Bruno Gasnier

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.

Cystinosin, the protein defective in cystinosis, is a H+‐driven lysosomal cystine transporter

Cystinosis is an inherited lysosomal storage disease characterized by defective transport of cystine out of lysosomes. However, the causative gene, CTNS, encodes a seven transmembrane domain lysosomal protein, cystinosin, unrelated to known transporters. To investigate the molecular function of cystinosin, the protein was redirected from lysosomes to the plasma membrane by deletion of its C‐terminal GYDQL sorting motif (cystinosin‐ΔGYDQL), thereby exposing the intralysosomal side of cystinosin to the extracellular medium. COS cells expressing cystinosin‐ΔGYDQL selectively take up L‐cystine from the extracellular medium at acidic pH. Disruption of the transmembrane pH gradient or incubation of the cells at neutral pH strongly inhibits the uptake. Cystinosin‐ΔGYDQL is directly involved in the observed cystine transport, since this activity is highly reduced when the GYDQL motif is restored and is abolished upon introduction of a point mutation inducing early‐onset cystinosis. We conclude that cystinosin represents a novel H+‐driven transporter that is responsible for cystine export from lysosomes, and propose that cystinosin homologues, such as mammalian SL15/Lec35 and Saccharomyces cerevisiae ERS1, may perform similar transport processes at other cellular membranes.

Identification and characterization of a lysosomal transporter for small neutral amino acids

In eukaryotic cells, lysosomes represent a major site for macromolecule degradation. Hydrolysis products are eventually exported from this acidic organelle into the cytosol through specific transporters. Impairment of this process at either the hydrolysis or the efflux step is responsible of several lysosomal storage diseases. However, most lysosomal transporters, although biochemically characterized, remain unknown at the molecular level. In this study, we report the molecular and functional characterization of a lysosomal amino acid transporter (LYAAT-1), remotely related to a family of H+-coupled plasma membrane and synaptic vesicle amino acid transporters. LYAAT-1 is expressed in most rat tissues, with highest levels in the brain where it is present in neurons. Upon overexpression in COS-7 cells, the recombinant protein mediates the accumulation of neutral amino acids, such as γ-aminobutyric acid, l-alanine, and l-proline, through an H+/amino acid symport. Confocal microscopy on brain sections revealed that this transporter colocalizes with cathepsin D, an established lysosomal marker. LYAAT-1 thus appears as a lysosomal transporter that actively exports neutral amino acids from lysosomes by chemiosmotic coupling to the H+-ATPase of these organelles. Homology searching in eukaryotic genomes suggests that LYAAT-1 defines a subgroup of lysosomal transporters in the amino acid/auxin permease family.

The loading of neurotransmitters into synaptic vesicles.

Classical (non-peptide) transmitters are stored into secretory vesicles by a secondary active transporter driven by a V-type H(+)-ATPase. Five vesicular neurotransmitter uptake activities have been characterized in vitro and, for three of them, the transporters involved have been identified at the molecular level using cDNA cloning and/or Caenorhabditis elegans genetics. These transporters belong to two protein families, which are both unrelated to the Na(+)-coupled neurotransmitter transporters operating at the plasma membrane. The two isoforms of the mammalian vesicular monoamine transporter, VMAT1 and VMAT2, are related to the vesicular acetylcholine transporter (VACHT), while a novel, unrelated vesicular inhibitory amino acid transporter (VIAAT), also designated vesicular GABA transporter (VGAT), is responsible for the storage of GABA, glycine or, at some synapses, both amino acids into synaptic vesicles. The observed effects of experimentally altered levels of VACHT or VMAT2 on synaptic transmission and behavior, as well as the recent awareness that GABAergic or glutamatergic receptors are not always saturated at central synapses, suggest a potential role of vesicular loading in synaptic plasticity.

Gephyrin-independent clustering of postsynaptic GABA(A) receptor subtypes.

