Isoforms of the neuronal glutamate transporter gene, SLC1A1/EAAC1, negatively modulate glutamate uptake: relevance to obsessive-compulsive disorder.

The SLC1A1 gene, which encodes the neuronal glutamate transporter, EAAC1, has consistently been implicated in obsessive-compulsive disorder (OCD) in genetic studies. Moreover, neuroimaging, biochemical and clinical studies support a role for glutamatergic dysfunction in OCD. Although SLC1A1 is an excellent candidate gene for OCD, little is known about its regulation at the genomic level. Here, we report the identification and characterization of three alternative SLC1A1/EAAC1 mRNAs: a transcript derived from an internal promoter, termed P2 to distinguish it from the transcript generated by the primary promoter (P1), and two alternatively spliced mRNAs: ex2skip, which is missing exon 2, and ex11skip, which is missing exon 11. All isoforms inhibit glutamate uptake from the full-length EAAC1 transporter. Ex2skip and ex11skip also display partial colocalization and interact with the full-length EAAC1 protein. The three isoforms are evolutionarily conserved between human and mouse, and are expressed in brain, kidney and lymphocytes under nonpathological conditions, suggesting that the isoforms are physiological regulators of EAAC1. Moreover, under specific conditions, all SLC1A1 transcripts were differentially expressed in lymphocytes derived from subjects with OCD compared with controls. These initial results reveal the complexity of SLC1A1 regulation and the potential clinical utility of profiling glutamatergic gene expression in OCD and other psychiatric disorders.

Mice lacking synapsin III show abnormalities in explicit memory and conditioned fear.

Synapsin III is a neuron-specific phosphoprotein that plays an important role in synaptic transmission and neural development. While synapsin III is abundant in embryonic brain, expression of the protein in adults is reduced and limited primarily to the hippocampus, olfactory bulb and cerebral cortex. Given the specificity of synapsin III to these brain areas and because it plays a role in neurogenesis in the dentate gyrus, we investigated whether it may affect learning and memory processes in mice. To address this point, synapsin III knockout mice were examined in a general behavioral screen, several tests to assess learning and memory function, and conditioned fear. Mutant animals displayed no anomalies in sensory and motor function or in anxiety- and depressive-like behaviors. Although mutants showed minor alterations in the Morris water maze, they were deficient in object recognition 24 h and 10 days after training and in social transmission of food preference at 20 min and 24 h. In addition, mutants displayed abnormal responses in contextual and cued fear conditioning when tested 1 or 24 h after conditioning. The synapsin III knockout mice also showed aberrant responses in fear-potentiated startle. As synapsin III protein is decreased in schizophrenic brain and because the mutant mice do not harbor obvious anatomical deficits or neurological disorders, these mutants may represent a unique neurodevelopmental model for dissecting the molecular pathways that are related to certain aspects of schizophrenia and related disorders.

Characterization of transcripts from the synapsin III gene locus.

Synapsin III, the most recently described member of the synapsin gene family, displays a gene structure and protein domain structure similar to those of synapsins I and II. In this report, however, we describe major differences in the temporal- and tissue-specific expressions of synapsin III. Whereas synapsins I and II each give rise to two isoforms that are expressed predominantly in adult brain, there are at least six synapsin III transcripts (synapsin IIIa-IIIf) that differ with respect to tissue- and developmental stage-specific expression. Three of the neuronal transcripts are detected in fetal and to a lesser extent in adult brain (IIa-IIIc), whereas one (IIId) is detected only in fetal brain. Two additional transcripts (IIIe and IIIf) are detected only in nonneuronal tissues. A putative second promoter, which is contained within an intron in the synapsin III gene locus, appears to generate the nonneuronal synapsin IIIe and IIIf transcripts. This level of genome complexity is far greater than that described previously for the synapsin I and II genes and suggests that synapsin III may have functions distinct from those described for synapsins I and II.

Molecular evolution of the synapsin gene family.

Synapsins, a family of synaptic vesicle proteins, play a crucial role in the regulation of neurotransmission and synaptogenesis. They have been identified in a variety of invertebrate and vertebrate species, including human, rat (Rattus norvegicus), cow (Bos taurus), longfin squid (Loligo pealei), and fruit fly (Drosophila melanogaster). Here, synapsins were cloned from three additional species: frog (Xenopus laevis), lamprey (Lampetra fluviatilis), and nematode (Caenorhabditis elegans). Synapsin protein sequences from all these species were then used to explore the molecular phylogeny of these important neuronal phosphoproteins. The ancestral condition of a single synapsin gene probably gave rise to the vertebrate synapsin gene family comprised of at least three synapsin genes (I, II, and III) in higher vertebrates. Synapsins possess multiple domains, which have evolved at different rates throughout evolution. In invertebrate synapsins, the most conserved domains are C and E. During the evolution of vertebrates, at least two gene duplication events are hypothesized to have given rise to the synapsin gene family. This was accompanied by the emergence of an additional conserved domain, termed A. J. Exp. Zool. ( Mol. Dev. Evol. ) 285:360-377, 1999.

Cloning of cDNAs encoding human synapsins IIa and IIb.

