LC-MS/MS. Comparison of proteome datasets from proximity labeling with those from FACS will provide cross validation of these methods of neuronal and glial subcellular protein analysis. Thirdly, we have established sophisticated optical imaging toolsets to validate proteomic hits in both fixed samples and in vivo that will be implemented to further study protein targets identified by proteomics in both mouse models and human postmortem tissue, in the context of the axonopathy found around amyloid plaques in Alzheimer’s disease (AD). This proposal is of high impact because it will perfect and implement methodologies that are widely useful in neuroscience research. In addition, the results obtained from the proteomics analysis could have important implications on our understanding of AD pathophysiology and treatment design.

Addiction is a complex disease that involves drug-induced changes in synaptic plasticity due to alterations in gene transcription, protein synthesis, and cell signaling. Evidence suggests that drugs of abuse interact with and change a common network of signaling pathways that include phosphorylation mediated signaling by a subset of specific protein kinases. Addiction to drugs such as nicotine and cocaine primarily affects brain and the limbic system (ventral tegmental area (VTA), striatum, and NAc) that have rich protein kinase expression. Research has established that several kinases such as cAMP-dependent protein kinase, cyclin-dependent protein kinase, protein kinase C, calcium/calmodulin-dependent protein kinase II, Src, and Fyn tyrosine kinase play a role in the neuro-pathology of drug addiction. Identifying phospho-mediated signaling pathways that contribute to the addicted state may provide novel approaches for treatment of drug addiction. The analysis of protein phosphorylation sites has largely been restricted to studies at the single-protein level. Recently, larger-scale MS-based analyses have emerged. However, such studies have been challenging due to the immaturity of methods to enrich for low-abundance phosphoproteins or phosphopeptides. In this pilot project we will develop a high throughput, novel, convenient, sensitive, and cost-effective affinity reagent (SH2 superbinder) that will have wide applications in protein tyrosine phosphorylation analyses in drug addiction and neuronal signaling. This pilot project has two major aims: 1): Evaluate the specificity of SH2 superbinder in pTyr peptide enrichment in the rat brain. 2): Next, we will discover how cocaine exposure changes the tissue specific pTyr-proteome by comparing cocaine treated vs control rats. Considering that pTyr analysis in these small brain regions (Striatum, VTA, NAc) is difficult, we will address this technical challenge using a cutting-edge discovery proteomics technology that uses tandem mass tag (TMT) labeling.

Addiction only occurs in a subset of individuals who use drugs of abuse, and understanding the factors contributing to susceptibility is crucial for the prevention and treatment of addiction. Pre-existing biological factors may increase individual susceptibility to behaviors associated with compulsive drug-use. These factors may affect the transition from recreational drug use, to abuse, to addiction. Understanding and identifying biological pathways involved in the likelihood of becoming addicted will provide us with biomarkers to identify individuals with increased susceptibility to drug-abuse and determine therapeutic targets for addiction. In the Carlyle lab we are studying the role of neuropeptides in cognitive impairment; work by us and others have identified neuropeptides such as neurosecretory protein VGF (VGF) and chromogranin A (CHGA) to be consistently dysregulated in the brains and biofluids of patients with dementia. These neuropeptides have also been linked to psychiatric disorders including major depressive and bipolar disorder. We will investigate a putative role for neuropeptides in addiction vulnerability and consequence by combining our strengths in bioinformatics, biomarkers, and neuropeptide analysis with Dr. Stephanie Groman’s expertise in biobehavioral mechanisms of addiction at Yale. Dr. Groman has identified two decision-making parameters: one predictive of future drug-abuse (”vulnerability” Δ1 parameter), and one altered after chronic drug exposure (“consequence” Δ2 parameter). In Dr. Groman’s dataset, there was a trend-level correlation between VGF abundance in the nucleus accumbens and the vulnerability parameter highlighting a potential role of neuropeptides in drug abuse, pathophysiology of addiction and psychiatric disorders, given their established role in dementia and depression.

The functional form of neuropeptides is as secreted, protease-cleaved peptide “proteoforms”. The proteoform composition of processed peptides from VGF and CHGA is currently poorly understood, as are the proteases involved in their cleavage. In Aim 1, we propose to use data-dependent proteomic methods to map size-selected neuropeptide proteoforms in rat cerebrospinal fluid (CSF), to then build a targeted data-independent acquisition method to accurately quantify key proteoforms. In Aim 2a, Dr. Groman will take CSF immediately before sacrifice from drug naïve adult animals exposed to escalating doses of morphine (or water control) during gestation. We will quantify neuropeptide proteoforms using the method built in Aim 1 and relate proteoform levels to decision-making processes. We hypothesize that changes in specific neuropeptide proteoforms will be linked to addiction vulnerability and consequence. In Aim 2b, we will test whether rats exposed to morphine during gestation have altered decision-making processes and altered neuropeptide CSF proteoform profiles after oxycodone self-administration. This proposal will help train the PI on drug abuse and the pathophysiology of addiction, while receiving further training on both data-dependent and data-independent mass spectrometry techniques. VGF and CHGA could represent common targets to enhance cognition after impairment and play a critical role in decision-making. When these neuropeptides are impaired in psychiatric diseases and drug abuse, this could lead to the aberrant decision-making processes that are observed in people diagnosed with psychiatric diseases such as addiction.

Addiction is a devastating disorder that is exceptionally difficult to treat due to the high vulnerability of patients to relapse even long after terminating drug use. Throughout extended abstinence, cravings for drugs of abuse remain strong and even intensify, or “incubate”, over time. Craving is thought to be mediated by dysregulated signaling in the nucleus accumbens (NAc) that primes motivational circuits to initiate drug-seeking behaviour. Understanding how drug-induced synaptic changes within the NAc contribute to craving may be the key to developing effective treatments for substance abuse. The goal of the present studies is to identify protein networks that stabilize synaptic changes within the NAc following cocaine self- administration that may sensitize the system for relapse to drug-seeking behaviour. To accomplish this, we will perform cutting-edge iTRAQ discovery proteomics on synaptosomes isolated from the NAc of mice following either short- (24h) or long-term (45 days) withdrawal from cocaine self- administration. One caveat to using the iTRAQ approach is that a broad screen of the protein content of the synaptosomal fractions may not detect changes to low-abundance proteins. Therefore, an additional aim of these studies is to perform targeted analysis of protein changes within the synapse during withdrawal using the PSD/PRM approach developed by the Yale/NIDA Neuroproteomics Center. This PSD/PRM approach enables a detailed characterization of targeted protein changes within the synapse by monitoring a pre-determined set of proteins specific to the synapse. Together, these studies will identify novel changes to proteins that regulate cellular and physiological processes that can be targeted to better understand the neural mechanisms of drug craving. The results of these studies will have broad utility as a reference proteome of NAc synapses during withdrawal for future studies from our lab and others, and the use of mice provides a foundation on which to manipulate protein targets in a cell-type specific manner.

