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In vitro culture media are being developed to understand how host site-specific nutrient profiles influence microbial pathogenicity and ecology. To mimic the cystic fibrosis (CF) lung environment, a variety of artificial sputum media (ASM) have been created. However, the composition of these ASM vary in the concentration of key nutrients, including amino acids, lipids, DNA, and mucin. In this work, we used feature-based molecular networking (FBMN) to perform comparative metabolomics of Pseudomonas aeruginosa, the predominant opportunistic pathogen infecting the lungs of people with CF, cultured in nine different ASM. We found that the concentration of aromatic amino acids and iron from mucin added to the media contributes to differences in the production of P. aeruginosa virulence-associated secondary metabolites. IMPORTANCE Different media formulations aiming to replicate in vivo infection environments contain different nutrients, which affects interpretation of experimental results. Inclusion of undefined components, such as commercial porcine gastric mucin (PGM), in an otherwise chemically defined medium can alter the nutrient content of the medium in unexpected ways and influence experimental outcomes.

Author(s): Petras, Daniel; Phelan, Vanessa V; Acharya, Deepa; Allen, Andrew E; Aron, Allegra T; Bandeira, Nuno; Bowen, Benjamin P; Belle-Oudry, Deirdre; Boecker, Simon; Cummings, Dale A; Deutsch, Jessica M; Fahy, Eoin; Garg, Neha; Gregor, Rachel; Handelsman, Jo; Navarro-Hoyos, Mirtha; Jarmusch, Alan K; Jarmusch, Scott A; Louie, Katherine; Maloney, Katherine N; Marty, Michael T; Meijler, Michael M; Mizrahi, Itzhak; Neve, Rachel L; Northen, Trent R; Molina-Santiago, Carlos; Panitchpakdi, Morgan; Pullman, Benjamin; Puri, Aaron W; Schmid, Robin; Subramaniam, Shankar; Thukral, Monica; Vasquez-Castro, Felipe; Dorrestein, Pieter C; Wang, Mingxun | Abstract: AbstractAccess to web-based platforms has enabled scientists to perform research remotely. A critical aspect of mass spectrometry data analysis is the inspection, analysis, and visualization of the raw data to validate data quality and confirm statistical observations. We developed the GNPS Dashboard, a web-based data visualization tool, to facilitate synchronous collaborative inspection, visualization, and analysis of private and public mass spectrometry data remotely.

Site-specific in vitro culture media are being developed to investigate microbial pathogenicity and ecology in nutrient environments that are more reflective of disease. For microbial ecology research in cystic fibrosis (CF), different artificial sputum media (ASM) formulations have been created to recapitulate the nutrient availability of the CF lung environment. However, these ASM formulations vary in concentration of amino acids, mucin, and other niche-specific compounds. Here, we measured the differential production of small molecule virulence factors by Pseudomonas aeruginosa, the predominant pathogen in CF pulmonary infections, cultured in nine different ASM formulations via liquid chromatography tandem mass spectrometry (LC-MS/MS) and molecular networking. We show that different ASM formulations lead to different phenotypes and metabolic profiles of P. aeruginosa and commercial porcine gastric mucin (PGM) contains a myriad of contaminants, including iron, which affect P. aeruginosa physiology.IMPORTANCEDifferent media formulations aiming to replicate in vivo infection environments contain different nutrients, which affects interpretation of experimental results. Inclusion of undefined components, such as commercial porcine gastric mucin (PGM), in an otherwise chemically defined medium can alter the nutrient content of the medium in unexpected ways and influence experimental outcomes.

Neuronal alternative splicing is a key gene regulatory mechanism in the brain. However, the spliceosome machinery is insufficient to fully specify splicing complexity. In considering the role of the epigenome in activity-dependent alternative splicing, we and others find the histone modification H3K36me3 to be a putative splicing regulator. In this study, we found that mouse cocaine self-administration caused widespread differential alternative splicing, concomitant with the enrichment of H3K36me3 at differentially spliced junctions. Importantly, only targeted epigenetic editing can distinguish between a direct role of H3K36me3 in splicing and an indirect role via regulation of splice factor expression elsewhere on the genome. We targeted Srsf11, which was both alternatively spliced and H3K36me3 enriched in the brain following cocaine self-administration. Epigenetic editing of H3K36me3 at Srsf11 was sufficient to drive its alternative splicing and enhanced cocaine self-administration, establishing the direct causal relevance of H3K36me3 to alternative splicing of Srsf11 and to reward behavior.

