Region-specific alterations of A-to-I RNA editing of serotonin 2c receptor in the cortex of suicides with major depression.

Brain region-specific abnormalities in serotonergic transmission appear to underlie suicidal behavior. Alterations of RNA editing on the serotonin receptor 2C (HTR2C) pre-mRNA in the brain of suicides produce transcripts that attenuate 5-HT2CR signaling by impairing intracellular G-protein coupling and subsequent intracellular signal transduction. In brain, the distribution of RNA-editing enzymes catalyzing deamination (A-to-I modification) shows regional variation, including within the cerebral cortex. We tested the hypothesis that altered pre-mRNA 5-HT2CR receptor editing in suicide is region-specific. To this end, we investigated the complete 5-HT2CR mRNA-editing profile in two architectonically distinct cortical areas involved in mood regulation and decision-making in a clinically well-characterized cohort of age- and sex-matched non-psychiatric drug-free controls and depressed suicides. By using an original biochemical detection method, that is, capillary electrophoresis single-stranded conformational polymorphism (CE-SSCP), we corroborated the 5-HT2CR mRNA-editing profile previously described in the dorsolateral prefrontal cortex (Brodmann area 9 (BA9)). Editing of 5-HT2CR mRNA displayed clear regional difference when comparing dorsolateral prefrontal cortex (BA9) and anterior cingulate cortex (BA24). Compared with non-psychiatric control individuals, alterations of editing levels of 5-HT2CR mRNA were detected in both cortical areas of depressed suicides. A marked increase in editing on 5-HT2CR was especially observed in the anterior cingulate cortex in suicides, implicating this cortical area in suicide risk. The results suggest that region-specific changes in RNA editing of 5-HT2CR mRNA and deficient receptor function likely contribute to the etiology of major depressive disorder or suicide.

PP2A-Bgamma subunit and KCNQ2 K+ channels in bipolar disorder.

Many bipolar affective disorder (BD) susceptibility loci have been identified but the molecular mechanisms responsible for the disease remain to be elucidated. In the locus 4p16, several candidate genes were identified but none of them was definitively shown to be associated with BD. In this region, the PPP2R2C gene encodes the Bgamma-regulatory subunit of the protein phosphatase 2A (PP2A-Bgamma). First, we identified, in two different populations, single nucleotide polymorphisms and risk haplotypes for this gene that are associated to BD. Then, we used the Bgamma subunit as bait to screen a human brain cDNA library with the yeast two-hybrid technique. This led us to two new splice variants of KCNQ2 channels and to the KCNQ2 channel itself. This unusual K+ channel has particularly interesting functional properties and belongs to a channel family that is already known to be implicated in several other monogenic diseases. In one of the BD populations, we also found a genetic association between the KCNQ2 gene and BD. We show that KCNQ2 splice variants differ from native channels by their shortened C-terminal sequences and are unique as they are active and exert a dominant-negative effect on KCNQ2 wild-type (wt) channel activity. We also show that the PP2A-Bgamma subunit significantly increases the current generated by KCNQ2wt, a channel normally inhibited by phosphorylation. The kinase glycogen synthase kinase 3 beta (GSK3beta) is considered as an interesting target of lithium, the classical drug used in BD. GSK3beta phosphorylates the KCNQ2 channel and this phosphorylation is decreased by Li+.

Molecular cloning and characterization of a transcription factor for the copia retrotransposon with homology to the BTB-containing lola neurogenic factor.

By transfection experiments, we previously identified a 72-bp enhancer sequence within the Drosophila copia retrotransposon which is involved in the control of the transcription level of this mobile element in cells in culture. Gel shift assays with nuclear extracts from Drosophila hydei-derived DH-33 cells further demonstrated specific interactions of at least two nuclear factors with this enhancer sequence. Using this sequence as a probe for the screening of an expression cDNA library that we constructed from DH-33 cells RNA, we have isolated a cDNA clone encoding a 110-kDa protein with features common to those of known transcription factors; these include a two-zinc-finger motif at the C terminus, three glutamine-rich domains in the presumptive activation domain of the protein, and an N-terminal domain which shares homology with the Bric-à-brac, Tramtrack, and Broad-Complex BTB boxes. The precise DNA recognition sequence for this transcription factor has been determined by both gel shift assays and footprinting experiments with a recombinant protein made in bacteria. The functionality of the cloned element was demonstrated upon transcriptional activation of copia reporter genes, as well as of a minimal promoter coupled with the identified target DNA sequence, in cotransfection assays in cells in culture with an expression vector for the cloned factor. Southern blot and nucleotide sequence analyses revealed a related gene in Drosophila melanogaster (the lola gene) previously identified by a genetic approach as involved in axon growth and guidance. Transfection assays in cells in culture with lola gene expression vectors and in situ hybridization experiments with lola gene mutants finally provided evidence that the copia retrotransposon is regulated by this neurogenic gene in D.melanogaster, with a repressor effect in the central nervous systems of the embryos.