A strong connection between the status of the intestinal environment and the function of the central nervous system (CNS) is increasingly being recognized. The bidirectional feedback loop commonly referred to as ‘gut-brain’ axis facilitates two-way communication between the central and the enteric nervous system, linking brain emotional and cognitive centers with peripheral intestinal functions (1). It has recently been reported that alterations in gut microbiota can affect behavioral responses to drugs of abuse (2). Major alterations in gastrointestinal (GI) physiology such as those resulting from bariatric surgery can affect reward/addiction behaviors (3). However, much remains to be discovered regarding the dialogue between the CNS and the GI system (4) and how it can contribute to drug addiction. Given that addictive drugs usurp brain reward circuitry including that which is committed to feeding behavior, it is rationale to hypothesize that altered gut-brain communication may impact formation, maintenance or relapse of the addicted state.

Inflammatory bowel diseases (IBD) represent a major health concern with continuously rising incidence and prevalence (5). Reports have suggested that persons with IBD may be at an increased risk for substance abuse and dependence (6-8). Furthermore, the risk of IBD is higher in persons with alcohol addiction (9). In addition, anxiety and depression related symptoms are frequently comorbid in patients with IBD (10). Such comorbidity may also substantially increase the risk of addiction as both mood disorders and drug addiction are associated with major disruptions in brain reward circuitry (11) and it is well established that mood disorders may motivate individuals to resort to drugs of abuse to cope with their negative affective states (12). However, the mechanistic link between gastrointestinal inflammation and addictive behavior remains poorly understood.

The nucleus accumbens (NAc) serves as a major input structure of the basal ganglia and integrates information from cortical and limbic structures to mediate goal-directed behaviors. Exposure to drugs of abuse disrupts plasticity in this region, allowing drug-associated cues to give rise to a pathologic motivation for drug seeking (13). Very little is known as to how GI inflammation may impact this brain region that plays a central role in the reward circuitry of the brain. Our preliminary data suggest that intestinal inflammation is associated with alterations in NAc dependent behavior as well as changes in synaptic function and phosphorylation patterns of some key neuronal signaling proteins in this brain region. Here, we propose to use a well-characterized mouse model of IBD to comprehensively investigate the effects of intestinal inflammation on the NAc proteome using state of the art proteomics approaches. Since, protein phosphorylation serves as a major posttranslational modification involved in a broad array of physiological functions we will also examine the possible role of these regulatory mechanisms in altering reward circuitry following intestinal inflammation. Furthermore, we will investigate the effect of bowel inflammation on behavioral responses to morphine and corresponding proteomic changes in the NAc. The study will generate novel insights on how gut-brain cross talk may potentially play a role in addiction.

Literature Cited:

1. Zhu et al (2017) Oncotarget 10;8(32):53829-53838.
2. Drew et al (2016) Sci Rep. 6: 35455.
3. Li et al (2016) J Psychiatr Res. 76:16-29
4. Madelyn et al (2017) NPJ Parkinsons Dis. 3: 3.
5. Duijvestein et al (2018) Curr Treat Options Gastroenterol. 16(1):129-146
6. Weiss et al (2015) Drug Alcohol Depend. 156:84-89.
7. Ravikoff et al (2013) Inflamm Bowel Dis. 19(13):2809-14.
8. Buckley et al (2015) Clin Gastroenterol Hepatol. 13(2):310-315.
9. Hsu et al (2016) PLoS One.11(11):e0165411
10. Filipovic et al (2014) World J Gastroenterol 20(13): 3552-3563
11. Russo et al (2013) Nat Rev Neurosci. 14(9):609-25
12. Quello et al (2005) Sci Pract Perspect. 3(1): 13–21
13. Floresco et al (2015) Annu Rev Psychol. 66:25-

Substance use disorders (SUD), defined as the dependence to substances such as but not limited to alcohol, nicotine and illicit drugs, affect as high as 9% of Americans [1]. Mounting evidence suggests that the dysregulation of innate immune system plays a role in the pathophysiology of SUD [2-4]. Stress, a major risk factor for the development of addiction, has been shown to promote drug seeking and dependence relapse in several pre-clinical, population-based and epidemiology studies [5]. Stress-induced risk for SUD may be partially mediated by stress-induced dysregulation in innate immune pathways. High mobility group box protein 1 (HMGB1), an endogenous ligand for innate immune receptors, has been implicated in SUD and is significantly elevated in the brains of lifelong addicts [4].

HMGB1 upregulation and extracellular signaling is mediated by stress [6, 7]. Chronic stress has been shown to induce persistent dysregulation of HMGB1 expression and release in resident microglia [8], which may contribute to the pathogenesis of addiction. Here we propose to identify the long-lasting effects of stress on HMGB1-mediated signaling by identifying persistent stress-induced HMGB1 post-translational modifications in microglia. In addition, we aim to identify differences in the proteomic signature of resident microglia, the innate immune cells of the brain, in the presence and absence of microglial HMGB1 signaling to identify alterations in protein expression that may increase risk for the addiction.

Literature Cited

1. Tolliver, B.K. and R.F. Anton, Assessment and treatment of mood disorders in the context of substance abuse. Dialogues Clin Neurosci, 2015. 17(2): 181-90.
2. Bachtell, R.K., et al., Glial and neuroinflammatory targets for treating substance use disorders. Drug Alcohol Depend, 2017. 180: 156-170.
3. Crews, F.T., et al., The role of neuroimmune signaling in alcoholism. Neuropharmacology, 2017. 122: 56-73.
4. Crews, F.T., et al., Toll-like receptor signaling and stages of addiction. Psychopharmacology (Berl), 2017. 234(9-10): 1483-1498.
5. Sinha, R., Chronic stress, drug use, and vulnerability to addiction. Ann N Y Acad Sci, 2008. 1141: 105-30.
6. Franklin, T.C., C. Xu, and R.S. Duman, Depression and sterile inflammation: Essential role of danger associated molecular patterns. Brain Behav Immun, 2017.
7. Wohleb, E.S., et al., Integrating neuroimmune systems in the neurobiology of depression. Nat Rev Neurosci, 2016. 17(8): 497-511.
8. Franklin, T.C., et al., Persistent Increase in Microglial RAGE Contributes to Chronic Stress-Induced Priming of Depressive-like Behavior. Biol Psychiatry, 2018. 83(1): 50-60.
   
