Circuit-Wide Gene Network Analysis Reveals Sex-Specific Roles for Phosphodiesterase 1b in Cocaine Addiction.

Cocaine use disorder is a significant public health issue without an effective pharmacological treatment. Successful treatments are hindered in part by an incomplete understanding of the molecular mechanisms that underlie long-lasting maladaptive plasticity and addiction-like behaviors. Here, we leverage a large RNA sequencing dataset to generate gene coexpression networks across six interconnected regions of the brain's reward circuitry from mice that underwent saline or cocaine self-administration. We identify phosphodiesterase 1b (Pde1b), a Ca2+/calmodulin-dependent enzyme that increases cAMP and cGMP hydrolysis, as a central hub gene within a nucleus accumbens (NAc) gene module that was bioinformatically associated with addiction-like behavior. Chronic cocaine exposure increases Pde1b expression in NAc D2 medium spiny neurons (MSNs) in male but not female mice. Viral-mediated Pde1b overexpression in NAc reduces cocaine self-administration in female rats but increases seeking in both sexes. In female mice, overexpressing Pde1b in D1 MSNs attenuates the locomotor response to cocaine, with the opposite effect in D2 MSNs. Overexpressing Pde1b in D1/D2 MSNs had no effect on the locomotor response to cocaine in male mice. At the electrophysiological level, Pde1b overexpression reduces sEPSC frequency in D1 MSNs and regulates the excitability of NAc MSNs. Lastly, Pde1b overexpression significantly reduced the number of differentially expressed genes (DEGs) in NAc following chronic cocaine, with discordant effects on gene transcription between sexes. Together, we identify novel gene modules across the brain's reward circuitry associated with addiction-like behavior and explore the role of Pde1b in regulating the molecular, cellular, and behavioral responses to cocaine.

Cell-Type-Specific Neuroproteomics of Synapses.

In the last two decades, our knowledge of synaptic proteomes and their relationship to normal brain function and neuropsychiatric disorders has been expanding rapidly through the use of more powerful neuroproteomic approaches. However, mass spectrometry (MS)-based neuroproteomic studies of synapses still require cell-type, spatial, and temporal proteome information. With the advancement of sample preparation and MS techniques, we have just begun to identify and understand proteomes within a given cell type, subcellular compartment, and cell-type-specific synapse. Here, we review the progress and limitations of MS-based neuroproteomics of synapses in the mammalian CNS and highlight the recent applications of these approaches in studying neuropsychiatric disorders such as major depressive disorder and substance use disorders. Combining neuroproteomic findings with other omics studies can generate an in-depth, comprehensive map of synaptic proteomes and possibly identify new therapeutic targets and biomarkers for several central nervous system disorders.

Chemically targeting the redox switch in AP1 transcription factor ΔFOSB.

The AP1 transcription factor ΔFOSB, a splice variant of FOSB, accumulates in the brain in response to chronic insults such as exposure to drugs of abuse, depression, Alzheimer's disease and tardive dyskinesias, and mediates subsequent long-term neuroadaptations. ΔFOSB forms heterodimers with other AP1 transcription factors, e.g. JUND, that bind DNA under control of a putative cysteine-based redox switch. Here, we reveal the structural basis of the redox switch by determining a key missing crystal structure in a trio, the ΔFOSB/JUND bZIP domains in the reduced, DNA-free form. Screening a cysteine-focused library containing 3200 thiol-reactive compounds, we identify specific compounds that target the redox switch, validate their activity biochemically and in cell-based assays, and show that they are well tolerated in different cell lines despite their general potential to bind to cysteines covalently. A crystal structure of the ΔFOSB/JUND bZIP domains in complex with a redox-switch-targeting compound reveals a deep compound-binding pocket near the DNA-binding site. We demonstrate that ΔFOSB, and potentially other, related AP1 transcription factors, can be targeted specifically and discriminately by exploiting unique structural features such as the redox switch and the binding partner to modulate biological function despite these proteins previously being thought to be undruggable.

