Project Details

Addiction is a national epidemic. Focusing only on animal models of self-administration and signaling cascades that mediate addiction-related behavior may not provide us with complete solutions. A better understanding of maladaptations underlying addiction may be reached by considering intersystemic biology in the context of brain circuitry function. Toward this goal we have been exploring how gastrointestinal immune state affects brain reward/aversion circuitry. In collaboration with the Yale/NIDA Neuroproteomics Center we have derived signaling libraries from male and female mouse cohorts with chemically-induced bowel inflammation, from which potential targets are now being screened with circuitry and neuron type-specific analysis. We are also conducting neuron type-specific polyribosomal transcriptomics that will allow multi-omic systems level integration to better understand the relationship between GI and reward circuitry state. These studies combined with assessment of effects on addiction behavior may provide new clinically relevant insights. 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 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 recently, we had worked primarily on LTPs that act at ER-plasma membrane contact sites, including the Extended-Synaptotagmins and TMEM24, a protein selectively enriched in neurons. More recently we have started to work on LTPs that function at contact between the ER and either mitochondria or endosomes/lysosomes, such as VPS13 family proteins and PDZD8. VPS13 family proteins (4 in mammals) are of special interest, 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. A collaboration with the laboratory of Karin Reinisch (a structural biologist, Yale Department of Cell Biology) has suggested that VPS13 family protein transfer lipids by a completely novel mechanisms (tunneling lipid from one membrane to another), making these studies of special interest.

In addition to studies of lipid dynamics, we are also continuing studies of membrane traffic at synapses. In this area we currently focus on the role of phase separation mechanisms in the assembly of membranous organelles. The recently developed concept that macromolecules, such as proteins and RNAs, can self-assemble within the cytoplasm into distinct liquid phases is having a major impact in cell biology. We have found that even membranous organelles can self-organize into liquid phase (phases in which the organelles are clustered, yet motile within the clusters) and we are investigating the role of these mechanisms in the generation and dynamics of synaptic vesicle clusters at synapses.

Opiates are the most widely used analgesics for the treatment of pain; however, prolonged treatment with opioids leads to the development of tolerance. Genetic and pharmacological inhibition of PKC in rodents demonstrated that PKC is a key player in inducing morphine tolerance. Mu opioid receptors (MORs) contain more than 15 serine, threonine, and tyrosine residues that are accessible to protein kinases. We intend to characterize differential phosphorylation of MORs and other PKC substrates upon activation by morphine versus fentanyl. In addition, we intend to identify PKC targets leading to opioid tolerance using a combination of novel tools and modern proteomics approaches. Previous studies have suggested that protein kinase C (PKC) is a key player in inducing morphine tolerance and addiction. In order to investigate the dynamics of PKC activity in native tissue we generated antibodies to several substrates of PKC and used these in combination with conformational-sensitive antibodies that detect time- and ligand-mediated activation of native PKC and explored the dynamics of PKC activity. In cells treated with morphine or fentanyl, we find that PKC activation induced by morphine is rapid and transient whereas the activation by fentanyl is slow and sustained. In animals with morphine administration we detect robust activation of PKC in the striatum. Using the Center’s Discovery Proteomics Core we intend to identify the PKC phosphorylated sites on mu opioid receptor activated by morphine/fentanyl.

In a related project we find that prolyl endopeptidase-like enzyme (PREPL) plays an important role in opioid receptor endocytosis. Specifically, in cells with the knock-down of PREPL protein, we find a decrease in the rate and extent of ligand-induced receptor internalization. A critical question in the field has been the mechanism by which PREPL mediates this function. Towards this end, we would like to characterize the substrates of PREPL. We are currently evaluating PREPL’s enzyme activity and will use the Center’s expertise to characterize the substrates of PREPL.

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 Anissa Bara 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. 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 addition. 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 complete our studies on the interactions of KNa1.1 with several signaling molecules that 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 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 Core to perform proteomic profiling of synaptically expressed receptor populations using cell-surface biotinylation combined with quantitative mass spectrometry.

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 analysis 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.

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 that can treat a number of pregnancy-related maladies. We are interested in how chronic use of THC (an active component of cannabis) and CBD affect the developing brain from prenatal days to adolescence.

Benzodiazepines are potent anxiolytic, anticonvulsant, hypnotic and sedative agents that are among the most widely used drugs to treat both psychiatric and neurological disorders. However, their long-term use results in tolerance and addiction. Accordingly, benzodiazepines are amongst the most highly abused class of prescription drugs. Benzodiazepines exert their behavioral effects via allosteric potentiation of ?-aminobutyric acid (GABA) type A receptors, the principal mediators of synaptic and tonic inhibition in the brain. GABA type A receptors are also the molecular targets for barbiturates, another widely abused class of hypnotic and sedative drugs, further highlighting their roles in drug addiction.