   

Not all individuals that use drugs of abuse will develop an addiction. The transition from drug use to abuse and, eventually, to dependence may be mediated by biological factors that impact the likelihood of individuals to develop an addiction. Identification of proteins that mediate addiction vulnerability, therefore, could lead to the development of novel prophylactic treatments for addiction.

We have been interested in using decision-making as a behavioral biomarker for understanding addiction pathophysiology and recently identified a computationally-derived decision-making parameter that predicts future drug-taking behaviors. Specifically, the ability of rats to use positive outcomes to guide their decision-making prior to any drug use predicted future drug-taking behaviors. Using this behavioral biomarker in drug-naïve and drug-exposed animals, we conducted a pilot study using label-free proteomics and identified three protein targets as potential addiction susceptibility proteins: sorting nexin 1 (Snx1), ryanodine receptor 2 (Ryr2), and ataxin 2-like (Atxn2l). Remarkably, genes encoding these same proteins have been linked to addiction in humans. These exciting results suggest that differential expression of these proteins may underlie addiction vulnerability and Aim 1 of this proposal will test this hypothesis by examining decision-making processes and expression of Snx1, Ryr2, and Atxn2l proteins in animals exposed to morphine during gestation – a known risk factor for addiction.

Self-administration of drugs of abuse, however, disrupts decision-making processes, regardless of pre-existing decision-making abilities. We have found that the drug-induced decision-making deficits are due to reductions in the ability of rats to use negative outcomes to guide their decision-making, suggesting that the behavioral processes that mediate an indivuial's risk for developing an addiction differ from the behavioral processes that are disrupted by drug use. Indeed, our label-free proteomic study identified a single protein responsible for the drug-induced decision-making deficits: ras-related protein Rab3B. It is unclear, however, if prenatal exposure to morphine modulates drug-induced changes in decision-making and Rab3B expression. In Aim 2 of this proposal, decision-making will be assessed in rats exposed to morphine during gestation before and after they are trained to self-administer methamphetamine (or saline). Expression of Rab3B will be quantified using targeted proteomics to determine if expression of Rab3B is differentially affected by drugs of abuse in addiction vulnerable populations.

The proteomic data collected in this proposal will provide the experimental evidence needed to support our discovery-based results that have implicated specific protein targets in different aspects of addiction pathophysiology. We believe that these data will lead to the development of therapeutic tools that can modulate the expression of addiction-risk proteins and, consequently, attenuate addiction liability.

Tobacco smoking continues to be the leading cause of premature death in the United States, and a significant public health burden worldwide. Addiction to tobacco is driven by nicotine, the primary psychoactive and addictive component in tobacco smoke. Studies demonstrate that nicotine addiction has a disproportionate impact on women, who are more likely to progress from initial use to dependence, and less likely to attain and maintain abstinence. The neurobiological mechanisms underlying sex differences in nicotine addiction have been studied through various experimental approaches, ranging from genetic and molecular to behavioral and neuroimaging techniques, in both humans and animal models. These studies have found sex differences in key brain regions of the mesocorticolimbic system, including the ventral tegmental area (VTA), striatum, and cortex, which have an established role in addiction to many drugs of abuse, including nicotine. However, no proteomic studies have investigated sex differences in nicotine addiction. One possibility is that underlying sex differences in the proteome of these structures result in baseline sex differences in neuronal activity and function that lead to differences in behavior. Additionally, nicotine exposure itself may differentially impact the proteome in males and females. With the cutting-edge technologies and expertise available through the Yale/NIDA Neuroproteomics Center, we propose to study sex differences in nicotine addiction by 1) evaluating sex differences in the proteome at baseline and after chronic nicotine administration in the mouse VTA, striatum and cortex and 2) evaluating sex differences in the proteome after a minimalistic, behaviorally relevant paradigm of nicotine exposure. Previous research into sex differences has overwhelmingly focused on neurobiological substrates that were selected as interesting targets based on initial studies conducted only in male subjects. Our approach will allow an unbiased investigation of sex differences in the proteome at baseline and after nicotine exposure, as well as a comparison between different nicotine regimens. Importantly, our results may identify new targets for future hypothesis-driven research into sex-specific mechanisms of nicotine addiction, specifically, or drug addictions more generally.

Prolonged treatment with opiates, the most widely used analgesics, leads to the development of tolerance and addiction. High-efficacy opiate agonists such as fentanyl activate the morphine receptor (MOR) leading to receptor translocation throughout the plasma membrane and phosphorylation by G protein receptor kinases, followed by receptor clustering to microdomains and transient nuclear ERK activation preceding receptor endocytosis and resensitization. Low-efficacy agonists such as morphine activate MOR leading to receptor phosphorylation by localized plasma membrane associated protein kinase C (PKC) and a restricted redistribution of MOR with sustained cytosolic ERK activation. Genetic and pharmacological inhibition of PKC in rodents has demonstrated PKC to be a key player responsible for morphine tolerance and addiction. The molecular mechanisms that lead to PKC mediated tolerance still remain to be elucidated. Dissecting PKC signaling is difficult due to the overlapping specificity of different members of the PKC family of kinases and the fact that kinase substrate interactions are normally transient. To this end, in the present project we intend to use a combination of novel tools (activation specific antibodies, biotin ligase fused PKC, & PKC substrate trapping mutants) and modern proteomics approaches to identify PKC substrates and binding partners involved in morphine induced tolerance both in cell culture systems and in animal models. The specific aims are: to use active state specific anti cPKC antibodies to identify PKC binding proteins (by mass spectrometry) and determine the dynamics of PKC activation in mice following morphine treatment (Aim 1), to employ the BioID method to identify PKC targets (by mass spectrometry) upon morphine induced MOR activation (Aim 2), and to utilize PKC substrate trapping mutants to identify targets (by mass spectrometry) upon morphine induced MOR activation (Aim 3). Together, the identification of key components involved in PKC mediated tolerance to morphine will contribute to the elucidation of mechanisms underlying morphine tolerance and the development of novel therapeutics for the treatment of tolerance and addiction.