Defective I elements introduced into Drosophila as transgenes can regulate reactivity and prevent I-R hybrid dysgenesis.

The I-R hybrid dysgenesis syndrome is characterized by a high level of sterility and I element transposition, occurring in the female offspring of crosses between males of inducer (I) strains, which contain full-length transposable I elements, and females of reactive (R) strains, devoid of functional I elements. The intensity of the syndrome in the dysgenic cross is essentially dependent on the reactivity level of the R females, which is ultimately controlled by still unresolved polygenic chromosomal determinants. In the work reported here, we have introduced a transposition-defective I element with a 2.6 kb deletion within its second open reading frame into a highly reactive R strain, by P-mediated transgenesis. We demonstrate that this defective I element gradually alters the level of reactivity in the three independent transgenic lines that were obtained, over several generations. After > 15 generations, the transgenic Drosophila show strongly reduced reactivity, and finally become refractory to hybrid dysgenesis, without, however, acquiring the inducer phenotype. Induction of a low reactivity level is reversible--reactivity again increases upon transgene removal--and is maternally inherited, as observed for the control of reactivity in natural R strains. These results demonstrate that defective I elements introduced as single-copy transgenes can act as regulators of reactivity, and suggest that some of the ancestral defective pericentromeric I elements that can be found in all reactive strains could be the molecular determinants of reactivity.

Identification of a strong transcriptional activator for the copia retrotransposon responsible for its differential expression in Drosophila hydei and melanogaster cell lines.

We have characterized the regulatory properties of a 72bp sequence located in the 5' untranslated domain of the Drosophila copia retrotransposon, 3' to the left LTR, by transient transfection assays with cell lines derived from either Drosophila hydei (DH33 cells) or Drosophila melanogaster (Schneider II and Kc cells). Reporter plasmids were constructed which contained the lacZ gene under the control of either the entire copia LTR with 5' untranslated domain, or a minimal heterologous promoter flanked with the identified copia regulatory sequences. Upon transfection into the copia-free DH33 cells, the presence of the 72bp sequence resulted for all reporter plasmids in a 100-700 fold increase in expression level -as well as in reporter gene RNA levels- whereas this sequence had no enhancing effect upon transfection of the same plasmids into the copia-containing Schneider II or Kc cells. Moreover, mobility shift assays with the 72bp enhancer sequence disclosed two specific bands of retarded mobility with whole-cell extracts from DH33 cells, whereas no retarded band could be detected, under identical conditions, with extracts from Schneider II cells. UV crosslinking experiments between the enhancer sequence and DH33 extracts revealed a single protein species -of app. mol. wt. 50kD- for both retarded bands, thus strongly suggesting that they simply correspond to the sequential binding of two identical factor molecules to the enhancer sequence. These data demonstrate that the copia-free D. hydei cells express a strong transcriptional activator for the copia element and possible interpretations for the absence of this factor in the copia-containing D. melanogaster cells are discussed in terms of a possible "adaptation" of the "host" (D. melanogaster) to an otherwise highly mutagenic "parasite" (copia with its transcription factor).

Retrotransposition of a marked Drosophila line-like I element in cells in culture.

We have marked a Drosophila transposable element--the LINE-like I element--with an intron-containing indicator gene inserted in place of a large deletion in the I element second ORF encompassing the reverse transcriptase domain, and this marked element was placed downstream to a potent actin promoter. An expression vector for the I element ORFs was also constructed, under the same heterologous promoter. The indicator gene contains a lacZ reporter gene the expression of which is conditioned by retrotransposition of the marked element, thus allowing detection of transposition events by testing for either beta-galactosidase expression or occurrence of spliced DNA molecules. The marked I element was introduced into Drosophila melanogaster cells in culture by transfection. Spliced DNA copies of the marked element and specifically stained beta-galactosidase-expressing cells were detected only upon co-transfection with the I expression vector, thus indicating that an ORF2-deleted element can be complemented in trans for transposition. This simple assay for retrotransposition in Drosophila cells in culture provides a tool for the rapid analysis of the mechanism of I transposition in its cis and trans sequence requirements.

The Drosophila copia retrotransposon contains binding sites for transcriptional regulation by homeoproteins.