Specificities of Gßγ subunits for the SNARE complex before and after stimulation of α2a-adrenergic receptors.

Ligand binding to G protein­coupled receptors (GPCRs), such as the α2a-adrenergic receptor (α2aAR), results in the activation of heterotrimeric G proteins, which consist of functionally distinct Gα subunits and Gßγ dimers. α2aAR-dependent inhibition of synaptic transmission regulates functions such as spontaneous locomotor activity, anesthetic sparing, and working memory enhancement and requires the soluble NSF attachment protein receptor (SNARE) complex, a Gßγ effector. To understand how the Gßγ-SNARE complex underlies the α2aAR-dependent inhibition of synaptic transmission, we examined the specificity of Gßγ subunits for the SNARE complex in adrenergic neurons, in which auto-α2aARs respond to epinephrine released from these neurons, and nonadrenergic neurons, in which hetero-α2aARs respond to epinephrine released from other neurons. We performed a quantitative, targeted multiple reaction monitoring proteomic analysis of Gß and Gγ subunits bound to the SNARE complex in synaptosomes from mouse brains. In the absence of stimulation of auto-α2aARs, Gß1 and Gγ3 interacted with the SNARE complex. However, Gß1, Gß2, and Gγ3 were found in the complex when auto-α2aARs were activated by epinephrine. Further understanding of the specific usage of distinct Gßγ subunits in vivo may provide insights into the homeostatic regulation of synaptic transmission and the mechanisms of dysfunction that occur in neurological diseases.

In vivo locus-specific editing of the neuroepigenome.

Studies over the past several decades have identified numerous epigenetic mechanisms associated with pathological states in psychiatric and neurological disease. Until recently, studies investigating chromatin-regulatory proteins, using overexpression or knockdown approaches, did not establish causal roles for epigenetic modifications at specific genes because these techniques typically affect hundreds or thousands of genomic loci. In this Review, we describe recent efforts in using locus-specific neuroepigenome editing in vivo to, for the first time, define causal relationships between a single chromatin modification at a specific gene in a defined cell population and downstream measures at the molecular, cellular, circuit and behavioural levels. We briefly introduce three epigenome-editing platforms: zinc-finger proteins, transcriptional activator-like effectors and clustered regularly interspaced short palindromic repeats (CRISPR). We then explore the development of in vivo neuroepigenome-editing tools and their applications to resolve epigenetic contributions to the pathophysiology of brain diseases. We also discuss technical considerations for in vivo neuroepigenome-editing experiments and ongoing innovations in the field, including new tools to investigate chromatin marks, manipulate chromatin topology and induce epigenetic modifications at multiple genes in the same cell. Lastly, we explore the potential clinical applications of in vivo neuroepigenome editing for treating brain pathology.

Author Correction: The in vivo specificity of synaptic Gß and Gγ subunits to the α2a adrenergic receptor at CNS synapses.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

The expanding roles and mechanisms of G protein-mediated presynaptic inhibition.

Throughout the past five decades, tremendous advancements have been made in our understanding of G protein signaling and presynaptic inhibition, many of which were published in the Journal of Biological Chemistry under the tenure of Herb Tabor as Editor-in-Chief. Here, we identify these critical advances, including the formulation of the ternary complex model of G protein-coupled receptor signaling and the discovery of Gßγ as a critical signaling component of the heterotrimeric G protein, along with the nature of presynaptic inhibition and its physiological role. We provide an overview for the discovery and physiological relevance of the two known Gßγ-mediated mechanisms for presynaptic inhibition: first, the action of Gßγ on voltage-gated calcium channels to inhibit calcium influx to the presynaptic active zone and, second, the direct binding of Gßγ to the SNARE complex to displace synaptotagmin downstream of calcium entry, which has been demonstrated to be important in neurons and secretory cells. These two mechanisms act in tandem with each other in a synergistic manner to provide more complete spatiotemporal control over neurotransmitter release.