Cocaine addiction is a chronic relapsing disorder that leads to significant morbidity, and for which there are currently no FDA-approved pharmacotherapies. In recent years, there has been a growing appreciation for the role of neuroimmune interactions in the pathogenesis of multiple psychiatric conditions – including substance use disorders. To further characterize the role of the innate immune system in models of cocaine use disorder we utilized a broad screen of serum cytokines following chronic cocaine treatment in mice. From this, we identified granulocyte-colony stimulating factor (G-CSF) as a cytokine that is upregulated both in the serum and in important limbic reward structures following cocaine treatment. Further experiments demonstrated G-CSF to be a potent modulator of neuronal and behavioral plasticity in response to cocaine, with the principle locus of this effect in the Nucleus Accumbens (NAc). While the effect of G-CSF on behavior is clear, the molecular mechanisms underlying its effect remain to be clarified. Multiple previous studies have demonstrated the G-CSF receptor to be expressed on neurons throughout the brain, and it is known to signal through multiple intracellular signaling cascades important for synaptic plasticity including Jak/STAT, Akt and ERK pathways. Here, we propose to utilize the cutting edge mass spectrometry protein profiling techniques available at the Yale/NIDA Neuroproteomics Center to directly assess the molecular effects of G-CSF on the NAc in two distinct aims: (1) Utilizing the iTRAQ proteomic profiling technology we will seek to perform whole-proteome discovery analysis to identify regulated proteins in whole NAc tissue punches from animals treated with ± G-CSF ± cocaine in a 2 x 2 design, and (2) To identify synapse-specific targets of G-CSF treatment we will utilize the PSD/MRM technology developed by the Neuroproteomics Center to perform targeted analyses of purified NAc synaptosomes from G-CSF and cocaine treated mice. Overall, the proposed research will clarify the molecular signaling underlying this newly identified potent modulator of neuronal and behavioral response to cocaine. These data will serve both to clarify the underlying neurobiology, and to develop targeted approaches to study this novel translationally-relevant signaling pathway.

Significance
We propose developing a novel strategy for protein identification and quantification in post mortem brain tissue of nicotine addicted individuals with Posttraumatic Stress Disorder (PTSD). Standard, single shot label free proteomic approaches do not interrogate the proteome at sufficient depth to see differences arising from low abundance mRNA transcripts. Offline fractionation of peptides allows for increased rates of protein identification, but quantification of proteins becomes a serious technical challenge with post fractionation. It is known that important proteomic changes are occurring at the level of low abundance proteins and so our focus is on developing technologies to identify these changes. By combining tandem mass tag labeling with offline fractionation, we can benefit from the increased depth of proteome coverage gained by fractionation, while using the TMT tags to allow between sample peptide quantification.

As protein forms the final output of the central dogma, it is important to develop proteomic technologies that move towards matching the sensitivity of RNA-based approaches. We have previously identified numerous mRNA changes in our PTSD cohorts, making integration of the transcriptome and proteome an important goal of this study. Our current post-mortem repository allows for a unique and powerful analysis of the human subgenual prefrontal cortex (PFC), not only measuring the protein changes occurring in PTSD smokers versus PTSD nonsmokers but also across cohorts. In addition to being the first systematic examination of proteomic changes occurring in nicotine dependent versus non-dependent PFC, this study will also be the first to examine proteomic changes in PTSD brain.

Specific Aim 1
In Aim1, we will assess the potential of using fractionated tandem mass tagged peptides from post mortem PFC in LC/MS-MS to detect changes in protein abundance. To increase the sensitivity of quantifying low abundance peptides, we propose using tandem mass tag (TMT) peptide labeling in combination with offline sample fractionation. These tags are designed to ensure that identical peptides labeled with different TMTs co-migrate throughout offline fractionation and online LC. These TMTs permit simultaneous determination of both the identity and relative abundance of peptides using a collision induced dissociation (CID)-based analysis method.

Specific Aim 2
In Aim 2, we will identify the proteomic changes in subgenual PFC of smokers with PTSD versus nonsmokers with PTSD. Building upon Aim 1, we will use fractionated TMT-LC-MS/MS to identify changes to the subgenual PFC proteome. A subset of our PTSD (~50%) and control cohorts (~33%) were using tobacco at the time of death. This will allow us to directly interrogate the changes in the cortical proteome of smoking subjects with PTSD versus nonsmoking PTSD patients. Additionally, we will be able to evaluate the proteomic changes in PFC in smoking and nonsmoking subjects with no mental health history.
 

Relapse to drug use, which can occur even after a long period of abstinence, is a critical problem in treating addiction. To study neurobiological mechanisms underlying drug relapse, we have been using a rodent model termed incubation of drug craving, which refers to the progressive intensification (‘incubation’) of cue-induced drug craving during abstinence/withdrawal from drug self-administration. Incubation of craving has been observed across several drug classes in rodents and more importantly, it has also been demonstrated in human drug users.

Previously, we and others have demonstrated the important role of glutamate transmission in striatum in incubation of craving to psychostimulants. For example, the Wolf lab has shown that incubation of craving for both cocaine and methamphetamine (meth) involves strengthening of excitatory synapses in nucleus accumbens (NAc) through incorporation of high conductance calcium permeable AMPA receptors (CP-AMPARs). However, Dr. Li’s postdoctoral work in the Shaham group has shown that the circuitry beyond the NAc appears to differ for cocaine and meth. For incubation of meth craving, Dr. Li’s work has recently demonstrated an important role for glutamatergic input from thalamus (anterior intralaminar nucleus of thalamus; AIT) to dorsomedial striatum (DMS). Additionally, important roles for prefrontal cortex (PFC) and basolateral amygdala (BLA) projections to the NAc have been demonstrated with cocaine.

In this pilot grant, we aim to characterize protein expression during incubation of craving to cocaine and Meth in a projection-specific manner. We will focus on projections strongly implicated in regulation of incubation for each psychostimulant (cocaine: BLA to NAc core; meth: AIT to DMS). We will achieve projection specificity at both the cell body and synaptic level. At the cell body level, we will combine retrograde transporting AAV virus and fluorescent-activated cell sorting to label and sort BLA and AIT neurons that project into NAc core or DMS, respectively. At the synaptic level, we will combine anterograde transporting AAV virus expressing a membrane-tethered extracellular tag, subcellular fractionation and immunoprecipitation to enrich for synaptoneurosomes postsynaptic to a particular pathway (synaptoneurosomes in NAc postsynaptic to BLA inputs; synaptoneurosomes in DMS postsynaptic to thalamic inputs). Overall, this proposal will help us gain insight into cellular mechanisms underlying the persistent vulnerability to drug relapse that makes addiction so difficult to treat.

