The adhesion-GPCR BAI3, a gene linked to psychiatric disorders, regulates dendrite morphogenesis in neurons.

Adhesion-G protein-coupled receptors (GPCRs) are a poorly studied subgroup of the GPCRs, which have diverse biological roles and are major targets for therapeutic intervention. Among them, the Brain Angiogenesis Inhibitor (BAI) family has been linked to several psychiatric disorders, but despite their very high neuronal expression, the function of these receptors in the central nervous system has barely been analyzed. Our results, obtained using expression knockdown and overexpression experiments, reveal that the BAI3 receptor controls dendritic arborization growth and branching in cultured neurons. This role is confirmed in Purkinje cells in vivo using specific expression of a deficient BAI3 protein in transgenic mice, as well as lentivirus driven knockdown of BAI3 expression. Regulation of dendrite morphogenesis by BAI3 involves activation of the RhoGTPase Rac1 and the binding to a functional ELMO1, a critical Rac1 regulator. Thus, activation of the BAI3 signaling pathway could lead to direct reorganization of the actin cytoskeleton through RhoGTPase signaling in neurons. Given the direct link between RhoGTPase/actin signaling pathways, neuronal morphogenesis and psychiatric disorders, our mechanistic data show the importance of further studying the role of the BAI adhesion-GPCRs to understand the pathophysiology of such brain diseases.

Klf9 is necessary and sufficient for Purkinje cell survival in organotypic culture.

During their phase of developmental programmed cell death (PCD), neurons depend on target-released trophic factors for survival. After this period, however, they critically change as their survival becomes target-independent. The molecular mechanisms underlying this major transition remain poorly understood. Here, we investigated, which transcription factors (TFs) might be responsible for the closure of PCD. We used Purkinje cells as a model since their PCD is restricted to the first postnatal week in the mouse cerebellum. Transcriptome analysis of Purkinje cells during or after PCD allowed the identification of Krüppel like factor 9 (Klf9) as a candidate for PCD closure, given its high increase of expression at the end of the 1st postnatal week. Klf9 function was tested in organotypic cultures, through lentiviral vector-mediated manipulation of Klf9 expression. In absence of trophic factors, the Purkinje cell survival rate is of 40%. Overexpression of Klf9 during PCD dramatically increases the Purkinje cell survival rate from 40% to 88%, whereas its down-regulation decreases it to 14%. Accordingly, in organotypic cultures of Klf9 knockout animals, Purkinje cell survival rate is reduced by half as compared to wild-type mice. Furthermore, the absence of Klf9 could be rescued by Purkinje cell trophic factors, Insulin growth factor-1 and Neurotrophin3. Altogether, our results ascribe a clear role of Klf9 in Purkinje cell survival. Thus, we propose that Klf9 might be a key molecule involved in turning off the phase of Purkinje PCD.

Placental lactogens induce serotonin biosynthesis in a subset of mouse beta cells during pregnancy.

AIMS/HYPOTHESIS: Upregulation of the functional beta cell mass is required to match the physiological demands of mother and fetus during pregnancy. This increase is dependent on placental lactogens (PLs) and prolactin receptors, but the mechanisms underlying these events are only partially understood. We studied the mRNA expression profile of mouse islets during pregnancy to gain a better insight into these changes. METHODS: RNA expression was measured ex vivo via microarrays and quantitative RT-PCR. In vivo observations were extended by in vitro models in which ovine PL was added to cultured mouse islets and MIN6 cells. RESULTS: mRNA encoding both isoforms of the rate-limiting enzyme of serotonin biosynthesis, tryptophan hydroxylase (TPH), i.e. Tph1 and Tph2, were strongly induced (fold change 25- to 200-fold) during pregnancy. This induction was mimicked by exposing islets or MIN6 cells to ovine PLs for 24 h and was dependent on janus kinase 2 and signal transducer and activator of transcription 5. Parallel to Tph1 mRNA and protein induction, islet serotonin content increased to a peak level that was 200-fold higher than basal. Interestingly, only a subpopulation of the beta cells was serotonin-positive in vitro and in vivo. The stored serotonin pool in pregnant islets and PL-treated MIN6 cells was rapidly released (turnover once every 2 h). CONCLUSIONS/INTERPRETATION: A very strong lactogen-dependent upregulation of serotonin biosynthesis occurs in a subpopulation of mouse islet beta cells during pregnancy. Since the newly formed serotonin is rapidly released, this lactogen-induced beta cell function may serve local or endocrine tasks, the nature of which remains to be identified.