We have identified in the 5' untranslated region of the Drosophila copia retrotransposon, 3' to the left LTR, a sequence for transcriptional regulation by homeoproteins. Co-transfection assays using expression vectors for homeoproteins and reporter vectors containing the lacZ gene under the control of either the entire copia LTR with 5' untranslated sequence, or a minimal heterologous promoter flanked with a 130 bp fragment containing the copia untranslated region, disclosed both positive and negative modulations of promoter activity in Drosophila cells in culture: a 5-10 fold decrease with engrailed, even-skipped and zerknüllt in DH33 cells, and a 10-30 fold increase with fushi tarazu and zerknüllt in Schneider II cells. In all cases, the regulatory effects were abolished with reporter plasmids deleted for a 58 bp fragment encompassing the putative homeoprotein binding sites. Mobility shift assays with a purified homeodomain-containing peptide demonstrated direct interaction with the 58 bp fragment, with an affinity in the 1-10 nM range as reported with the same peptide for other well characterized homeodomain binding regulatory sites. Foot-printing experiments with the extended LTR demonstrated protection of 'consensus' sequences, located within the 58 bp fragment. These homeodomain binding sites could be involved in the developmental regulation of the copia retrotransposon.

Target lysis by human LAK cells is critically dependent upon target binding properties, but LFA-1, LFA-3 and ICAM-1 are not the major adhesion ligands on targets.

The cytotoxicity mediated by the CD2+ CD3- lymphocyte subset, either NK or LAK, is puzzling since no specific antigen recognition structures, equivalent to the CD3-associated heterodimer T-cell receptor, have been recognized on these cells so far. The possibility exists that the CD3- cytotoxic effectors recognize their targets through non-specific adhesion mechanisms. The goal of this study was: (a) to examine the correlation between binding properties and susceptibility to lysis of 6 informative target cell lines; (b) to evaluate the role, as ligands on these targets, of adhesion molecules such as LFA-1, LFA-3 and ICAM-1. The effectors used in this study were IL-2-activated LGL, predominantly CD3-, or highly purified CD3- lymphocytes from normal human donors. The 6 target lines studied included 2 pairs of EBV-transformed B-cell lines (721 LCL vs. 721.134, and MM vs. MM-10F2) in which the parental lines were resistant to lysis while HLA variants were susceptible. A third pair was the Daudi Burkitt cell line, susceptible to LAK lysis, and an HLA-positive transfected Daudi line which was more resistant to lysis. The binding properties of these targets to LAK effectors (conjugate formation) were evaluated using a sensitive double fluorescence flow cytometry method. In each pair examined, the susceptible targets formed more conjugates and were surrounded by more cytotoxic LAK effectors than their resistant counterparts, indicating that the conjugation properties of targets are closely correlated with their susceptibility to LAK lysis. The expression of adhesion molecules on the informative targets was examined by indirect immunofluorescence and their role was evaluated by inhibition of lysis after pre-coating the targets with the relevant antibodies. The differences in the expression of the classical cell-cell adhesion molecules LFA-1, LFA-3 and ICAM-1 on the target surfaces were only marginal, insufficient to explain the striking differences in susceptibility to lysis and in binding properties. Coating the target cells with antibodies directed against these adhesion determinants had no effects on the lysis of susceptible target cells. The same antibodies reacting with the LAK effectors did inhibit lysis. Taken together, these results suggest that, on the targets, presently undefined membrane adhesion structures may have a major role in conjugate formation between target and CD3- effectors and determine the susceptibility of the targets to lysis.

An improved double fluorescence flow cytometry method for the quantification of killer cell/target cell conjugate formation.

We have developed an improved method to analyse stable associations (conjugate formation) between effector and target cells. Hydroethidine (red) stained lymphoblastoid target cells were cocentrifuged with carboxyfluorescein diacetate acetoxymethylester (green) stained human IL-2 activated cytotoxic cells (LAK). In the present studies either enriched or purified CD3 negative large granular lymphocytes (LGL) were used as cytotoxic cells. These fluorescent vital dyes localize intracellularly and therefore do not modify the cell to cell contact which eventually leads to the lytic events. Both dyes can be excited at a common wavelength (488 nm) using a single argon laser. Effectors firmly bound to target(s) (stable conjugates) were detected as two color fluorescent events (red and green). This method has several features: (a) the number of conjugates is recorded with reference to a fixed number of target cells; (b) the composition of conjugates (number of effectors or targets per conjugate) can be studied by analysis of the fluorescence intensities (red or green); (c) conjugate formation can be studied at E:T ratios comparable to those used in the classical 51Cr release cytotoxic assay; (d) it gives reproducible results and permits the study of very weak differences in binding properties. This method was used to study conjugate formation between human IL-2-activated cytotoxic cells (or purified CD3 negative LGL) and various lymphoblastoid target cells. We were able to demonstrate that cell lines susceptible to lysis formed more conjugates and were surrounded by more LAK effectors than their resistant counterparts and that no conjugate contained more than one target.