Preclinical and clinical evidence on the approach-avoidance conflict evaluation as an integrative tool for psychopathology.

The approach-avoidance conflict (AAC), i.e. the competing tendencies to undertake goal-directed actions or to withdraw from everyday life challenges, stands at the basis of humans' existence defining behavioural and personality domains. Gray's Reinforcement Sensitivity Theory posits that a stable bias toward approach or avoidance represents a psychopathological trait associated with excessive sensitivity to reward or punishment. Optogenetic studies in rodents and imaging studies in humans associated with cross-species AAC paradigms granted new emphasis to the hippocampus as a hub of behavioural inhibition. For instance, recent functional neuroimaging studies show that functional brain activity in the human hippocampus correlates with threat perception and seems to underlie passive avoidance. Therefore, our commentary aims to (i) discuss the inhibitory role of the hippocampus in approach-related behaviours and (ii) promote the integration of functional neuroimaging with cross-species AAC paradigms as a means of diagnostic, therapeutic, follow up and prognosis refinement in psychiatric populations.

LSD1 is an environmental stress-sensitive negative modulator of the glutamatergic synapse.

Along with neuronal mechanisms devoted to memory consolidation -including long term potentiation of synaptic strength as prominent electrophysiological correlate, and inherent dendritic spines stabilization as structural counterpart- negative control of memory formation and synaptic plasticity has been described at the molecular and behavioral level. Within this work, we report a role for the epigenetic corepressor Lysine Specific Demethylase 1 (LSD1) as a negative neuroplastic factor whose stress-enhanced activity may participate in coping with adverse experiences. Constitutively increasing LSD1 activity via knocking out its dominant negative splicing isoform neuroLSD1 (neuroLSD1KO mice), we observed extensive structural, functional and behavioral signs of excitatory decay, including disrupted memory consolidation. A similar LSD1 increase, obtained with acute antisense oligonucleotide-mediated neuroLSD1 splicing knock down in primary neuronal cultures, dampens spontaneous glutamatergic transmission, reducing mEPSCs. Remarkably, LSD1 physiological increase occurs in response to psychosocial stress-induced glutamatergic signaling. Since this mechanism entails neuroLSD1 splicing downregulation, we conclude that LSD1/neuroLSD1 ratio modulation in the hippocampus is instrumental to a negative homeostatic feedback, restraining glutamatergic neuroplasticity in response to glutamate. The active process of forgetting provides memories with salience. With our work, we propose that softening memory traces of adversities could further represent a stress-coping process in which LSD1/neuroLSD1 ratio modulation may help preserving healthy emotional references.

Identification and Characterization of a Phase-Variable Element That Regulates the Autotransporter UpaE in Uropathogenic Escherichia coli.

Uropathogenic Escherichia coli (UPEC) is the most common etiologic agent of uncomplicated urinary tract infection (UTI). An important mechanism of gene regulation in UPEC is phase variation that involves inversion of a promoter-containing DNA element via enzymatic activity of tyrosine recombinases, resulting in biphasic, ON or OFF expression of target genes. The UPEC reference strain CFT073 has five tyrosine site-specific recombinases that function at two previously characterized promoter inversion systems, fimS and hyxS Three of the five recombinases are located proximally to their cognate target elements, which is typical of promoter inversion systems. The genes for the other two recombinases, IpuA and IpuB, are located distal from these sites. Here, we identified and characterized a third phase-variable invertible element in CFT073, ipuS, located proximal to ipuA and ipuB The inversion of ipuS is catalyzed by four of the five CFT073 recombinases. Orientation of the element drives transcription of a two-gene operon containing ipuR, a predicted LuxR-type regulator, and upaE, a predicted autotransporter. We show that the predicted autotransporter UpaE is surface located and facilitates biofilm formation as well as adhesion to extracellular matrix proteins in a K-12 recombinant background. Consistent with this phenotype, the ipuS ON condition in CFT073 results in defective swimming motility, increased adherence to human kidney epithelial cells, and a positive competitive kidney colonization advantage in experimental mouse UTIs. Overall, the identification of a third phase switch in UPEC that is regulated by a shared set of recombinases describes a complex phase-variable virulence network in UPEC.IMPORTANCE Uropathogenic Escherichia coli (UPEC) is the most common cause of urinary tract infection (UTI). ON versus OFF phase switching by inversion of small DNA elements at two chromosome sites in UPEC regulates the expression of important virulence factors, including the type 1 fimbria adhesion organelle. In this report, we describe a third invertible element, ipuS, in the UPEC reference strain CFT073. The inversion of ipuS controls the phase-variable expression of upaE, an autotransporter gene that encodes a surface protein involved in adherence to extracellular matrix proteins and colonization of the kidneys in a murine model of UTI.