Structurally GABA type A receptors are heteropentamers that can be assembled from 7 subunit classes; a1-6, ß1-3, ?1-3, d, e, ?, and p-subunits, providing the molecular basis for extensive receptor structural heterogeneity throughout the brain. Consensus opinion suggests that synaptic benzodiazepine GABA type A receptor subtypes are arranged from a1-3, ß1-3 and ?2 subunits, and the activation of subtypes containing a1 and/or a2 subunits play essential roles in mediating their rewarding effects on behavior. In this proposal, we will assess if persistent exposure of mice to benzodiazepines induces sustained effects on the composition of these distinct receptor subtypes, their association with components of the inhibitory proteome, or modifies their phosphorylation. To perform these experiments, we will make use of recently developed knock-in mice in which distinct fluorescent proteins and reporter epitopes have been inserted between amino acids 4 and 5 within the N-terminus of the mature a1 and a2 subunits; mKate-a1 and pHluorin-a2, respectively. Importantly these modifications are functionally silent and do not compromise the formation of inhibitory synapses, neuronal architecture, or animal behavior (Nakamura et al., 2016). However, these additions facilitate the isolation of native multiple protein complexes containing individual GABA type A receptors subtypes. They will be used here in combination with quantitative proteomics to assess if benzodiazepine exposure modifies the composition of a1 and a2 subunit-containing GABA type A receptors in addition to their phosphorylation status.

Since joining the center in 2019 we have made progress on deciphering the structurally heterogeneity of GABA_AR subunits in the brain as described in two recent publications (Hines et al., 2018; Nathanson et al., 2019).

Literature Cited

Hines, R.M., Maric, H.M., Hines, D.J., Modgil, A., Panzenelli, P., Nakamura, Y., Nathanson, A., Cross, A., Brandon, N., Davies, P., Fritschy, J.M., Schindelin, H., and Moss S.J. (2018). Decreasing the interaction of the GABAAR a2 subunit with collybistin results in spontaneous seizures and premature death. Nat Comm 9(1):3130.

Nakamura, Y., Morrow, D.H., Modgil, A., Huyghe, D., Deeb, T.Z., Lumb, M.J., Davies, P.A. and Moss, S.J. (2016). Proteomic characterization of inhibitory synapses using a novel pHluorin-tagged GABA type A receptor a2 subunit knockin mouse. J. Biol. Chem. 291(23):12394-407.

Nathanson, A.J., Zhang, Y., Smalley, J.S. Thomas A. Ollerhead, O.A., Rodriguez Santos, M.A. Peter M. Andrews, P.M., Bope, C., Brandon, N.J., Hines, R.M., Davies, P.A. and Moss S.J. (2019). Identification of a core amino acid motif within the a subunit of GABAARs that promotes inhibitory synaptogenesis and resilience to seizures. Cell Reports 28(3):670-681.e8. doi: 10.1016/j.celrep.2019

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: Over the last funding period we focused on identifying the high affinity nicotinic acetylcholine receptor (b2*-nAChR)-associated proteome, the phosphorylation sites on the a4 and b2 nAChR subunits, and the effects of nicotine on both. In pilot studies, we also started to evaluate sex differences in the proteome of mouse brain areas important for behaviors related to nicotine addiction, including the ventral tegmental area (VTA), nucleus accumbens (NAc) and the basolateral amygdala (BLA), as well as the effects of either locomotor stimulating or rewarding regimens of nicotine administration in vivo. These pilot studies have shown that we can use 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 nicotine-dependent differences in the VTA proteome to be identified with high reliability and sensitivity. Our proposal going forward will be to establish the baseline and nicotine-dependent proteome in male and female mice of 2 different genetic backgrounds with opposite locomotor responses to acute nicotine challenge (C57BL/6, decreased and C3H, increased) to identify baseline sex differences in the VTA, NAc and BLA that are robust across mouse strains and that differ depending on sensitivity to acute nicotine challenge. We will also evaluate the effects of chronic nicotine exposure, a regimen that leads to nicotine dependence, b2*-nAChR upregulation and locomotor activation in mice, or a subchronic regimen that leads to nicotine conditioned place preference, on the sex-dependent proteome in these structures.

Our preliminary data implicate pathways associated with dopamine signaling and protein kinase A (PKA) signaling in the VTA with nicotine-dependent effects in male, but not female, mice. A number of different pathways are emerging as different between male and female VTA. These analyses will be carried forward in the next funding period.

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.

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

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

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

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

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

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

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