Trauma can cause dysfunctional fear regulation leading some to develop disorders like post-traumatic stress disorder (PTSD). The amygdala regulates fear, and, PACAP and PAC1 receptors are linked to PTSD symptom severity at genetic/epigenetic levels, with a strong link in females with PTSD. We discovered a PACAPergic projection from the basomedial amygdala (BMA) to the medial intercalated cells (mICCs). In vivo optogenetic stimulation of this pathway increased cfos expression in mICCs, decreased fear retention and increased fear extinction. Selective deletion of PAC1 receptors from the mICCs in females reduced fear acquisition, but enhanced fear generalization and reduced fear extinction in males. Optogenetic stimulation of the BMA-mICCs PACAPergic pathway produced excitatory postsynaptic currents (EPSCs) in mICCs neurons, which was enhanced by PAC1 receptor antagonist, PACAP 6-38. Our findings show that mICCs modulate contextual fear in a dynamic and sex-dependent manner via the microcircuit containing the BMA and mICCs, dependent on behavioral state.

Mechanisms by which epigenetic modifications regulate alternative splicing are largely unexplored. Differential alternative splicing and histone modification enrichment are key mechanisms for neuronal gene regulation. Both are grossly altered in mouse brain following investigator-administered cocaine. Our group and others have identified the histone modification, H3K36me3, as a putative splicing regulator. In the current study, we found that mouse cocaine self-administration caused widespread differential alternative splicing, concomitant with enrichment of H3K36me3 at differentially spliced junctions. The splice factor motif for Srsf11 was enriched in splice junctions of cocaine induced alternative exons, yet Srsf11 expression was unchanged by cocaine treatment. Rather, Srsf11 was both differentially spliced and enriched in H3K36me3. Epigenetic editing showed that H3K36me3 functions directly in alternative splicing of Srsf11. Finally, both Srsf11-targeted and global H3K36me3 enrichment enhanced cocaine self-administration. These findings established the direct causal relevance of H3K36me3 to alternative splicing of Srsf11 and to reward behavior.

Alternative splicing is a key mechanism for neuronal gene regulation, and is grossly altered in mouse brain reward regions following investigator-administered cocaine. It is well established that cocaine epigenetically regulates transcription, yet mechanism(s) by which cocaine-induced epigenetic modifications regulate alternative splicing is largely unexplored. Our group and others have previously identified the histone modification, H3K36me3, as a putative splicing regulator. However, it has not yet been possible to establish the direct causal relevance of this modification to alternative splicing in brain or any other context. We found that mouse cocaine self-administration caused widespread alternative splicing, concomitant with enrichment of H3K36me3 at splice junctions. Differentially spliced genes were enriched in the motif for splice factor, Srsf11, which was both differentially spliced and enriched in H3K36me3. Epigenetic editing led us to conclude that H3K36me3 functions directly in alternative splicing of Srsf11, and that Set2 mediated H3K36me3 bidirectionally regulates cocaine intake.

Proteasomal-regulated chromatin remodeler mediates cocaine relapse during abstinence., Neuroadaptations in the nucleus accumbens (NAc) underlie cue-induced cocaine craving that intensifies (“incubates”) during abstinence and is believed to contribute to persistent relapse vulnerability. Changes in gene expression often govern perpetual behavioral abnormalities, but epigenetic plasticity during prolonged abstinence from drug exposure is poorly understood. We examined how E3 ubiquitin ligase TRIM3 dysregulates chromatin remodeler INO80 to mediate cocaine craving during prolonged abstinence. We found that INO80 expression increased in the NAc on abstinence day 30 (AD30) but not on AD1 following cocaine self-administration. Furthermore, TRIM3, which mediates degradation of INO80, was reduced on AD30, along with TRIM3-INO80 interaction. Viral-mediated gene transfer of INO80 or TRIM3 governed cocaine craving during prolonged abstinence. Lastly, chromatin immunoprecipitation followed by massively parallel DNA sequencing identified INO80-mediated transcriptional regulation of predicted pathways associated with cocaine plasticity. Together, these results demonstrate a novel ubiquitin-proteasomal-epigenetic mechanism by which TRIM3-INO80 mediates cocaine craving during prolonged abstinence.