Disabling the Gßγ-SNARE interaction disrupts GPCR-mediated presynaptic inhibition, leading to physiological and behavioral phenotypes.

G protein-coupled receptors (GPCRs) that couple to Gi/o proteins modulate neurotransmission presynaptically by inhibiting exocytosis. Release of Gßγ subunits from activated G proteins decreases the activity of voltage-gated Ca2+ channels (VGCCs), decreasing excitability. A less understood Gßγ-mediated mechanism downstream of Ca2+ entry is the binding of Gßγ to SNARE complexes, which facilitate the fusion of vesicles with the cell plasma membrane in exocytosis. Here, we generated mice expressing a form of the SNARE protein SNAP25 with premature truncation of the C terminus and that were therefore partially deficient in this interaction. SNAP25Δ3 homozygote mice exhibited normal presynaptic inhibition by GABAB receptors, which inhibit VGCCs, but defective presynaptic inhibition by receptors that work directly on the SNARE complex, such as 5-hydroxytryptamine (serotonin) 5-HT1b receptors and adrenergic α2a receptors. Simultaneously stimulating receptors that act through both mechanisms showed synergistic inhibitory effects. SNAP25Δ3 homozygote mice had various behavioral phenotypes, including increased stress-induced hyperthermia, defective spatial learning, impaired gait, and supraspinal nociception. These data suggest that the inhibition of exocytosis by Gi/o-coupled GPCRs through the Gßγ-SNARE interaction is a crucial component of numerous physiological and behavioral processes.

The in vivo specificity of synaptic Gß and Gγ subunits to the α2a adrenergic receptor at CNS synapses.

G proteins are major transducers of signals from G-protein coupled receptors (GPCRs). They are made up of α, ß, and γ subunits, with 16 Gα, 5 Gß and 12 Gγ subunits. Though much is known about the specificity of Gα subunits, the specificity of Gßγs activated by a given GPCR and that activate each effector in vivo is not known. Here, we examined the in vivo Gßγ specificity of presynaptic α2a-adrenergic receptors (α2aARs) in both adrenergic (auto-α2aARs) and non-adrenergic neurons (hetero-α2aARs) for the first time. With a quantitative MRM proteomic analysis of neuronal Gß and Gγ subunits, and co-immunoprecipitation of tagged α2aARs from mouse models including transgenic FLAG-α2aARs and knock-in HA-α2aARs, we investigated the in vivo specificity of Gß and Gγ subunits to auto-α2aARs and hetero-α2aARs activated with epinephrine to understand the role of Gßγ specificity in diverse physiological functions such as anesthetic sparing, and working memory enhancement. We detected Gß2, Gγ2, Gγ3, and Gγ4 with activated auto α2aARs, whereas we found Gß4 and Gγ12 preferentially interacted with activated hetero-α2aARs. Further understanding of in vivo Gßγ specificity to various GPCRs offers new insights into the multiplicity of genes for Gß and Gγ, and the mechanisms underlying GPCR signaling through Gßγ subunits.

GPCR regulation of secretion.

Modulation of neurotransmitter exocytosis by activated Gi/o coupled G-protein coupled receptors (GPCRs) is a universal regulatory mechanism used both to avoid overstimulation and to influence circuitry. One of the known modulation mechanisms is the interaction between Gßγ and the soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNAREs). There are 5 Gß and 12 Gγ subunits, but specific Gßγs activated by a given GPCR and the specificity to effectors, such as SNARE, in vivo are not known. Although less studied, Gßγ binding to the exocytic fusion machinery (i.e. SNARE) provides a more direct regulatory mechanism for neurotransmitter release. Here, we review some recent insights in the architecture of the synaptic terminal, modulation of synaptic transmission, and implications of G protein modulation of synaptic transmission in diseases. Numerous presynaptic proteins are involved in the architecture of synaptic terminals, particularly the active zone, and their importance in the regulation of exocytosis is still not completely understood. Further understanding of the Gßγ-SNARE interaction and the architecture and mechanisms of exocytosis may lead to the discovery of novel therapeutic targets to help patients with various disorders such as hypertension, attention-deficit/hyperactivity disorder, post-traumatic stress disorder, and acute/chronic pain.

