Drosophila modifier screens to identify novel neuropsychiatric drugs including aminergic agents for the possible treatment of Parkinson's disease and depression.

Small molecules that increase the presynaptic function of aminergic cells may provide neuroprotection in Parkinson's disease (PD) as well as treatments for attention deficit hyperactivity disorder (ADHD) and depression. Model genetic organisms such as Drosophila melanogaster may enhance the detection of new drugs via modifier or 'enhancer/suppressor' screens, but this technique has not been applied to processes relevant to psychiatry. To identify new aminergic drugs in vivo, we used a mutation in the Drosophila vesicular monoamine transporter (dVMAT) as a sensitized genetic background and performed a suppressor screen. We fed dVMAT mutant larvae ∼ 1000 known drugs and quantitated rescue (suppression) of an amine-dependent locomotor deficit in the larva. To determine which drugs might specifically potentiate neurotransmitter release, we performed an additional secondary screen for drugs that require presynaptic amine storage to rescue larval locomotion. Using additional larval locomotion and adult fertility assays, we validated that at least one compound previously used clinically as an antineoplastic agent potentiates the presynaptic function of aminergic circuits. We suggest that structurally similar agents might be used to development treatments for PD, depression and ADHD, and that modifier screens in Drosophila provide a new strategy to screen for neuropsychiatric drugs. More generally, our findings demonstrate the power of physiologically based screens for identifying bioactive agents for select neurotransmitter systems.

Two invariant tryptophans on the alpha1 subunit define domains necessary for GABA(A) receptor assembly.

Two invariant tryptophan residues on the N-terminal extracellular region of the rat alpha1 subunit, Trp-69 and Trp-94, are critical for the assembly of the GABA(A) (gamma-aminobutyric acid, type A) receptor into a pentamer. These tryptophans are common not only to all GABA(A) receptor subunits, but also to all ligand-gated ion channel subunits. Converting each Trp residue to Phe and Gly by site-directed mutagenesis allowed us to study the role of these invariant tryptophan residues. Mutant alpha1 subunits, coexpressed with beta2 subunits in baculovirus-infected Sf9 cells, displayed high affinity binding to [(3)H]muscimol, a GABA site ligand, but no binding to [(35)S]t-butyl bicyclophosphorothionate, a ligand for the receptor-associated ion channel. Neither [(3)H]muscimol binding to intact cells nor immunostaining of nonpermeabilized cells gave evidence of surface expression of the receptor. When expressed with beta2 and gamma2 polypeptides, the mutant alpha1 polypeptides did not form [(3)H]flunitrazepam binding sites though wild-type alpha1 polypeptides did. The distribution of the mutant receptors on sucrose gradients suggests that the effects on ligand binding result from the inability of the mutant alpha1 subunits to form pentamers. We conclude that Trp-69 and Trp-94 participate in the formation of the interface between alpha and beta subunits, but not of the GABA binding site.

Cyclic AMP decreases the expression of a neuronal marker (GAD67) and increases the expression of an astroglial marker (GFAP) in C6 cells.

C6 cells express proteins and mRNAs that are characteristic of both glia and neurons. Agents that increase intracellular levels of cyclic AMP (cAMP) decrease the enzymatic activity of glutamate decarboxylase (GAD), a neuronal marker, and the mRNA levels for one of the two GAD isoenzymes, GAD67. This reduction is accompanied by increased levels of glial fibrillary acidic protein (GFAP) mRNA, an astrocyte marker. Transient transfection assays, in which a 2-kb upstream regulatory region of the human GFAP gene was linked to a reporter gene, indicate that at least some of the cAMP-mediated increase of GFAP mRNA levels is due to increased transcription. Increases in intracellular cAMP appear to induce differentiation of C6 cells toward a more mature astrocyte phenotype.

Molecular organization of the human CD3 gene family on chromosome 11q23.

The genes encoding three invariant components of the human T-cell antigen receptor, the CD3 delta, gamma, and epsilon chains, are located on human chromosome 11 at band q23. We isolated cosmid clones containing the human CD3 delta and gamma chain genes in vectors designed for rapid and efficient chromosome "walking". The human CD3 epsilon gene was located in the region immediately downstream of the CD3 delta and gamma genes using synthetic oligonucleotide probes and the localization of this gene confirmed by DNA sequencing. Detailed restriction mapping of the CD3 locus demonstrated that all three CD3 subunits are encoded within 60 kb of DNA with the CD3 epsilon gene located 26 kb downstream of the CD3 delta and gamma genes. Analysis of genomic DNA on pulsed field gels using probes isolated from these cosmid clones defined a physical map of 750 kb spanning the CD3 locus on human chromosome 11q23. The CD3 genes thus comprise a multigene family encoding cell surface components important for transmembrane signaling on T lymphocytes. The arrangement of these genes suggest that they may share common regulatory elements for the control of gene expression during T-cell ontogeny.

The mouse Thy-1.2 glycoprotein gene: complete sequence and identification of an unusual promoter.

Recombinant bacteriophage and cosmid clones containing the gene for the mouse Thy-1.2 glycoprotein were isolated and characterized. The complete sequence of the gene was determined, including a previously unidentified exon located 2.1 kb upstream of the portion of the gene encoding the Thy-1.2 glycoprotein. The transcriptional initiation site was located by S1 nuclease protection mapping in both T lymphocytes and neural cells and was found to be located immediately upstream of this exon. S1 nuclease protection mapping was also used to define the 3' end of the Thy-1.2 transcription unit, and no evidence for alternate mRNA processing was found. Thus, the mouse Thy-1.2 gene is 5447 base pairs in length, including promoter sequences, rather than 2094 as previously described. The mouse and rat Thy-1 genes are highly homologous in both introns and exons. However, the mouse Thy-1 cDNA and rat Thy-1 cDNA differ significantly in sequence in the 5' untranslated region. This suggests that the transcriptional initiation site of the mouse and rat genes may be located at different positions within the genomic sequence and may be related to the differing tissue distribution of Thy-1 in the two species.