Bruno Giros

Mutations of the X-linked genes encoding neuroligins NLGN3 and NLGN4 are associated with autism

Many studies have supported a genetic etiology for autism. Here we report mutations in two X-linked genes encoding neuroligins NLGN3 and NLGN4 in siblings with autism-spectrum disorders. These mutations affect cell-adhesion molecules localized at the synapse and suggest that a defect of synaptogenesis may predispose to autism.

Localization of dopamine D3 receptor mRNA in the rat brain using in situ hybridization histochemistry: comparison with dopamine D2 receptor mRNA

The messenger RNA (mRNA) of the recently characterized D3 dopamine receptor was visualized on rat brain sections using in situ hybridization with a 32P-labeled ribonucleic acid probe corresponding to a major part of the third cytoplasmic loop, a domain in which D2 and D3 dopamine receptors display little homology. For the purpose of comparison, D2 receptor mRNA was also specifically visualized on adjacent sections. The areas that expressed D2 and/or D3 receptors were also compared with those previously detected using [125I]iodosulpride, a ligand that binds to both D2 and D3 receptors with a similar affinity. The localization of D3 receptor mRNa markedly differs from that of D2 receptor mRNA. Whereas D2 receptor mRNA is expressed in all major brain areas receiving dopaminergic projections, particularly in the whole striatal complex, D3 receptor mRNA is expressed in a more restricted manner. It is mainly detected in telencephalic areas receiving dopaminergic inputs from the A10 cell group, e.g. accumbens nucleus, islands of Calleja, bed nucleus of the stria terminalis and other limbic areas such as the hippocampus and the mammillary nuclei. D2 and D3 receptor mRNAs were also detected at the level of the substantia nigra, suggesting that these receptors function as both autoreceptor and postsynaptic receptors. In several dopaminergic projection areas, e.g. ventral straitum, septal or mammillary nuclei, the distributions of D2 and D3 receptor mRNAs appeared complementary without overlap. The distribution of [125I]iodosulpride binding sites generally overlapped that of D2 or D3 receptor mRNAs, the latter being most abundant in dopaminergic areas known to be associated with cognitive and emotional functions.

Molecular characterization of the dopamine transporter

Neurotransmission, which represents chemical signalling between neurons, usually takes place at highly differentiated anatomical structures called synapses. To fulfill both the time and space confinements required for optimal neurotransmission, highly specialized proteins, known as transporters or uptake sites, occur and operate at the presynaptic plasma membrane. Using the energy provided by the Na+ gradient generated by the Na+/K(+)-transporting ATPase, these transporters reuptake the neurotransmitters soon after their release, thereby regulating their effective concentrations at the synaptic cleft and the availability of neurotransmitters for a time-dependent activation of both pre- and postsynaptic receptors. The key role these proteins play in normal neurotransmission is further emphasized when the physiological and social consequences of drugs that interfere with the function of these transporters, such as the psychostimulants (e.g. amphetamine and cocaine) or the widely prescribed antidepressant drugs, are considered. In this review, Bruno Giros and Marc Caron elaborate on the potential consequences of the recent molecular cloning of the dopamine and related transporters and summarize some of the interesting properties that are emerging from this growing family of Na(+)- and Cl(-)-dependent transporters.

Cocaine self-administration in dopamine-transporter knockout mice.

The plasma membrane dopamine transporter (DAT) is responsible for clearing dopamine from the synapse. Cocaine blockade of DAT leads to increased extracellular dopamine, an effect widely considered to be the primary cause of the reinforcing and addictive properties of cocaine. In this study we tested whether these properties are limited to the dopaminergic system in mice lacking DAT. In the absence of DAT, these mice exhibit high levels of extracellular dopamine, but paradoxically still self-administer cocaine. Mapping of the sites of cocaine binding and neuronal activation suggests an involvement of serotonergic brain regions in this response. These results demonstrate that the interaction of cocaine with targets other than DAT, possibly the serotonin transporter, can initiate and sustain cocaine self-administration in these mice.

Pharmacology of human dopamine D3 receptor expressed in a mammalian cell line : comparison with D2 receptor

Two cell lines were created by transfecting cDNAs of the human D2 receptor or the recently cloned human D3 receptor to CHO cells, and the properties of [125I]iodosulpride binding to membranes of these cells were compared. In cell lines expressing the D2 receptor subtype where the selectable marker, a phleomycin-resistance gene, was cotransfected in a different plasmid, a stable expression could be maintained for only few passages. In cell lines expressing the D3 receptor subtype, the selectable marker, a dihydrofolate reductase gene, was cotransfected in the same plasmid and a stable expression could be obtained. In addition, the D3 receptor gene could be amplified in these latter cell lines and a high expression level reached (up to 10(6) binding sites per cell). Sodium and, to a lesser extent, lithium similarly increased [125I]iodosulpride binding to D2 and D3 receptors. In the absence of guanylnucleotide, dopamine had a 24-fold higher apparent affinity at D3 than at D2 receptors. Gpp(NH)p induced rightward shift and steepening of dopamine competition curves at either subtype but the effects were more marked at D2 than at D3 receptors. Several agonists and antagonists, previously regarded as autoreceptor-selective, displayed higher affinities at D3 than at D2 receptors. Although most antagonists used as antipsychotics displayed high affinities at the D3 receptor, all were more potent at the D2 receptor. However, the ratio of Ki values varied over about 10-fold among these compounds, suggesting that they realize differential dopamine receptor subtype occupancy during treatments and that this might be reflected in their clinical profile.

Cloning and functional characterization of a cocaine-sensitive dopamine transporter.