Quantitative Multiple-Reaction Monitoring Proteomic Analysis of Gß and Gγ Subunits in C57Bl6/J Brain Synaptosomes.

Gßγ dimers are one of the essential signaling units of activated G protein-coupled receptors (GPCRs). There are five Gß and 12 Gγ subunits in humans; numerous studies have demonstrated that different Gß and Gγ subunits selectively interact to form unique Gßγ dimers, which in turn may target specific receptors and effectors. Perturbation of Gßγ signaling can lead to impaired physiological responses. Moreover, previous targeted multiple-reaction monitoring (MRM) studies of Gß and Gγ subunits have shown distinct regional and subcellular localization patterns in four brain regions. Nevertheless, no studies have quantified or compared their individual protein levels. In this study, we have developed a quantitative MRM method not only to quantify but also to compare the protein abundance of neuronal Gß and Gγ subunits. In whole and fractionated crude synaptosomes, we were able to identify the most abundant neuronal Gß and Gγ subunits and their subcellular localizations. For example, Gß1 was mostly localized at the membrane while Gß2 was evenly distributed throughout synaptosomal fractions. The protein expression levels and subcellular localizations of Gß and Gγ subunits may affect the Gßγ dimerization and Gßγ-effector interactions. This study offers not only a new tool for quantifying and comparing Gß and Gγ subunits but also new insights into the in vivo distribution of Gß and Gγ subunits, and Gßγ dimer assembly in normal brain function.

A Presynaptic Group III mGluR Recruits Gßγ/SNARE Interactions to Inhibit Synaptic Transmission by Cone Photoreceptors in the Vertebrate Retina.

G-protein ßγ subunits (Gßγ) interact with presynaptic proteins and regulate neurotransmitter release downstream of Ca2+ influx. To accomplish their roles in sensory signaling, photoreceptor synapses use specialized presynaptic proteins that support neurotransmission at active zone structures known as ribbons. While several G-protein coupled receptors (GPCRs) influence synaptic transmission at ribbon synapses of cones and other retinal neurons, it is unknown whether Gßγ contributes to these effects. We tested whether activation of one particular GPCR, a metabotropic glutamate receptor (mGluR), can reduce cone synaptic transmission via Gßγ in tiger salamander retinas. In recordings from horizontal cells, we found that an mGluR agonist (L-AP4) reduced cone-driven light responses and mEPSC frequency. In paired recordings of cones and horizontal cells, L-AP4 slightly reduced cone ICa (∼10%) and caused a larger reduction in cone-driven EPSCs (∼30%). Proximity ligation assay revealed direct interactions between SNAP-25 and Gßγ subunits in retinal synaptic layers. Pretreatment with the SNAP-25 cleaving protease BoNT/A inhibited L-AP4 effects on synaptic transmission, as did introduction of a peptide derived from the SNAP-25 C terminus. Introducing Gßγ subunits directly into cones reduced EPSC amplitude. This effect was inhibited by BoNT/A, supporting a role for Gßγ/SNAP-25 interactions. However, the mGluR-dependent reduction in ICa was not mimicked by Gßγ, indicating that this effect was independent of Gßγ. The finding that synaptic transmission at cone ribbon synapses is regulated by Gßγ/SNAP-25 interactions indicates that these mechanisms are shared by conventional and ribbon-type synapses. Gßγ liberated from other photoreceptor GPCRs is also likely to regulate synaptic transmission.SIGNIFICANCE STATEMENT Dynamic regulation of synaptic transmission by presynaptic G-protein coupled receptors shapes information flow through neural circuits. At the first synapse in the visual system, presynaptic metabotropic glutamate receptors (mGluRs) regulate cone photoreceptor synaptic transmission, although the mechanisms and functional impact of this are unclear. We show that mGluRs regulate light response encoding across the cone synapse, accomplished in part by triggering G-protein ßγ subunits (Gßγ) interactions with SNAP-25, a core component of the synaptic vesicle fusion machinery. In addition to revealing a role in visual processing, this provides the first demonstration that Gßγ/SNAP-25 interactions regulate synaptic function at a ribbon-type synapse, contributing to an emerging picture of the ubiquity of Gßγ/SNARE interactions in regulating synaptic transmission throughout the nervous system.