Chronic stress is a key risk factor for mood disorders like depression, but the stress-induced changes in brain circuit function and gene expression underlying depression symptoms are not completely understood, hindering development of novel treatments. Because of its projections to brain regions regulating reward and anxiety, the ventral hippocampus is uniquely poised to translate the experience of stress into altered brain function and pathological mood, though the cellular and molecular mechanisms of this process are not fully understood. Here, we use a novel method of circuit-specific gene editing to show that the transcription factor ΔFosB drives projection-specific activity of ventral hippocampus glutamatergic neurons causing behaviorally diverse responses to stress. We establish molecular, cellular, and circuit-level mechanisms for depression- and anxiety-like behavior in response to stress and use circuit-specific gene expression profiling to uncover novel downstream targets as potential sites of therapeutic intervention in depression., Chronic stress is a risk factor for mood disorders, yet the molecular and circuit mechanisms of stress-induced changes are not well understood. Here, the authors report the role of the transcription factor ΔFosB in driving activity changes in response to stress in glutamatergic neurons in the ventral hippocampus that project to nucleus accumben.

We have examined catalytic oxidations of non-benzylic alcohols under solvent-free conditions with the goal of discovering greener alternatives to current synthetic protocols for these transformations. An iron(III) complex bearing a bicyclic pentadentate 14,28-[1,3-diiminoisoindolinato]phthalocyaninato (diiPc) ligand and an axial water ligand has been shown to function as a very effective catalyst in oxidation reactions of three secondary aliphatic alcohols (2-pentanol, 2,4-dimethyl-3-pentanol and cyclohexanol), one primary aliphatic alcohol (1-pentanol), and one bifunctional allyl alcohol (5-hydroxymethylfurfural) with tert-butylhydroperoxide (TBHP) in the absence of added organic solvent. All reactions proceed with high turnover numbers and turnover frequency relative to related systems. Selectivity for the desired aldehyde and ketone products is excellent. The presence of only water as the monodentate axial ligand in the catalyst provides solubility in non-aromatic substrates, in contrast to diiPc complexes of FeIII where the axial monodentate ligand is a mixture of methanol and water strongly favoring methanol.

SummaryStress resilience involves numerous brain-wide transcriptional changes. Determining the organization and orchestration of these transcriptional events may reveal novel antidepressant targets, but this remains unexplored. Here, we characterize the resilient transcriptome with co-expression analysis and identify a single transcriptionally-active uniquely-resilient gene network. Zfp189, a previously unstudied zinc finger protein, is the top network key driver and its overexpression in prefrontal cortical (PFC) neurons preferentially activates this network, alters neuronal activity and promotes behavioral resilience. CREB, which binds Zfp189, is the top upstream regulator of this network. To probe CREB-Zfp189 interactions as a network regulatory mechanism, we employ CRISPR-mediated locus-specific transcriptional reprogramming to direct CREB selectively to the Zfp189 promoter. This single molecular interaction in PFC neurons recapitulates the pro-resilient Zfp189-dependent downstream effects on gene network activity, electrophysiology and behavior. These findings reveal an essential role for Zfp189 and a CREB-Zfp189 regulatory axis in mediating a central transcriptional network of resilience.