We report the cloning of a rat cDNA encoding a functional dopamine transporter. This cDNA, derived from an intron‐containing gene, encodes a protein of 620 amino acids. Hydropathicity analysis of the protein sequence suggests the presence of 12 putative transmembrane domains. The protein displays considerable identity with transporters for noradrenaline and GABA (64 and 30%, respectively). Transient expression of the cDNA in COS7 cells directs the expression of dopamine uptake activity with appropriate pharmacology and in a sodium‐dependent fashion. In situ hybridization reveals that the mRNA for this transporter is expressed in the substantia nigra and ventral tegmental area, regions that contain dopaminergic cell bodies.

Cloning of a functional vesicular GABA and glycine transporter by screening of genome databases

The unc‐47 locus of Caenorhabditis elegans has been suggested to encode a synaptic vesicle GABA transporter. Here we used hydropathy plot analysis to identify a candidate vesicular GABA transporter in genomic sequences derived from a region of the physical map comprising unc‐47. A mouse homologue was identified and cloned from EST database information. In situ hybridization in rat brain revealed codistribution with both GABAergic and glycinergic neuronal markers. Moreover, expression in COS‐7 and PC12 cells induced an intracellular, glycine‐sensitive GABA uptake activity. These observations, consistent with previous data on GABA and glycine uptake by synaptic vesicles, demonstrate that the mouse clone encodes a vesicular inhibitory amino acid transporter.

Linkage and association of the glutamate receptor 6 gene with autism.

A genome scan was previously performed and pointed to chromosome 6q21 as a candidate region for autism. This region contains the glutamate receptor 6 (GluR6 or GRIK2) gene, a functional candidate for the syndrome. Glutamate is the principal excitatory neurotransmitter in the brain and is directly involved in cognitive functions such as memory and learning. We used two different approaches, the affected sib-pair (ASP) method and the transmission disequilibrium test (TDT), to investigate the linkage and association between GluR6 and autism. The ASP method, conducted with additional markers on the 51 original families and in eight new sibling pairs, showed a significant excess of allele sharing, generating an elevated multipoint maximum LOD score (ASPEX MLS = 3.28). TDT analysis, performed in the ASP families and in an independent data set of 107 parent-offspring trios, indicated a significant maternal transmission disequilibrium (TDTall P = 0.0004). Furthermore, TDT analysis (with only one affected proband per family) showed significant association between GluR6 and autism (TDT association P = 0.008). In contrast to maternal transmission, paternal transmission of GluR6 alleles was as expected in the absence of linkage, suggesting a maternal effect such as imprinting. Mutation screening was performed in 33 affected individuals, revealing several nucleotide polymorphisms (SNPs), including one amino acid change (M867I) in a highly conserved domain of the intracytoplasmic C-terminal region of the protein. This change is found in 8% of the autistic subjects and in 4% of the control population and seems to be more maternally transmitted than expected to autistic males (P = 0.007). Taken together, these data suggest that GluR6 is in linkage disequilibrium with autism.

Serotonin transporter gene polymorphisms and hyperserotonemia in autistic disorder

Tryptophan hydroxylase isoform 2 (TPH2) is expressed in serotonergic neurons in the raphe nuclei, where it catalyzes the rate-limiting step in the synthesis of the neurotransmitter serotonin. In search for functional polymorphisms within the TPH2 gene locus, we measured allele-specific expression of TPH2 mRNA in sections of human pons containing the dorsal and median raphe nuclei. Differences in allelic mRNA expression – referred to as allelic expression imbalance (AEI) – are a measure of cis-acting regulation of gene expression and mRNA processing. Two marker SNPs, located in exons 7 and 9 of TPH2 (rs7305115 and rs4290270, respectively), served for quantitative allelic mRNA measurements in pons RNA samples from 27 individuals heterozygous for one or both SNPs. Significant AEI (ranging from 1.2- to 2.5-fold) was detected in 19 out of the 27 samples, implying the presence of cis-acting polymorphisms that differentially affect TPH2 mRNA levels in pons. For individuals heterozygous for both marker SNPs, the results correlated well (r=0.93), validating the AEI analysis. AEI is tightly associated with the exon 7 marker SNP, in 17 of 18 subjects. Remarkably, expression from the minor allele exceeded that of the major allele in each case, possibly representing a gain-of-function. Genotyping of 20 additional TPH2 SNPs identified a haplotype block of five tightly linked SNPs for which heterozygosity is highly correlated with AEI and overall expression of TPH2 mRNA. These results reveal the presence of a functional cis-acting polymorphism, with high frequency in normal human subjects, resulting in increased TPH2 expression levels. The SNPs that correlate with AEI are closely linked to TPH2 SNPs previously shown to associate with major depression and suicide.

Anterior Pituitary Hypoplasia and Dwarfism in Mice Lacking the Dopamine Transporter

Deletion of the dopamine transporter (DAT) results in increased dopaminergic tone, anterior pituitary hypoplasia, dwarfism, and an inability to lactate. DAT elimination alters the spatial distribution and dramatically reduces the numbers of lactotrophs and somatotrophs in the pituitary. Despite having normal circulating levels of growth hormone and prolactin in blood, hypoplastic glands from DAT-/- mice fail to respond to secretagog stimulation. The effects of DAT deletion on pituitary function result from elevated DA levels that down-regulate the lactotroph D2 DA receptors and depress hypothalamic growth hormone-releasing hormone content. These results reveal an unexpected and important role or DA in the control of developmental events in the pituitary gland and assign a critical role for hypothalamic DA reuptake in regulating these events.