Structure of a murine alpha interferon pseudogene with a repetitive R-type sequence in the 3' flanking region.

A murine alpha interferon pseudogene was identified in a mouse genomic library. The nucleotide sequence revealed several in-phase termination codons within the gene and repetitive oligonucleotides in the flanking regions. The nucleotide sequences and the amino acids of the peptide signal sequences were compared with known human alpha interferon genes and the pseudogene.

Structure and characterization of a murine chromosomal fragment containing the interferon beta gene.

In this paper we report on the cloning and characterization of the murine interferon (IFN) beta gene. We have isolated and sequenced a 2.8 kb genomic fragment containing the murine IFN beta gene flanked by 1.2 kb 5' and 1 kb 3' untranslated regions (1 kb = 10(3) base-pairs). The mRNA cap site has been defined. An extensive analysis of the flanking sequence is provided and points out striking features such as: the presence of A + T-rich motifs characteristic of transiently expressed mRNAs, and homologies to repetitive R-type element flanks and to hormone-responsive elements. Comparison of the MuIFN beta 5' flanking region with those from other species reveals similarities in the sequences required for the regulated expression of such inducible genes. Computer analysis of the 130 base-pairs preceding the cap site has revealed TGAAAG motifs and shows that the presence of such elements and their permutants have biological significance, according to statistical calculations. Thus, the comparison between the mouse promoter reported here and the promoters from other species highlights the region containing the hexanucleotide blocks, which is strongly conserved.

Isolation and characterization of a novel interferon-alpha-encoding gene, IFN-alpha 11, within a murine IFN cluster.

Two recombinant cosmids containing three complete murine interferon-alpha-encoding genes (Mu IFN-alpha) have been isolated from a mouse cosmid library. The cluster organization of these genes has been determined. A new Mu IFN-alpha gene (Mu IFN-alpha 11) has been isolated and studied with respect to its structure and inducible transcription pattern. The nucleotide and deduced amino acid sequences of the Mu IFN-alpha 11 gene, as compared to the two other IFN-alpha 7 and IFN-alpha 8 genes, show that, albeit highly homologous, these genes are all different. The transient expression of the three genes gave rise to proteins showing antiviral properties which were neutralized with murine anti-IFN-alpha antibodies. The transcription of the Mu IFN-alpha genes was studied in two uninduced or Newcastle-disease-virus-induced murine cell types. Mu IFN-alpha 11, as well as alpha 7 and alpha 8, are not expressed in L929 nor in C243 cells upon viral induction and therefore constitute an interesting model to study Mu IFN-alpha gene repression.

Gabra5-gene haplotype block associated with behavioral properties of the full agonist benzodiazepine chlordiazepoxide.

The gabra5 gene is associated with pharmacological properties (myorelaxant, amnesic, anxiolytic) of benzodiazepines. It is tightly located (0.5 cM) close to the pink-eyed dilution (p) locus which encodes for fur color on mouse chromosome 7. We tested the putative role of the gabra5 gene in pharmacological properties of the full non specific agonist chlordiazepoxide (CDP), using behavioral and molecular approaches in mutated p/p mice and wild type F2 from crosses between two multiple markers inbred strain ABP/Le and C57BL/6By strain. From our results, using rotarod, light-dark box, elevated maze and radial arm maze tests, we demonstrate that p/p mice are more sensitive than WT to the sensory motor, anxiolytic and amnesic effect of CDP. This is associated with the presence of a haplotypic block on the murine chromosome 7 and with an up regulation of gabra5 mRNAs in hippocampi of p/p F2 mice.

Sequence and expression of a novel murine interferon alpha gene--homology with enhancer elements in the regulatory region of the gene.

A murine interferon gene (MuIFN alpha) has been isolated from a cosmid library. The sequence of a 1.2-kb HindIII-PstI fragment revealed a new MuIFN alpha gene which has not yet been described and which was termed MuIFN alpha 7. The coding sequence produced biologically active IFN when expressed in monkey cells under the control of an SV40 promoter. A comparison of the MuIFN alpha 7 gene with the known interferon genes in their coding and flanking sequences shows homologies between enhancer elements found in the 5' upstream region of the coding gene. The core element common to all known viral enhancers, GTGG(AAA/TTT)G is repeated four times in the MuIFN alpha 7 5'-flanking region, as in all known MuIFN alpha genes.