Wnt proteins have emerged as transmembrane signaling molecules that regulate learning and memory as well as synaptic plasticity at central synapses (Inestrosa and Arenas (2010) Nat Rev Neurosci 11:77-86; Maguschak and Ressler (2011) J Neurosci 31:13057-13067; Tabatadze et al. (2012) Hippocampus 22: 1228-1241; Fortress et al. (2013) J Neurosci 33:12619-12626). For example, there is both a training-selective and Wnt isoform-specific increase in Wnt 7 levels in hippocampus seven days after spatial learning in rats (Tabatadze et al. (2012) Hippocampus 22: 1228-1241). Despite growing interest in Wnt signaling pathways in the adult brain, intracellular distribution and release of Wnt molecules from synaptic compartments as well as their influence on synaptic strength and connectivity remain less well understood. As a first step in such an analysis, we show here that Wnt 7 levels in primary hippocampal cells are elevated by potassium or glutamate activation in a time-dependent manner. Subsequent Wnt 7 elevation in dendrites suggests selective somato-dendritic trafficking followed by transport from dendrites to their spines. Wnt 7 elevation is also TTX-reversible, establishing that its elevation is indeed an activity-dependent process. A second stimulation given 6 h after the first significantly reduces Wnt 7 levels in dendrites 3 h later as compared to non-stimulated controls suggesting activity-dependent Wnt 7 release from dendrites and spines. In a related experiment designed to mimic the release of Wnt 7, exogenous recombinant Wnt 7 increased the number of active zones in presynaptic terminals as indexed by bassoon. This suggests the formation of new presynaptic release sites and/or presynaptic terminals. Wnt signaling inhibitor sFRP-1 completely blocked this Wnt 7-induced elevation of bassoon cluster number and cluster area. We suggest that Wnt 7 is a plasticity-related protein involved in the regulation of presynaptic plasticity via a retrograde signaling mechanism as previously proposed (Routtenberg (1999) Trends in Neuroscience 22:255-256). These findings provide support for this proposal, which offers a new perspective on the synaptic tagging mechanism (Redondo and Morris (2011) Nat Rev Neurosci 12:17-30).

Depression and anxiety disorders are more prevalent in females, but the majority of research in animal models, the first step in finding new treatments, has focused predominantly on males. Here we report that exposure to subchronic variable stress (SCVS) induces depression-associated behaviors in female mice, whereas males are resilient as they do not develop these behavioral abnormalities. In concert with these different behavioral responses, transcriptional analysis of nucleus accumbens (NAc), a major brain reward region, by use of RNA sequencing (RNA-seq) revealed markedly different patterns of stress regulation of gene expression between the sexes. Among the genes displaying sex differences was DNA methyltransferase 3a (Dnmt3a), which shows a greater induction in females after SCVS. Interestingly, Dnmt3a expression levels were increased in the NAc of depressed humans, an effect seen in both males and females. Local overexpression of Dnmt3a in NAc rendered male mice more susceptible to SCVS, whereasDnmt3aknock-out in this region rendered females more resilient, directly implicating this gene in stress responses. Associated with this enhanced resilience of female mice upon NAc knock-out ofDnmt3awas a partial shift of the NAc female transcriptome toward the male pattern after SCVS. These data indicate that males and females undergo different patterns of transcriptional regulation in response to stress and that a DNA methyltransferase in NAc contributes to sex differences in stress vulnerability.SIGNIFICANCE STATEMENTWomen have a higher incidence of depression than men. However, preclinical models, the first step in developing new diagnostics and therapeutics, have been performed mainly on male subjects. Using a stress-based animal model of depression that causes behavioral effects in females but not males, we demonstrate a sex-specific transcriptional profile in brain reward circuitry. This transcriptional profile can be altered by removal of an epigenetic mechanism, which normally suppresses DNA transcription, creating a hybrid male/female transcriptional pattern. Removal of this epigenetic mechanism also induces behavioral resilience to stress in females. These findings shed new light onto molecular factors controlling sex differences in stress response.

Alterations in motivation have been implicated in the pathophysiology of several psychiatric disorders, including substance abuse and depression. Repeated exposure to drugs of abuse or stress is known to persistently induce the transcription factor deltaFosB in the nucleus accumbens (NAc) and dorsal striatum, effects hypothesized to contribute to neuroadaptations in dopamine-regulated signaling. Little is known, however, about the specific involvement of deltaFosB in dysregulation of appetitively motivated behaviors. We show here that inducible overexpression of deltaFosB in NAc and dorsal striatum of bitransgenic mice, or specifically in the NAc core of rats by use of viral-mediated gene transfer, enhanced food-reinforced instrumental performance and progressive ratio responding. Very similar behavioral effects were found after previous repeated exposure to cocaine, amphetamine, MDMA [(+)-3,4-methylenedioxymethamphetamine], or nicotine in rats. These results reveal the powerful regulation of motivational processes by deltaFosB, and provide evidence that drug-induced alterations in gene expression via induction of deltaFosB within the NAc core may play a critical role in the impact of motivational influences on instrumental behavior.