The synapsins are a family of neuronal phosphoproteins that are specifically associated with the cytoplasmic surface of synaptic vesicles. In mammals, distinct genes for synapsins I, II, and III give rise to members of the synapsin family. The synapsins are implicated in neurotransmitter release and synaptogenesis, processes believed to be aberrant in several neuropsychiatric diseases. The characterization of human synapsins is therefore important for evaluating the possible role of synapsins in human neuropathology. In this report, we describe the cloning and sequence of human synapsins IIa and IIb, products of the synapsin II gene. Human synapsins IIa and IIb conform to the previously described domain model of the synapsins, and the most conserved protein domains are A, C, and E.

A third member of the synapsin gene family.

Synapsins are a family of neuron-specific synaptic vesicle-associated phosphoproteins that have been implicated in synaptogenesis and in the modulation of neurotransmitter release. In mammals, distinct genes for synapsins I and II have been identified, each of which gives rise to two alternatively spliced isoforms. We have now cloned and characterized a third member of the synapsin gene family, synapsin III, from human DNA. Synapsin III gives rise to at least one protein isoform, designated synapsin IIIa, in several mammalian species. Synapsin IIIa is associated with synaptic vesicles, and its expression appears to be neuron-specific. The primary structure of synapsin IIIa conforms to the domain model previously described for the synapsin family, with domains A, C, and E exhibiting the highest degree of conservation. Synapsin IIIa contains a novel domain, termed domain J, located between domains C and E. The similarities among synapsins I, II, and III in domain organization, neuron-specific expression, and subcellular localization suggest a possible role for synapsin III in the regulation of neurotransmitter release and synaptogenesis. The human synapsin III gene is located on chromosome 22q12-13, which has been identified as a possible schizophrenia susceptibility locus. On the basis of this localization and the well established neurobiological roles of the synapsins, synapsin III represents a candidate gene for schizophrenia.

Brain specific proteins binding to the 3' UTR of the 5-HT2C receptor mRNA.

The 5-HT2C receptor2 is a prominent serotonin receptor that is uniquely expressed in the central nervous system and has been implicated in a variety of psychiatric diseases. While characterizing the 5-HT2C receptor gene, we observed that the mRNA contains a long 3' untranslated region that binds multiple brain proteins. Two proteins, molecular weights 55 and 58 kDa, were of particular interest because they were detected only in brain regions known to express the 5-HT2C receptor abundantly, namely, the hippocampus and cortex. These proteins bind with high affinity to the 5-HT2C receptor mRNA at its extreme 3' end (Kd = 1.8 nM), and binding can be specifically competed by selected regions of the 3' UTR. Furthermore, binding of the 55 and 58 kDa proteins to the mRNA is directionally specific and shows preference for an AU-rich loop containing 6 to 7 nucleotides. These results suggest the possibility that these two brain specific proteins may play a role in the post-transcriptional regulation of the 5-HT2C receptor, and that post-transcriptional control of 5-HT2C receptor expression may be an important regulatory mechanism which has not been previously reported for this serotonin receptor subtype.

Immunoglobulin heavy-chain enhancer is required to maintain transfected gamma 2A gene expression in a pre-B-cell line.

The immunoglobulin heavy-chain (IgH) enhancer serves to activate efficient and accurate transcription of cloned IgH genes when introduced into B lymphomas or myelomas. The role of this enhancer after gene activation, however, is unclear. The endogenous IgH genes in several cell lines, for example, have lost the IgH enhancer by deletion and yet continue to be expressed. This might be explained if the role of the enhancer were to establish high-level gene transcription but not to maintain it. Alternatively, other enhancers might lie adjacent to endogenous IgH genes, substituting their activity for that of the lost IgH enhancer. To address both of these alternatives, we searched for enhancer activity within the flanking regions of one of these IgH enhancer-independent genes and designed an experiment that allowed us to consider separately the establishment and maintenance of expression of a transfected gene. For the latter experiment we generated numerous pre-B cell lines stably transformed with a gamma 2a gene. In this gene, the IgH enhancer lay at a site outside the heavy-chain transcription unit, between DH and JH gene segments. After expression of the transfected gene was established, selective conditions were chosen for the outgrowth of subclones that had undergone D-J joining and thus IgH enhancer deletion. Measurements of gamma 2a expression before and after enhancer deletion revealed that the enhancer was required for maintenance of expression of the transfected gene. The implication of this finding for models of enhancer function in endogenous genes is discussed.

Role of the octamer motif in hybrid cell extinction of immunoglobulin gene expression: extinction is dominant in a two enhancer system.

We have shown previously that genes activated by the immunoglobulin heavy chain (IgH) enhancer or promoter in mouse myeloma cells are extinguished upon fusion of the myeloma with a mouse T cell lymphoma. Here we show that the conserved octamer sequence shared by the IgH enhancer and promoter, when multimerized to form a tissue-specific enhancer, can also render a gene extinguishable under the same experimental conditions. Extinction, however, is not correlated with either absence of the tissue-specific transcription factor OTF-2 or loss of its ability to bind the octamer sequence. It was also found that extinction mediated by the IgH enhancer is dominant to transcriptional activation by the SV40 enhancer. We propose, therefore, that the T cell-negative regulator responsible for IgH gene extinction does not simply prevent IgH enhancer activation but interferes with gene expression more directly, perhaps by disrupting the transcription complex established as a result of tissue-specific enhancer activation.