Synapses are specialized cellular junctions that connect neurons into the brain circuits that underlie cognitive processes and behavior. Synaptic junctions are heterogeneous in molecular composition and function, reflecting diverse health and disease states. Aberrations in neuronal connectivity occur in brain disorders, and synaptic changes in addiction-relevant brain regions are correlated with drug seeking and relapse. Progress over the past years has shown that trans-synaptic adhesion molecules mediate synapse formation and differentiation, including in brain regions relevant for addiction, and we recently reported that a mouse model lacking a synaptogenic adhesion protein exhibits altered addiction-relevant behaviors. This agrees with human genetic data that support that mutations in synaptic adhesion proteins can be linked to addictive behaviors.

Aiming to define the mechanisms that control synapse maturation and remodeling, we hypothesize that synaptic cell adhesion molecules modulate the molecular composition of synapse-organizing complexes in the synaptic cleft when synapses undergo experience-dependent changes. Further, we hypothesize that disease states and exposure to psychostimulants impact the molecular makeup of the cleft to alter signaling and synaptic function. The objective of this pilot project is to apply a proteomic approach to define the cleft composition of synapse populations. To test our hypothesis, we aim to use cleft reporter proteins that allow for the labeling of synaptic surface proteins. We will combine this molecular labeling method with affinity purification and unbiased analysis by quantitative proteomics. Labeled synaptic membrane protein targets will be purified and subjected to quantitative proteomics for identification at the Yale/NIDA Neuroproteomics Center.

This approach has the potential to probe the synaptic cleft as a compartment rather than individual protein-protein interactions, and to untangle the heterogeneity of synapses in different brain regions. Once established, the molecular remodeling of the synaptic cleft during plasticity can be investigated. Further, this approach can be applied to diverse neuronal populations. This includes neurons in addiction-relevant brain regions which can determine how drugs of abuse may impact the synaptic cleft and alter neuronal communication. We expect this to reveal novel candidate proteins that can underlie acute and chronic synaptic changes after psychostimulant exposure.

Significance

Basic and clinical research strongly suggests there are extensive bidirectional interactions between circadian rhythms and addiction. Disruptions to the circadian system, either by environmental or genetic perturbation, may increase the vulnerability to addiction, while chronic drug use could lead to circadian disruptions that persist during abstinence, contributing to relapse. Although these are intriguing, very little is known about the cellular and molecular mechanisms underlying these relationships. We have identified a novel mechanism for the circadian transcription factor, neuronal PAS domain protein 2 (NPAS2), in the regulation of cocaine reward, potentially via interactions with an unknown co-activator, or co-factors, selectively within dopamine receptor 1 expression (D1+) medium spiny neurons (MSNs) of the nucleus accumbens (NAc). Ongoing studies are investigating the downstream transcriptional targets of NPAS2 using ChIP-seq and RNA-seq, and whether NPAS2 also influences cocaine self-administration. Preliminary data indicates that chronic cocaine increases the expression and activity of NPAS2 in the NAc and prevents the typical interaction between NPAS2 and BMAL1 to activate circadian-driven gene transcription. We propose a novel mechanism by which NPAS2 binds another co-activator to initiate transcription in response to cocaine, since NPAS2 alone cannot drive gene transcription.

Specific Aims

The aims of this proposal will investigate the following: 1) Identify the potential binding partners and co-factors of NPAS2 in response to cocaine in the NAc across the circadian cycle; and 2) Identify the proteins in D1+ and D2+ MSNs of the NAc that are altered by cocaine across the circadian cycle, along with potential proteins downstream of NPAS2 in an effort to integrate with ongoing whole-genome sequencing studies. These pilot funds will be important for investigating a potential novel mechanism by which circadian rhythms influences
motivation and reward by identifying circadian regulated protein networks in specific cell types within a brain region that is a key substrate for integrating motivation and reward signals. The proposed project is directly related to the theme of the Yale/NIDA Neuroproteomics Center to identify altered protein signaling pathways associated with the acute and chronic effects of drugs of abuse as related to addiction.

Addiction to nicotine is mediated through nicotinic acetylcholine receptors containing the α4 and β2 subunits (α4β2* nAChRs). Upon nicotine exposure, α4β2* nAChRs are activated, then rapidly desensitized. Chronic nicotine exposure, as would be observed in the brain of a smoker, is also associated with a robust upregulation of α4β2* nAChR expression, which is post-translationally mediated. As such, the molecular mechanisms of α4β2* nAChR regulation are paramount to our understanding of the physiological and behavioral effects of nicotine. In vitro and cell biological studies have demonstrated that the assembly, trafficking and desensitization of nicotinic receptors are all regulated by phosphorylation of the receptor by PKA and PKC. However, phosphorylation of α4β2* nAChRs has not been demonstrated in vivo and other important brain kinases, like CaMKII, have not been evaluated. In this study, we will utilize the cutting edge mass spectrometry techniques and expertise at the Yale/NIDA Neuroproteomics Center to directly assess phosphorylation of α4β2* nAChRs with two major aims: (1) To evaluate phosphorylation of α4β2* nAChRs in the mouse brain at baseline and following acute and chronic nicotine exposure, and (2) To assess the ability of CaMKIIα to phosphorylate α4 and β2 nAChR subunits. The results from this study will provide a necessary extension of previous studies of α4β2 nAChR regulation by characterizing receptor subunit phosphorylation in vivo. Additionally, we will, for the first time, evaluate the effects of nicotine on nAChR phosphorylation and explore the role of a novel interacting kinase, CaMKIIα. Results from these studies will improve our understanding of the cell signaling mechanisms underlying nicotine addiction and may also inform studies of other disorders linked to nAChR dysfunction, including schizophrenia, depression and other addictions.

Sustained abstinence from cocaine use is frequently compromised by exposure to environmental stimuli that have previously been strongly associated with drug taking. Such cues trigger memories of the effects of the drug, leading to craving and potential relapse. Our previous work has demonstrated that manipulating cocaine-cue memories by destabilizing them through interfering with the reconsolidation process is one potential therapeutic tool by which to prolong abstinence. We have recently successfully used the naturally-occurring histone acetyltransferase (HAT) inhibitor, garcinol, to block the reconsolidation of a cocaine-cue memory in a manner that is specific to the reactivated memory only, cue-specific, long-lasting and temporally constrained. Additionally, we have found that intra-amygdala garcinol infusion is sufficient to block a cocaine-cue memory. Collectively, these data support the hypothesis that garcinol may be a useful novel therapeutic tool by which to interfere with the reconsolidation of cocaine-cue memories. To enhance the translational potential of this idea, we aim to investigate the signaling transduction pathways in the lateral amygdala (LA) following memory retrieval and garcinol administration by examining neuronal proteomic profiles.