Isolation of generalized transducing bacteriophages for uropathogenic strains of Escherichia coli.

The traditional genetic procedure for random or site-specific mutagenesis in Escherichia coli K-12 involves mutagenesis, isolation of mutants, and transduction of the mutation into a clean genetic background. The transduction step reduces the likelihood of complications due to secondary mutations. Though well established, this protocol is not tenable for many pathogenic E. coli strains, such as uropathogenic strain CFT073, because it is resistant to known K-12 transducing bacteriophages, such as P1. CFT073 mutants generated via a technique such as lambda Red mutagenesis may contain unknown secondary mutations. Here we describe the isolation and characterization of transducing bacteriophages for CFT073. Seventy-seven phage isolates were acquired from effluent water samples collected from a wastewater treatment plant in Madison, WI. The phages were differentiated by a host sensitivity-typing scheme with a panel of E. coli strains from the ECOR collection and clinical uropathogenic isolates. We found 49 unique phage isolates. These were then examined for their ability to transduce antibiotic resistance gene insertions at multiple loci between different mutant strains of CFT073. We identified 4 different phages capable of CFT073 generalized transduction. These phages also plaque on the model uropathogenic E. coli strains 536, UTI89, and NU14. The highest-efficiency transducing phage, ΦEB49, was further characterized by DNA sequence analysis, revealing a double-stranded genome 47,180 bp in length and showing similarity to other sequenced phages. When combined with a technique like lambda Red mutagenesis, the newly characterized transducing phages provide a significant development in the genetic tools available for the study of uropathogenic E. coli.

Regulation of neuronal traits by a novel transcriptional complex.

The transcriptional repressor, REST, helps restrict neuronal traits to neurons by blocking their expression in nonneuronal cells. To examine the repercussions of REST expression in neurons, we generated a neuronal cell line that expresses REST conditionally. REST expression inhibited differentiation by nerve growth factor, suppressing both sodium current and neurite growth. A novel corepressor complex, CoREST/HDAC2, was shown to be required for REST repression. In the presence of REST, the CoREST/HDAC2 complex occupied the native Nav1.2 sodium channel gene in chromatin. In neuronal cells that lack REST and express sodium channels, the corepressor complex was not present on the gene. Collectively, these studies define a novel HDAC complex that is recruited by the C-terminal repressor domain of REST to actively repress genes essential to the neuronal phenotype.

Neuronal and extraneuronal expression and regulation of the human alpha5 nicotinic receptor subunit gene.

The mRNA encoding the human alpha5 nicotinic subunit was detected in several structures of the nervous system but appeared to be mainly expressed in cerebellum, thalamus, and the autonomic ganglia. For the first time, the alpha5 transcript was also detected in several non-neuronal tissues, with maximal expressions being found throughout the gastrointestinal tract, thymus, and testis. Many other extraneuronal sites expressed alpha5, but there were also nonexpressing organs, such as the liver, spleen, and kidney. To understand the transcriptional mechanisms controlling such a diversified expression of alpha5 in neuronal and nonneuronal cells, we isolated the 5'-regulatory region of the human gene and characterized its properties. Here we identify the alpha5 core promoter and demonstrate that the DNA regions surrounding it contain elements (with positive or negative activities) that work in a tissue-specific fashion. In particular, the segment specifying the 5'-untranslated region in neuronal cells has most of the properties of an enhancer because it activates a heterologous promoter in a position- and orientation-independent fashion. We therefore conclude that the expression of alpha5 relies on a highly complex promoter that uses distinct regulatory elements to comply with the different functional and developmental requirements of the various tissues and organs.

Transcriptional regulation of the human alpha5 nicotinic receptor subunit gene in neuronal and non-neuronal tissues.

The human alpha5 nicotinic receptor subunit gene appears to be expressed in several structures of the nervous system, but also in a number of non-neuronal tissues, with maximal expressions occurring in the entire gastrointestinal tract, thymus and testis. To understand whether specific transcriptional mechanisms are involved in the tissue-specific expression of the alpha5 subunit in neuronal and non-neuronal cells, we isolated the 5'-regulatory region of the human gene and characterized its functional properties. We demonstrate that specific DNA elements, with positive or negative activities depending on the cell type, are responsible for the diversified expression of the alpha5 subunit in different tissues. We therefore conclude that the expression of the alpha5 subunit relies on a highly complex promoter that uses distinct regulatory elements to comply with the different functional and developmental requirements of the various tissues and organs.

The co-repressor mSin3A is a functional component of the REST-CoREST repressor complex.