Gephyrin has been shown to be essential for the synaptic localization of the inhibitory glycine receptor and major GABA(A) receptor (GABA(A)R) subtypes. However, in retina certain GABA(A)R subunits are found at synaptic sites in the absence of gephyrin. Here, we quantitatively analyzed GABA(A)R alpha1, alpha2, alpha3, alpha5, beta2/3, and gamma2 subunit immunoreactivities in spinal cord sections derived from wild-type and gephyrin-deficient (geph -/-) mice. The punctate staining of GABA(A)R alpha1 and alpha5 subunits was unaltered in geph -/- mice, whereas the numbers of alpha2-, alpha3-, beta2/3-, and gamma2-subunit-immunoreactive synaptic sites were significantly or even strikingly reduced in the mutant animals. Immunostaining with an antibody specific for the vesicular inhibitory amino acid transporter revealed that the number of inhibitory presynaptic terminals is unaltered upon gephyrin deficiency. These data show that in addition to gephyrin other clustering proteins must exist that mediate the synaptic localization of selected GABA(A)R subtypes.

The vesicular glutamate transporter VGLUT3 synergizes striatal acetylcholine tone

Three subtypes of vesicular transporters accumulate glutamate into synaptic vesicles to promote its vesicular release. One of the subtypes, VGLUT3, is expressed in neurons, including cholinergic striatal interneurons, that are known to release other classical transmitters. Here we showed that disruption of the Slc17a8 gene (also known as Vglut3) caused an unexpected hypocholinergic striatal phenotype. Vglut3−/− mice were more responsive to cocaine and less prone to haloperidol-induced catalepsy than wild-type littermates, and acetylcholine release was decreased in striatum slices lacking VGLUT3. These phenotypes were associated with a colocalization of VGLUT3 and the vesicular acetylcholine transporter (VAChT) in striatal synaptic vesicles and the loss of a synergistic effect of glutamate on vesicular acetylcholine uptake. We propose that this vesicular synergy between two transmitters is the result of the unbalanced bioenergetics of VAChT, which requires anion co-entry for continuing vesicular filling. Our study reveals a previously unknown effect of glutamate on cholinergic synapses with potential functional and pharmacological implications.

Identification of a putative lysosomal cobalamin exporter altered in the cblF defect of vitamin B12 metabolism

Vitamin B12 (cobalamin) is essential in animals for metabolism of branched chain amino acids and odd chain fatty acids, and for remethylation of homocysteine to methionine. In the cblF inborn error of vitamin B12 metabolism, free vitamin accumulates in lysosomes, thus hindering its conversion to cofactors. Using homozygosity mapping in 12 unrelated cblF individuals and microcell-mediated chromosome transfer, we identified a candidate gene on chromosome 6q13, LMBRD1, encoding LMBD1, a lysosomal membrane protein with homology to lipocalin membrane receptor LIMR. We identified five different frameshift mutations in LMBRD1 resulting in loss of LMBD1 function, with 18 of the 24 disease chromosomes carrying the same mutation embedded in a common 1.34-Mb haplotype. Transfection of fibroblasts of individuals with cblF with wild-type LMBD1 rescued cobalamin coenzyme synthesis and function. This work identifies LMBRD1 as the gene underlying the cblF defect of cobalamin metabolism and suggests that LMBD1 is a lysosomal membrane exporter for cobalamin.

VGLUT3 (Vesicular Glutamate Transporter Type 3) Contribution to the Regulation of Serotonergic Transmission and Anxiety

Three different subtypes of H+-dependent carriers (named VGLUT1–3) concentrate glutamate into synaptic vesicles before its exocytotic release. Neurons using other neurotransmitter than glutamate (such as cholinergic striatal interneurons and 5-HT neurons) express VGLUT3. It was recently reported that VGLUT3 increases acetylcholine vesicular filling, thereby, stimulating cholinergic transmission. This new regulatory mechanism is herein designated as vesicular-filling synergy (or vesicular synergy). In the present report, we found that deletion of VGLUT3 increased several anxiety-related behaviors in adult and in newborn mice as early as 8 d after birth. This precocious involvement of a vesicular glutamate transporter in anxiety led us to examine the underlying functional implications of VGLUT3 in 5-HT neurons. On one hand, VGLUT3 deletion caused a significant decrease of 5-HT1A-mediated neurotransmission in raphe nuclei. On the other hand, VGLUT3 positively modulated 5-HT transmission of a specific subset of 5-HT terminals from the hippocampus and the cerebral cortex. VGLUT3- and VMAT2-positive serotonergic fibers show little or no 5-HT reuptake transporter. These results unravel the existence of a novel subset of 5-HT terminals in limbic areas that might play a crucial role in anxiety-like behaviors. In summary, VGLUT3 accelerates 5-HT transmission at the level of specific 5-HT terminals and can exert an inhibitory control at the raphe level. Furthermore, our results suggest that the loss of VGLUT3 expression leads to anxiety-associated behaviors and should be considered as a potential new target for the treatment of this disorder.