The proposed experiments will provide the groundwork necessary for future studies to further understand how garcinol exerts its effects in the brain. Because of the potential therapeutic use of this novel compound in humans, we believe it is vital to first characterize the underlying signaling transduction pathways by examining large-scale protein expression profiles in the LA, a key site of action of garcinol. Other future studies could also employ a genome-wide expression analysis using RNA sequencing following retrieval and systemic garcinol. While we plan to initially examine changes in proteins, this approach will allow for the identification of potentially novel genes associated with the reconsolidation of cocaine-associated memories. Thus, the proposed series of experiments will not only enhance our current knowledge of how garcinol impairs reconsolidation but will also provide further evidence for its use in the treatment of addictive disorders making this project relevant to the Yale/NIDA Neuroproteomics Center’s theme of Proteomics of Altered Signaling in Addiction.

The specific aim of this project is to investigate how garcinol alters the cocaine-cue memory retrieval-induced neuroproteomic expression profile in the LA. We will first investigate which proteins are differentially expressed in the LA following memory retrieval and garcinol administration by utilizing a high resolution LC MS/MS Label Free Quantitative (LFQ) approach. Next, we will examine levels of Lysine acetylation on proteins in the LA under these conditions utilizing an LFQ MS workflow in conjunction with an acetyl-Lysine Motif enrichment protocol.

Substance use disorders produce an enormous burden, yet our understanding of their underlying pathophysiology remains remarkably limited. Drugs of abuse, such as cocaine, evoke long-lasting synaptic changes which may underlie the development and persistence of substance use disorders. Thus, understanding drug-induced synaptic adaptations that increase vulnerability to substance abuse may be critical in order to identify novel treatment strategies. We recently identified a novel role for synaptic pH and acid-sensing ion channel-1A (ASIC1A) in opposing drug-seeking behaviors involving cocaine and morphine and further suggest a role for ASICA in regulating dendritic spine morphology and synaptic plasticity in medium spiny neurons in the nucleus accumbens (NAc) (Kreple et al., 2014). This pilot project seeks to increase our understanding of how ASIC1A regulates synaptic physiology by testing the effects of Asic1a disruption on the NAc neuroproteome. We will isolate proteins from the NAc and use an unbiased proteomics approach to identify proteins that change in response to ASIC1A-dependent synaptic activity and cocaine withdrawal. We will then extend these analyses to analyze fractions enriched in synaptic terminals. Follow-up validation studies will hopefully be able to use targeted proteomic methods to analyze post-synaptic proteins. Comparing the proteomes of naïve and cocaine withdrawn Asic1a+/+ and Asic1a-/- mice may help reveal mechanisms by which ASIC1A regulates synapse morphology and plasticity and predisposes animals toward enhanced cocaine sensitivity. Thus, these studies may provide insight into the therapeutic potential of targeting ASIC1A for the treatment of substance abuse.

Literature Cited

Kreple, C.J., et al., (2014) Acid-sensing ion channels contribute to synaptic transmission and inhibit cocaine-evoked plasticity. Nat. Neurosci. 17(8): 1083-91.

Recent research on addiction has paid increasing attention to the neurotransmitter glutamate. A primary class of glutamate receptors are metabotropic glutamate receptors (mGluRs), belonging to family CG protein-coupled receptors. They mediate slower, modulatory glutamate transmission. Considerable efforts thus have been directed at targeting mGluRs for treatments of drug addiction and many other diseases, such as Alzheimer’s disease, fragile X mental retardation, and schizophrenia.

Metabotropic glutamate receptor I (mGluR1) is a typical mGluR family member, existing as a homodimer linked by a single disulfide bridge in the extracellular ligand-binding domain (LBD), and extensive non-covalent interactions at the dimer interface. The LBD functions as a sensor of glutamate. Each subunit in the LBD dimer adopts a Venus flytrap (VFT) fold with one glutamate binding site in its cleft. The dimeric LBD exhibits negative cooperativity for glutamate binding, with an elusive molecular mechanism. We thus aim to understand the underlying mechanism of the cooperativity and how homodimerization modulate the sensitivity and dynamic range of ligand sensing for mGluR1. We will (1) express and purify the extracellular LBD of native dimeric mGluR1 and monomeric mGluR1 with mutations at the cysteine site of the disulfide bridge, (2) use multiangle light scattering to quantify the monomer/dimer equilibrium in the mGluR1 LBD mutants, and (3) use fluorescence spectroscopy to measure glutamate response for wild type and mutated mGluR1 LBD to assess the role of dimerization in the molecular mechanism of glutamate binding. The study will in turn guide the design of drugs that are precise modulators of the conformational equilibria of receptors and downstream responses of the cells.

Significance

The vast intercellular and intracellular heterogeneity of the brain presents major challenges for proteomic analysis. Regulatory events are localized to specific neuronal cell types or subcellular compartments, resulting in discrete patterns of protein expression and activity. Quantifying the proteome in brain regions with respect to this localization is therefore extremely difficult. The striatum, a region critically important in the development of addictive drug-related behavior, is a perfect example of this complexity; as it contains two major cell types (D1 and D2 type dopaminoceptive neurons) whose cell bodies and processes are intermixed throughout the region. While the use of D1 and D2 mice for ribosome immunoprecipitation (TRAP) experiments has shown significant differences in cocaine-induced mRNA expression changes between these two cell populations, little is known about changes in the levels of protein expression or modification in these neuronal populations. We are currently working to overcome these issues of cellular heterogeneity and shape by using D1 and D2 TRAP mice to assess neural protein translation dynamics through cell type specific ribosomal footprinting. However, this approach does not allow for assessment of the entire cellular proteome, only measuring rates of protein production and disallowing assessment of post-translational modifications that form the final functional state of the neural proteome. In this pilot project we propose complementary methods to address the question of the complete neural proteome.

Specific Aim 1

We will evaluate the use of fluorescence-activated cell sorting to isolate GFP-L10a positive neurons from D1 and D2 mice for proteomic assessment. FACS will first be performed on isolated nuclei from these animals. Assessing protein changes in other irregularly shaped cellular compartments poses greater challenges. We have optimized a sort method that enables assessment of 24,000 D1 neuron positive events, which we presume includes the nucleus, some cytoplasm, and rough endoplasmic reticulum from the surrounding soma. We will evaluate the proteome of these events from D1 compared to D2 mice.