Using peptide arrays created by the SPOT method for defining protein-protein interactions.

Evaluating sites of protein-protein interactions can be an arduous task involving extensive mutagenesis work and attempts to express and purify individual proteins in sufficient quantities. Peptide mapping is a useful alternative to traditional methods as it allows rapid detection of regions and/or individual residues important for binding, and it can be readily applied to numerous proteins at once. Here we describe the use of the ResPep SL SPOT method to evaluate protein-protein binding interactions such as that between G-protein ßγ subunits and SNARE proteins, identifying both regions of interest and subsequently individual residues which can then be manipulated in further biochemical assays to confirm their validity.

Gßγ inhibits exocytosis via interaction with critical residues on soluble N-ethylmaleimide-sensitive factor attachment protein-25.

Spatial and temporal regulation of neurotransmitter release is a complex process accomplished by the exocytotic machinery working in tandem with numerous regulatory proteins. G-protein ßγ dimers regulate the core process of exocytosis by interacting with the soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) proteins soluble N-ethylmaleimide-sensitive factor attachment protein-25 (SNAP-25), syntaxin 1A, and synaptobrevin. Gßγ binding to ternary SNAREs overlaps with calcium-dependent binding of synaptotagmin, inhibiting synaptotagmin-1 binding and fusion of the synaptic vesicle. To further explore the binding sites of Gßγ on SNAP-25, peptides based on the sequence of SNAP-25 were screened for Gßγ binding. Peptides that bound Gßγ were subjected to alanine scanning mutagenesis to determine their relevance to the Gßγ-SNAP-25 interaction. Peptides from this screen were tested in protein-protein interaction assays for their ability to modulate the interaction of Gßγ with SNAP-25. A peptide from the C terminus, residues 193 to 206, significantly inhibited the interaction. In addition, Ala mutants of SNAP-25 residues from the C terminus of SNAP-25, as well as from the amino-terminal region decreased binding to Gß1γ1. When SNAP-25 with eight residues mutated to alanine was assembled with syntaxin 1A, there was significantly reduced affinity of this mutated t-SNARE for Gßγ, but it still interacted with synaptotagmin-1 in a Ca²âº -dependent manner and reconstituted evoked exocytosis in botulinum neurotoxin E-treated neurons. However, the mutant SNAP-25 could no longer support 5-hydroxytryptamine-mediated inhibition of exocytosis.

Localization and quaternary structure of the PKA RIß holoenzyme.

Specificity for signaling by cAMP-dependent protein kinase (PKA) is achieved by both targeting and isoform diversity. The inactive PKA holoenzyme has two catalytic (C) subunits and a regulatory (R) subunit dimer (R(2):C(2)). Although the RIα, RIIα, and RIIß isoforms are well studied, little is known about RIß. We show here that RIß is enriched selectively in mitochondria and hypothesized that its unique biological importance and functional nonredundancy will correlate with its structure. Small-angle X-ray scattering showed that the overall shape of RIß(2):C(2) is different from its closest homolog, RIα(2):C(2). The full-length RIß(2):C(2) crystal structure allows us to visualize all the domains of the PKA holoenzyme complex and shows how isoform-specific assembly of holoenzyme complexes can create distinct quaternary structures even though the R(1):C(1) heterodimers are similar in all isoforms. The creation of discrete isoform-specific PKA holoenzyme signaling "foci" paves the way for exploring further biological roles of PKA RIß and establishes a paradigm for PKA signaling.