To better understand the molecular mechanisms of depression and antidepressant action, we administered chronic social defeat stress followed by chronic imipramine (a tricyclic antidepressant) to mice and studied adaptations at the levels of gene expression and chromatin remodeling of five brain-derived neurotrophic factor (Bdnf) splice variant mRNAs (I-V) and their unique promoters in the hippocampus. Defeat stress induced lasting downregulation of Bdnf transcripts III and IV and robustly increased repressive histone methylation at their corresponding promoters. Chronic imipramine reversed this downregulation and increased histone acetylation at these promoters. This hyperacetylation by chronic imipramine was associated with a selective downregulation of histone deacetylase (Hdac) 5. Furthermore, viral-mediated HDAC5 overexpression in the hippocampus blocked imipramine's ability to reverse depression-like behavior. These experiments underscore an important role for histone remodeling in the pathophysiology and treatment of depression and highlight the therapeutic potential for histone methylation and deacetylation inhibitors in depression.

The inhibitory activity associated with myelin is a major obstacle for successful axon regeneration in the adult mammalian central nervous system (CNS)1,2. In addition to myelin-associated glycoprotein (MAG)3,4 and Nogo-A5,6,7, available evidence suggests the existence of additional inhibitors in CNS myelin8. We show here that a glycosylphosphatidylinositol (GPI)-anchored CNS myelin protein, oligodendrocyte-myelin glycoprotein (OMgp), is a potent inhibitor of neurite outgrowth in cultured neurons. Like Nogo-A, OMgp contributes significantly to the inhibitory activity associated with CNS myelin. To further elucidate the mechanisms that mediate this inhibitory activity of OMgp, we screened an expression library and identified the Nogo receptor (NgR)9 as a high-affinity OMgp-binding protein. Cleavage of NgR and other GPI-linked proteins from the cell surface renders axons of dorsal root ganglia insensitive to OMgp. Introduction of exogenous NgR confers OMgp responsiveness to otherwise insensitive neurons. Thus, OMgp is an important inhibitor of neurite outgrowth that acts through NgR and its associated receptor complex. Interfering with the OMgp/NgR pathway may allow lesioned axons to regenerate after injury in vivo.

The oxidation of four benzylic alcohols employing hydrogen peroxide and TBHP as oxidants, catalyzed by an iron(III) complex bearing a 14,28-[1,3-diiminoisoindolinato]phthalocyaninato (diiPc) ligand has been studied and found to proceed with good selectivity, high turnover numbers, and high turnover frequencies in the absence of organic solvents other than the substrates themselves.

Eph receptors transduce short-range repulsive signals for axon guidance by modulating actin dynamics within growth cones. We report the cloning and characterization of ephexin, a novel Eph receptor-interacting protein that is a member of the Dbl family of guanine nucleotide exchange factors (GEFs) for Rho GTPases. Ephrin-A stimulation of EphA receptors modulates the activity of ephexin leading to RhoA activation, Cdc42 and Rac1 inhibition, and cell morphology changes. In addition, expression of a mutant form of ephexin in primary neurons interferes with ephrin-A-induced growth cone collapse. The association of ephexin with Eph receptors constitutes a molecular link between Eph receptors and the actin cytoskeleton and provides a novel mechanism for achieving highly localized regulation of growth cone motility.