The vesiicular monoamine transporter: from chromaffin granule to brain

All characterized monoaminergic cells utilize the same transport system for the vesicular accumulation of monoamines prior to their release. This system operates in neuronal (catecholaminergic, serotoninergic or histaminergic) as well as in endocrine or neuroendocrine cells. For several decades, chromaffin granules from bovine adrenal medulla have been used as a model system, allowing progress in the understanding of the biophysics, the biochemistry and the pharmacology of the monoamine vesicular transporter. The transporters from rat, bovine and man have been cloned. Surprisingly, two genes encode different isoforms of the protein which are differentially expressed in monoaminergic systems. The conjunction of recombinant DNA techniques and expression in secretory or non-secretory cells with the large body of data obtained on the chromaffin granule transporter has allowed rapid progress in the study of the protein. But interestingly enough, this progress has open new possibilities in the study of biological problems, especially in the brain. The transporter is useful for the determination of the relationship between small and large dense core vesicles, for the understanding of the mechanism of the drugs such as 1-methyl-4-phenylpyridinium (MPP+), tetrabenazine or amphetamines, and as a marker in brain development. The possibility of regulations at the vesicular transporter level and of their effect on the quantum size has to be investigated. The vesicular monoamine transporter is also an important target for brain imaging.

Postnatal maturation of Na+, K+, 2Cl– cotransporter expression and inhibitory synaptogenesis in the rat hippocampus: an immunocytochemical analysis

GABA, a major inhibitory neurotransmitter, depolarizes hippocampal pyramidal neurons during the first postnatal week. These depolarizations result from an efflux of Cl– through GABAA‐gated anion channels. The outward Cl– gradient that provides the driving force for Cl– efflux might be generated and maintained by the Na+, K+, 2Cl– cotransporter (NKCC) that keeps intracellular Cl– concentration above electrochemical equilibrium. The developmental pattern of expression of the cotransporter in the hippocampus is not known. We studied the postnatal distribution pattern of NKCC in the hippocampus using a monoclonal antibody (T4) against a conserved epitope in the C‐terminus of the cotransporter molecule. We also examined the temporal relationships between the developmental pattern of NKCC expression and the formation of perisomatic GABAergic synapses. This study was aimed at determining, with antivesicular inhibitory amino acid transporter (VIAAT) antibodies, whether perisomatic GABAergic synapses are formed preferentially at the time when GABA is depolarizing. During the first postnatal week, NKCC immunolabelling was restricted to cell bodies in the pyramidal cell layer and in the strata oriens and radiatum. In contrast, at postnatal day 21 (P21) and in adult animals little or no labelling occurred in cell bodies; instead, a prominent dendritic labelling appeared in both pyramidal and nonpyramidal neurons. The ultrastructural immunogold study in P21 rat hippocampi corroborated the light‐microscopy results. In addition, this study revealed that a portion of the silver‐intensified colloidal gold particles were located on neuronal plasmalemma, as expected for a functional cotransporter. The formation of inhibitory synapses on perikarya of the pyramidal cell layer was a late process. The density of VIAAT‐immunoreactive puncta in the stratum pyramidale at P21 reached four times the P7 value in CA3, and six times the P7 value in CA1. Electron microscopy revealed that the number of synapses per neuronal perikaryal profile in the stratum pyramidale of the CA3 area at P21 was three times higher than at P7, even if a concomitant 20% increase in the area of these neuronal perikaryal profiles occurred. It is concluded that, in hippocampal pyramidal cells, there is a developmental shift in the NKCC localization from a predominantly somatic to a predominantly dendritic location. The presence of NKCC during the first postnatal week is consistent with the hypothesis that this transporter might be involved in the depolarizing effects of GABA. The depolarizing effects of GABA may not be required for the establishment of the majority of GABAergic synapses in the stratum pyramidale, because their number increases after the first postnatal week, when GABA action becomes hyperpolarizing.