Isolation and characterization of a cDNA clone encoding testis protamine Z1 from the dog-fish Scylliorhinus caniculus.

A clone containing a 445-bp cDNA insert was isolated from a cDNA library synthesized from dog-fish testes mRNA. The nucleotide sequence was determined and corresponded to a 50-amino-acid protein. The known five-amino-acid N-terminal sequence corresponded exactly to our deduced amino acid sequence. After in vitro transcription of this cDNA using SP6 RNA polymerase, the translated polypeptide comigrated with the Z1 scylliorhinine marker. Analysis of the cDNA 3' flanking region of our Scylliorhinus protamine Z1 revealed an inverted repeat sequence, an ACAA motif and a CAGGAAAGA box known as regulatory signals for transcription termination in histone genes. In addition, sequences homologous to the simian virus (SV 40) and polyoma virus core enhancer elements were identified in the 5' and 3' flanking regions.

Nucleotide sequence of a cDNA clone encoding Scylliorhinus caniculus protamine Z2.

A cDNA library was constructed from a protamine-enriched fraction of dogfish (Scylliorhinus caniculus) mRNA. The nucleotide sequence of a 440-bp insert was determined, and its produced protein sequence confirmed its identification as a cysteine-rich protamine Z2 [Martinage, A., Gusse, M., Belaiche, D., Sautiere, P. and Chevaillier, P. (1985) Biochim. Biophys. Acta 831, 172-178]. The frequency of utilization of the different triplets coding for arginine, which represents 30-70% of the total amino acid residues for trout, mouse and dogfish protamines, is discussed. An alternative repetitive sequence of CGC-AGG was found in the N terminus of the protein. Analysis of the 3' flanking region after the mRNA-terminating TAA codon identified an inverted repeat sequence and an ACCA sequence, which may be possible vestiges of a histone-like termination signal.

Repression of the murine interferon alpha 11 gene: identification of negatively acting sequences.

The uninducible murine interferon alpha 11 gene (Mu IFN-alpha 11) shows strong homology with the highly inducible Mu IFN-alpha 4 gene in the promoter region. Negative regulatory sequences located between positions -470 and -145 were characterized in the Mu IFN-alpha 11 promoter. The removal of these sequences leads to virus-inducibility of Mu IFN-alpha 11 while their insertion in Mu IFN-alpha 4 corresponding region significantly reduced the inducibility of Mu IFN-alpha 4 promoter. On the other hand, the virus-responsive element (VRE) of the Mu IFN-alpha 11 differs by a single nucleotide substitution at position -78 from the VRE alpha 4. Constructions carrying either VRE alpha 11 or VRE alpha 4 upstream a heterologous promoter displayed different virus inducibilities. The -78 A/G substitution affects the inducibility by decreasing the affinity of VRE-binding trans-regulators. Our results suggest that the combined effect of the negative regulatory sequences and of the mutation in the VRE alpha 11, completely silences the Mu IFN-alpha 11 gene.

Isolation and characterization of a functional murine interferon alpha gene which is not expressed in fibroblasts upon virus induction.

A mouse genomic segment containing three new members of the murine interferon alpha (MuIFN-alpha) gene family was isolated from a fibroblastic cosmid library. A 4 kb EcoRI fragment contained a new MuIFN-alpha gene named MuIFN-alpha 8. The nucleotide sequence of the coding and flanking regions of this gene showed a high level of homology to those of known members of the MuIFN-alpha family. Transient expression of the MuIFN-alpha 8 gene in COS cells and oocyte translation of in vitro transcripts both led to a biologically active protein. The antiviral activity was neutralized by monoclonal and polyclonal MuIFN-alpha antibodies. Although the 5' flanking sequence shows features characteristic of an IFN regulatory region, the MuIFN-alpha 8 gene is not expressed in murine fibroblasts treated with Newcastle disease virus or poly(I).poly(C).

Structure of mouse interferon-beta cDNA clones: sequence rearrangements in a cloned cDNA.