Specific Aim 2

The FACS-based isolation method depends on random shearing of cellular membrane and therefore may result in irregular sampling of the cytoplasm. For comparison, we will perform Laser Capture Microdissection using tissue from D1-Flag-DARPP-32 and D2-Myc-DARPP-32 mice. The advantage of using these mice is twofold: DARPP-32 acts as a superior volume label of the cell body than GFP ribosomes, and both D1 and D2 cells types can be collected from the same animal. It is estimated that LCM dissection of 1,000 or more cells can bring neural proteomes within reach, so by collecting ~10,000 cells from each experimental condition for proteomic assessment we expect to obtain very high quality data.
 

Significance

Neuronal refinement and stabilization in late adolescence are believed to confer resilience to poor decision making and addictive like behaviors. In previous work, we found that cocaine triggers dendritic spine destabilization on neurons in the orbital prefrontal cortex (oPFC). The Abl2/Arg nonreceptor tyrosine kinase acts downstream of integrin receptors to control cytoskeletal signaling pathways that stabilize dendrites and dendritic spines in the adolescent mouse brain. Loss of Arg function destabilizes dendritic spines in the oPFC, and this is accompanied by increased locomotor sensitization to cocaine and amphetamine, increased responding to reward-related cues, and decrements in instrumental reversal learning, all phenotypes implicated in addiction. Moreover, cocaine exposure led to spine enlargement in oPFC neurons in control animals, but this response is not observed following genetic or pharmacological inhibition of Arg.

Despite the clear implication of Arg-mediated signaling pathways in the refinement of oPFC neuron structure and determining the responsiveness to psychostimulants, we do not understand why loss of Arg function leads to reductions in oPFC stability or reduced structural plasticity in response to cocaine. We will use unbiased state-of-the-art proteomics approaches to identify cocaine- and Arg-regulated signaling processes that mediate oPFC stabilization and oPFC structural plasticity and assess their importance in regulating oPFC neuron structure and function.

Specific Aim 1

To identify candidate mediators of oPFC spine stabilization that are targeted by cocaine. We will use differential iTRAQ labeling and mass spectrometry of wild type and arg–/– mouse oPFC to identify novel proteins whose levels are altered upon loss of Arg function as candidate mediators of dendritic spine stabilization by Arg. We will also perform similar iTRAQ analysis on oPFC tissue from cocaine-treated wild type and arg–/– mice to identify potential pathways that mediate cocaine-induced dendritic spine plasticity. We will also conduct similar analyses on oPFC tissue from wild type mice treated chronically with corticosterone, which we have shown independently to destabilize oPFC spines. Comparison to the cocaine-treated mice will identify alterations that are specific to cocaine treatment.

Specific Aim 2

To characterize the role of candidate mediators on oPFC neuron structure and function we will use a complementary set of biochemical, cell-based, and whole animal assays to assess the relative roles for the candidate molecules in oPFC spine stabilization and cocaine-induced plasticity. First, we will assess how cocaine affects the levels, subcellular localization, or activity of the candidate proteins in wild type (WT) or arg–/– mice. We will also how knockdown of the candidate proteins in long-term established cortical cultures affects dendritic spine and dendrite stability. We will monitor dendritic spine density, size, and shape as well as dendrite arbor size and branching patterns using methods we have described previously. In cases where we observe effects, we will test whether a GFP-tagged shRNA-resistant version of the candidate rescues defects in dendrite and dendritic spine stability. These studies will identify which of the novel candidates regulate dendritic spine and dendrite stability. 

Cocaine administration leads to oxidative stress in the nucleus accumbens (NAc) and alters redox homeostasis. However, the role of redox-mediated signaling events in cocaine addiction is unknown. The glutathione (GSH) precursor, N-acetylcysteine, has been shown to restore glutamate homeostasis and reduce relapse to cocaine seeking. In addition to changes in GSH levels, chronic cocaine can also induce changes in S-glutathionylation, which is a post-translational modification (PTM) on cysteine residues following oxidative and/or nitrosative stress. S-glutathionylation of proteins can prevent further modification of cysteine residues to sulfinic and sulfonic acids and subsequent degradation. Target proteins for S-glutathionylation are considered redox switches and contribute to redox- mediated signaling events. Furthermore, the consequences of S-glutathionylation may be redox-mediated modifications of the structure/function of a target protein. Previously we have shown that after withdrawal from daily cocaine using a sensitization paradigm, an acute cocaine challenge elicited a marked increase in both NAc global protein S-glutathionylation and in the enzyme responsible for catalyzing the forward reaction of S-glutathionylation, glutathione-S-transferase Pi (GSTP), in rats. In addition, a single cocaine injection in saline withdrawn rats does not lead to a change in S-glutathionylation, suggesting that the observed changes can only be attributed to chronic cocaine exposure. Furthermore, genetic deletion or inhibition of GSTP increases cocaine sensitivity and cocaine self-administration in mice, implicating S-glutathionylation as a protective mechanism keeping neuronal plasticity in check. Taken together these data suggest an important role for protein S-glutathionylation in models of cocaine sensitization and self-administration. Known proteins that contain redox sensitive cysteines and have a regulatory role in cocaine addiction include cofilin, actin, and N-methyl-D-aspartate (NMDA) receptors. However, this small list of proteins will definitely be expanded through the application of novel PTM proteomic technologies. Given that S-glutathionylation of target proteins can result in altered protein signaling and is dysregulated after withdrawal from chronic cocaine, the proposed project has a direct relationship to the theme of the Center. This project will focus on the proteomic analysis of the NAc “glutathionome” after cocaine self-administration, withdrawal and cocaine-induced reinstatement in rats.

We will use powerful and unbiased iodoacetyl-activated Tandem Mass Tag (iodoTMT) approaches for de novo identification of candidate proteins and their S-glutathionylated modifications in the NAc. IodoTMT labeling will be used to quantitate both cysteine containing proteins and the cysteinyl modification, S-glutathionylation. For S-glutathionylation analysis we will use a sequential tagging method by which we first label all exposed cysteines with one of the irreversible iodoTMT labels. This step will be followed by reduction with the deglutathionylating enzyme, glutaredoxin, to remove GSH from modified cysteines (S-glutathionylated cysteines) and the newly exposed cysteines will be labeled with a second irreversible iodoTMT label (S-glutathionylation label). This will be followed by Lys-C/trypsin digestion and fractionation by Strong Cation Exchange prior to LC-MS/MS analysis on a Thermo Orbitrap Fusion Tribrid or Q Exactive Plus platform. The sequential labeling approach will allow us to generate quantitative data on both S-glutathionylated and non-modified cysteine-containing proteins as well as quantification of S-glutathionylated/total protein ratios.