Wnt proteins have emerged as transmembrane signaling molecules that regulate learning and memory as well as synaptic plasticity at central synapses (Inestrosa and Arenas (2010) Nat Rev Neurosci 11:77-86; Maguschak and Ressler (2011) J Neurosci 31:13057-13067; Tabatadze et al. (2012) Hippocampus 22: 1228-1241; Fortress et al. (2013) J Neurosci 33:12619-12626). For example, there is both a training-selective and Wnt isoform-specific increase in Wnt 7 levels in hippocampus seven days after spatial learning in rats (Tabatadze et al. (2012) Hippocampus 22: 1228-1241). Despite growing interest in Wnt signaling pathways in the adult brain, intracellular distribution and release of Wnt molecules from synaptic compartments as well as their influence on synaptic strength and connectivity remain less well understood. As a first step in such an analysis, we show here that Wnt 7 levels in primary hippocampal cells are elevated by potassium or glutamate activation in a time-dependent manner. Subsequent Wnt 7 elevation in dendrites suggests selective somato-dendritic trafficking followed by transport from dendrites to their spines. Wnt 7 elevation is also TTX-reversible, establishing that its elevation is indeed an activity-dependent process. A second stimulation given 6 h after the first significantly reduces Wnt 7 levels in dendrites 3 h later as compared to non-stimulated controls suggesting activity-dependent Wnt 7 release from dendrites and spines. In a related experiment designed to mimic the release of Wnt 7, exogenous recombinant Wnt 7 increased the number of active zones in presynaptic terminals as indexed by bassoon. This suggests the formation of new presynaptic release sites and/or presynaptic terminals. Wnt signaling inhibitor sFRP-1 completely blocked this Wnt 7-induced elevation of bassoon cluster number and cluster area. We suggest that Wnt 7 is a plasticity-related protein involved in the regulation of presynaptic plasticity via a retrograde signaling mechanism as previously proposed (Routtenberg (1999) Trends in Neuroscience 22:255-256). These findings provide support for this proposal, which offers a new perspective on the synaptic tagging mechanism (Redondo and Morris (2011) Nat Rev Neurosci 12:17-30).

Glutamatergic systems, including α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid receptors (AMPARs) are involved in opiate-induced neuronal and behavioral plasticity, although the mechanisms underlying these effects are not fully understood. In the present study, we investigated the effects of repeated morphine administration on AMPAR expression, synaptic plasticity, and context-dependent behavioral sensitization to morphine. We found that morphine treatment produced changes of synaptic AMPAR expression in the hippocampus, a brain area that is critically involved in learning and memory. These changes could be observed one week after the treatment, but only when mice developed context-dependent behavioral sensitization to morphine in which morphine treatment was associated with drug administration environment. Context-dependent behavioral sensitization to morphine was also associated with increased basal synaptic transmission and disrupted hippocampal long-term potentiation (LTP), whereas these effects were less robust when morphine administration was not paired with the drug administration environment. Interestingly, some effects may be related to the prior history of morphine exposure in the drug-associated environment, since alterations of AMPAR expression, basal synaptic transmission, and LTP were observed in mice that received a saline challenge one week after discontinuation of morphine treatment. Furthermore, we demonstrated that phosphorylation of GluA1 AMPAR subunit plays a critical role in the acquisition and expression of context-dependent behavioral sensitization, as this behavior is blocked by a viral vector that disrupts GluA1 phosphorylation. These data provide evidence that glutamatergic signaling in the hippocampus plays an important role in context-dependent sensitization to morphine and supports further investigation of glutamate-based strategies for treating opiate addiction.

Publisher Summary Glutamate or its analogs, such as homocysteate (HCA), induce nonreceptor-mediated toxicity in immature cortical neurons. Nonreceptor-mediated toxicity dominates, as functional glutamate receptors do not develop for up to a week after plating neurons from E17 rat embyros. Specifically, glutamate inhibits plasma membrane cystine transport, leading to depletion of glutathione and oxidative stress-induced cell death. The model has been used to probe mechanisms of oxidative death in neurons by a number of laboratories. Several laboratories have used glutamate-induced oxidative death as a model to elucidate the precise mechanisms by which oxidative stress can impact neuronal viability. The advantages of this model are numerous: (1) it involves primary postmitotic neurons, (2) at least 100–150 million cells can be obtained from each culture, facilitating biochemical and cell biological measurements, (3) the cells are harvested from brain at a stage in development when the cultures are predominantly neuronal (>85%), (4) oxidative stress is induced by the depletion of an endogenous antioxidant rather than the addition of high and possibly nonphysiological concentrations of an oxidant, and (5) after addition of glutamate, primary neurons do not commit to die until 8–12 hour later (depending on the antioxidant used), permitting primary oxidative events to be easily separated from those that occur as a consequence of cell death.