The repressor REST/NRSF restricts expression of a large set of genes to neurons by suppressing their expression in non-neural tissues. We find that REST repression involves two distinct repressor proteins. One of these, CoREST, interacts with the COOH-terminal repressor domain of REST (Andres, M. E., Burger, C., Peral-Rubio, M. J., Battaglioli, E., Anderson, M. E., Grimes, J., Dallmanm J., Ballas, N. , and Mandel, G. (1999) Proc. Natl. Acad. Sci. U. S. A. 96, 9873-9878). Here we show that the co-repressor mSin3A also interacts with REST. The REST-mSin3A association involves the NH(2)-terminal repressor domain of REST and the paired amphipathic helix 2 domain of mSin3A. REST forms complexes with endogenous mSin3A in mammalian cells, and both mSin3A and CoREST interact with REST in intact mammalian cells. REST repression is blocked in yeast lacking Sin3 and rescued in its presence. In mammalian cells, repression by REST is reduced when binding to mSin3A is inhibited. In mouse embryos, the distribution of mSin3A and REST transcripts is largely coincident. The pattern of CoREST gene expression is more restricted, suggesting that mSin3A is required constitutively for REST repression, whereas CoREST is recruited for more specialized repressor functions.

CoREST: a functional corepressor required for regulation of neural-specific gene expression.

Several genes encoding proteins critical to the neuronal phenotype, such as the brain type II sodium channel gene, are expressed to high levels only in neurons. This cell specificity is due, in part, to long-term repression in nonneural cells mediated by the repressor protein REST/NRSF (RE1 silencing transcription factor/neural-restrictive silencing factor). We show here that CoREST, a newly identified human protein, functions as a corepressor for REST. A single zinc finger motif in REST is required for CoREST interaction. Mutations of the motif that disrupt binding also abrogate repression. When fused to a Gal4 DNA-binding domain, CoREST functions as a repressor. CoREST is present in cell lines that express REST, and the proteins are found in the same immunocomplex. CoREST contains two SANT (SW13/ADA2/NCoR/TFIIIB B) domains, a structural feature of the nuclear receptor and silencing mediator for retinoid and thyroid human receptors (SMRT)-extended corepressors that mediate inducible repression by steroid hormone receptors. Together, REST and CoREST mediate repression of the type II sodium channel promoter in nonneural cells, and the REST/CoREST complex may mediate long-term repression essential to maintenance of cell identity.

Expression and transcriptional regulation of the human alpha3 neuronal nicotinic receptor subunit in T lymphocyte cell lines.

The expression of neurotransmitter receptors on the surface of immunocompetent cells is generally accepted as evidence that the nervous system can influence immune responses, even though many aspects of these interactions remain to be elucidated. In this article, we analyzed the expression of the alpha3 nicotinic receptor subunit in human cell lines of myeloid and lymphoid origin and show that the alpha3 mRNA and the receptor molecules containing this subunit are specifically expressed in T lymphocyte cell lines. We have previously characterized the structural properties of the human alpha3 nicotinic subunit gene promoter and defined its functional profile in neuronal cells; in this study, we analyzed the activity of the alpha3 promoter in T lymphocytes and found that the same minimal promoter located in the 0.16-kb BglII-AccIII fragment is responsible for the expression of the alpha3 mRNA in both neuronal and T lymphocyte cell lines. However, the alpha3 transcription initiation patterns in the two cell types were both qualitatively and quantitatively different, and the minimal promoter was differentially modulated by downstream and upstream regulatory elements. These findings suggest that distinct transcriptional mechanisms allow the same promoter to be regulated in a tissue-specific fashion, according to the different functional needs of the two cell types.

Structural and functional characterization of the human alpha3 nicotinic subunit gene promoter.

We describe the structural and functional features of the human alpha3 nicotinic receptor subunit promoter. A 0.35-kb region immediately upstream of the start codon was identified that when transfected in human neuroblastoma cells was able to drive the expression of the luciferase reporter gene with a strength comparable to that of the well-characterized simian virus 40 promoter/enhancer. This region displayed the features of a multistart-site, GC-rich, TATA-less, and CAAT-less promoter, containing many overlapping Sp1 and AP-2 putative binding sites. Further dissections of the 0.35-kb fragment revealed that its 3' region, specifying the 5' UT of the mRNA, plays a relevant positive effect in determining the strength of the promoter. This region contains putative cis-acting elements for AP-2, nuclear factor-kappaB, and the recently described multiple-start site element downstream-1. By mutation analysis, we showed that these sites are functional and when combined increase the promoter activity by 4-fold. The 0.35-kb promoter was found to be under the negative control of upstream sequences that include a modern Alu repeat. The alpha3 Alu repeat works as a composite region, containing both positive and negative elements that control the activity of the downstream promoter. Finally, we investigated the tissue-specific activity of the human alpha3 gene 5' regulatory sequences, showing that they are able to drive the expression of the reporter gene preferentially in neuronal cells.