A fraction enriched in interferon (IFN) mRNA was prepared from mouse C243-3 induced cells and was used for the construction of a cDNA library. Two plasmids were obtained after screening by differential colony hybridization and IFN mRNA hybridization-selection and translation. The nucleotide sequences of the cDNA inserts revealed that both were partial copies of IFN-beta mRNA. The cDNA 861 corresponds to the entire 3' nontranslated region of the mRNA while the cDNA 2939 consists of rearranged translated regions of IFN mRNA. A mechanism for the rearrangement events during cDNA synthesis is proposed. A chromosomal DNA fragment hybridizing to cDNA 2939 was identified by screening a mouse genomic library.

Involvement of NF-Y and Sp1 in basal and cAMP-stimulated transcriptional activation of the tryptophan hydroxylase (TPH ) gene in the pineal gland.

The expression of the tryptophan hydroxylase (TPH) gene, encoding the rate-limiting enzyme of serotonin biosynthesis, is tightly regulated both at the transcriptional and at the post-transcriptional levels. In the pineal gland, transcription of the gene is activated in response to an intracellular circadian increase of the cAMP concentration. We have previously shown that transcription of a 2.1-kb fragment of the human TPH promoter is induced by cAMP, although it lacks the canonical cAMP responsive element, CRE. The minimal promoter (-73/+29) has only weak transcriptional activity but is responsive to cAMP. It contains an inverted CCAAT box, which was demonstrated to be involved in this response. Here, we have extended our investigation to the functional features of the inverted CCAAT box in the -252/+29 TPH promoter, which has a higher basal activity. We show that an additional cis -acting sequence, the adjacent GC-rich region, cooperates with the inverted CCAAT box for the full activation of basal transcription, and that both elements are essential for the full cAMP response. We also show that in pinealocytes, NF-Y and Sp1 transactivators bind the inverted CCAAT box and GC-rich-region, respectively. These factors participate in a novel pathway for the cAMP-mediated response of the TPH promoter, which is independent of the canonical CRE-mediated response.

Identification of distal silencing elements in the murine interferon-A11 gene promoter.

The murine interferon-A11 (Mu IFN-A11) gene is a member of the IFN-A multigenic family. In mouse L929 cells, the weak response of the gene's promoter to viral induction is due to a combination of both a point mutation in the virus responsive element (VRE) and the presence of negatively regulating sequences surrounding the VRE. In the distal part of the promoter, the negatively acting E1E2 sequence was delimited. This sequence displays an inhibitory effect in either orientation or position on the inducibility of a virus-responsive heterologous promoter. It selectively represses VRE-dependent transcription but is not able to reduce the transcriptional activity of a VRE-lacking promoter. In a transient transfection assay, an E1E2-containing DNA competitor was able to derepress the native Mu IFN-A11 promoter. Specific nuclear factors bind to this sequence; thus the binding of trans-regulators participates in the repression of the Mu IFN-A11 gene. The E1E2 sequence contains an IFN regulatory factor (IRF)-binding site. Recombinant IRF2 binds this sequence and anti-IRF2 antibodies supershift a major complex formed with nuclear extracts. The protein composing the complex is 50 kDa in size, indicating the presence of IRF2 or antigenically related proteins in the complex. The Mu IFN-A11 gene is the first example within the murine IFN-A family, in which a distal promoter element has been identified that can negatively modulate the transcriptional response to viral induction.

Is RE1/NRSE a common cis-regulatory sequence for ChAT and VAChT genes?

Choline acetyltransferase (ChAT), the biosynthetic enzyme of acetylcholine, and the vesicular acetylcholine transporter (VAChT) are both required for cholinergic neurotransmission. These proteins are encoded by two embedded genes, the VAChT gene lying within the first intron of the ChAT gene. In the nervous system, both ChAT and VAChT are synthesized only in cholinergic neurons, and it is therefore likely that the cell type-specific expression of their genes is coordinately regulated. It has been suggested that a 2336-base pair genomic region upstream from the ChAT and VAChT coding sequences drives ChAT gene expression in cholinergic structures. We investigated whether this region also regulates VAChT gene transcription. Transfection assays showed that this region strongly represses the activity of the native VAChT promoters in non-neuronal cells, but has no major effect in neuronal cells whether or not they express the endogenous ChAT and VAChT genes. The silencer activity of this region is mediated solely by a repressor element 1 or neuron-restrictive silencer element (RE1/NRSE). Moreover, several proteins, including RE1-silencing transcription factor or neuron-restrictive silencer factor, are recruited by this regulatory sequence. These data suggest that this upstream region and RE1/NRSE co-regulate the expression of the ChAT and VAChT genes.