Significance

STriatal-Enriched protein tyrosine Phosphatase 61 kDa (STEP₆₁) normally opposes synaptic strengthening by dephosphorylation of regulatory tyrosine residues on its substrates. These substrates include GluN2B, GluA2, Fyn, Pyk2 and ERK1/2 (Snyder et al., 2005; Xu et al., 2009; Zhang et al., 2008, Zhang et al., 2011; Nguyen et al., 2002; Xu et al., 2012; Venkitaramani et al., 2009; Paul et al., 2003). All these molecules have been implicated in the neuroadaption that occurs in response to drugs of abuse (Cahill et al., 2014; Puhl et al., 2015). Recent studies have suggested a role of STEP₆₁ in cocaine-seeking behaviors. Acute or long-term self-administration of cocaine in rodent models results in increased STEP₆₁ protein and decreased Tyr phosphorylation of its substrates, suggesting over-activation of STEP₆₁ upon cocaine treatment (Sun et al., 2013; Chiodi et al., 2014).

Brain-derived neurotrophic factor (BDNF) regulates synaptic strengthening and memory consolidation (Lu et al., 2008). Altered BDNF expression is implicated in drug-induced long-term neuroadaptation, which is often disrupted by psychostimulants such as cocaine (McGinty et al., 2010). Infusion of BDNF into the dorsomedial prefrontal cortex (dmPFC) immediately following a final session of cocaine self-administration blocked cocaine-induced decreases in GluN2B and ERK phosphorylation and attenuated relapse to cocaine seeking for as long as three weeks (McGinty et al., 2014). The recent findings that BDNF signaling leads to degradation of STEP₆₁ suggest that STEP₆₁ may play a role in this process (Saavedra et al., 2015; Xu et al., 2015). Understanding the molecular basis for the cross-talk between BDNF/TrkB signaling and STEP₆₁ function will hopefully lead to better therapeutic strategies in treating drug abuse and addiction.

Specific Aim 1

Determine the residues involved in BDNF-induced phosphorylation of STEP₆₁. We hypothesize that BDNF/TrkB/PLCγ signaling leads to a PKC-mediated phosphorylation of STEP₆₁, which is required for its subsequent ubiquitination and degradation. We will perform PKC phosphorylation of STEP₆₁ in vitro and determine the sites by mass spectrometry. Mutational analyses will confirm these sites in neuronal cultures.

Specific Aim 2

Determine the lysine residues involved in BDNF-induced ubiquitination of STEP₆₁. We will treat rat cortical neurons with BDNF in the presence of the proteasome inhibitor MG-132. All STEP species will be pulled down by immunoprecipitation using anti-STEP antibody. Ubiquitinated STEP₆₁ peptides will be enriched using anti-K-GG antibody after trypsin digestion. Lysine sites that are modified will be determined by mass spectrometry.

References

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Chiodi V, Mallozzi C, Ferrante A, Chen JF, Lombroso PJ, Di Stasi AM, Popoli P, Domenici MR (2014) Cocaine-induced changes of synaptic transmission in the striatum are modulated by adenosine A2A receptors and involve the tyrosine phosphatase STEP. Neuropsychopharmacology 39:569-578.

Lu Y, Christian K, Lu B (2008) BDNF: a key regulator for protein synthesis-dependent LTP and long-term memory? Neurobiol Learn Mem 89:312-323.

McGinty JF, Whitfield TW Jr, Berglind WJ (2010) Brain-derived neurotrophic factor and cocaine addiction. Brain Res 1314:183-193.

McGinty JF, Zelek-Molik A, Sun WL (2014) Cocaine self-administration causes signaling deficits in corticostriatal circuitry that are reversed by BDNF in early withdrawal. Brain Res pii: S0006-8993(14)01299-2.

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Paul S, Nairn AC, Wang P, Lombroso PJ (2003) NMDA-mediated activation of the tyrosine phosphatase STEP regulates the duration of ERK signaling. Nat Neurosci 6: 34-42.

Puhl MD, Berg AR, Bechtholt AJ, Coyle JT (2015) Availability of N-Methyl-d-Aspartate Receptor Coagonists Affects Cocaine-Induced Conditioned Place Preference and Locomotor Sensitization: Implications for Comorbid Schizophrenia and Substance Abuse. J Pharmacol Exp Ther 353:465-470.

Saavedra A, Puigdellívol M, Tyebji S, Kurup P, Xu J, Ginés S, Alberch J, Lombroso PJ, Pérez-Navarro E (2015) BDNF Induces Striatal-Enriched Protein Tyrosine Phosphatase 61 Degradation Through the Proteasome. Mol Neurobiol doi:10.1007/s12035-015-9335-7. [Epub ahead of print]

Snyder EM, Nong Y, Almeida CG, Paul S, Moran T, Choi EY, Nairn AC, Salter MW, Lombroso PJ, Gouras GK, Greengard P (2005) Regulation of NMDA receptor trafficking by amyloid-beta. Nat Neurosci 8:1051-1058.

Sun WL, Zelek-Molik A, McGinty JF (2013) Short and long access to cocaine self-administration activates tyrosine phosphatase STEP and attenuates GluN expression but differentially regulates GluA expression in the prefrontal cortex. Psychopharmacology (Berl) 229:603-613.

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Xu J, Kurup P, Bartos JA, Patriarchi T, Hell JW, Lombroso PJ (2012) Striatal-enriched protein-tyrosine phosphatase (STEP) regulates Pyk2 kinase activity. J Biol Chem 287: 20942-20956.

Xu J, Kurup P, Azkona G, Baguley TD, Saavedra A, Nairn AC, Ellman JA, Pérez-Navarro E, Lombroso PJ (2015) Down-regulation of BDNF in cell and animal models increases striatal-enriched protein tyrosine phosphatase 61 (STEP61 ) levels. J Neurochem doi:10.1111/jnc.13295. [Epub ahead of print]

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Zhang Y, Kurup P, Xu J, Anderson GM, Greengard P, Nairn AC, Lombroso PJ (2011) Reduced levels of the tyrosine phosphatase STEP block beta amyloid-mediated GluA1/GluA2 receptor internalization. J Neurochem 119:664-672.

Zhang Y, Venkitaramani DV, Gladding CM, Zhang Y, Kurup P, Molnar E, Collingridge GL, Lombroso PJ (2008) The tyrosine phosphatase STEP mediates AMPA receptor endocytosis after metabotropic glutamate receptor stimulation. J Neurosci 28:10561-10566.