Jana Haase

Comparison of seven different heterologous protein expression systems for the production of the serotonin transporter.

The rat serotonin transporter (rSERT) is an N-glycosylated integral membrane protein with 12 transmembrane regions; the N-glycans improve the ability of the SERT polypeptide chain to fold into a functional transporter, but they are not required for the transmembrane transport of serotonin per se. In order to define the best system for the expression, purification and structural analysis of serotonin transporter (SERT), we expressed SERT in Escherichia coli, Pichia pastoris, the baculovirus expression system and in four different stable mammalian cell lines. Two stable cell lines that constitutively expressed SERT (Imi270 and Coca270) were constructed using episomal plasmids in HEK293 cells expressing the EBNA-1 antigen. SERT expression in the three different inducible stable mammalian cell lines was induced either by a decrease in temperature (cell line pCytTS-SERT), the addition of tetracycline to the growth medium (cell line T-REx-SERT) or by adding DMSO which caused the cells to differentiate (cell line MEL-SERT). All the mammalian cell lines expressed functional SERT, but SERT expressed in E. coli or P. pastoris was nonfunctional as assessed by 5-hydroxytryptamine uptake and inhibitor binding assays. Expression of functional SERT in the mammalian cell lines was assessed by an inhibitor binding assay; the cell lines pCytTS-SERT, Imi270 and Coca270 contained levels of functional SERT similar to that of the standard baculovirus expression system (250,000 copies per cell). The expression of SERT in induced T-REx-SERT cells was 400,000 copies per cell, but in MEL-SERT it was only 80,000 copies per cell. All the mammalian stable cell lines expressed SERT at the plasma membrane as assessed by [3H]-5-hydroxytryptamine uptake into whole cells, but the V(max) for the T-Rex-SERT cell line was 10-fold higher than any of the other cell lines. It was noticeable that the cell lines that constitutively expressed SERT grew extremely poorly, compared to the inducible cell lines whose growth rates were similar to the parental cell lines when not induced. In addition, the cell lines MEL-SERT, Imi270 and T-REx-SERT all expressed fully N-glycosylated SERT and no unglycosylated inactive protein, in contrast to the baculovirus expression system where the vast majority of expressed SERT was unglycosylated and nonfunctional.

Components of the RP4 Conjugative Transfer Apparatus Form an Envelope Structure Bridging Inner and Outer Membranes of Donor Cells: Implications for Related Macromolecule Transport Systems

During bacterial conjugation, the single-stranded DNA molecule is transferred through the cell envelopes of the donor and the recipient cell. A membrane-spanning transfer apparatus encoded by conjugative plasmids has been proposed to facilitate protein and DNA transport. For the IncPalpha plasmid RP4, a thorough sequence analysis of the gene products of the transfer regions Tra1 and Tra2 revealed typical features of mainly inner membrane proteins. We localized essential RP4 transfer functions to Escherichia coli cell fractions by immunological detection with specific polyclonal antisera. Each of the gene products of the RP4 mating pair formation (Mpf) system, specified by the Tra2 core region and by traF of the Tra1 region, was found in the outer membrane fraction with one exception, the TrbB protein, which behaved like a soluble protein. The membrane preparation from Mpf-containing cells had an additional membrane fraction whose density was intermediate between those of the cytoplasmic and outer membranes, suggesting the presence of attachment zones between the two E. coli membranes. The Tra1 region is known to encode the components of the RP4 relaxosome. Several gene products of this transfer region, including the relaxase TraI, were detected in the soluble fraction, but also in the inner membrane fraction. This indicates that the nucleoprotein complex is associated with and/or assembled facing the cytoplasmic site of the E. coli cell envelope. The Tra1 protein TraG was predominantly localized to the cytoplasmic membrane, supporting its potential role as an interface between the RP4 Mpf system and the relaxosome.

Subcellular Redistribution of the Serotonin Transporter by Secretory Carrier Membrane Protein 2

The serotonin transporter (SERT) belongs to the SLC6 family of sodium- and chloride-dependent neurotransmitter transporters responsible for uptake of amino acids and biogenic amines from extracellular spaces. Their activities and subcellular distributions are regulated by various cellular mechanisms, including interactions with other proteins. Using the yeast two-hybrid approach we screened a human brain cDNA library and identified secretory carrier membrane protein 2 (SCAMP2) as a novel SERT-interacting protein. GST-pulldown assays confirmed the physical interaction between SCAMP2 and the N-terminal domain of SERT. In addition, SERT was found to form a complex with SCAMP2 as demonstrated by co-immunoprecipitation from a heterologous expression system and from rat brain homogenate. Co-expression of SERT and SCAMP2 in mammalian cells results in the subcellular redistribution of SERT with a decrease in cell surface SERT and a concomitant reduction in 5-HT uptake activity. Using confocal microscopy we show that in neuronal cells endogenous SERT co-localizes with SCAMP2 in discrete structures also containing the lipid raft marker flotillin-1 and the SNARE protein syntaxin 1A. In contrast, SERT immunoreactivity is clearly segregated from transferrin receptor-containing endosomes. A single amino acid mutation, cysteine-201 to alanine, within the conserved cytoplasmic E peptide of SCAMP2, abolished SCAMP2-mediated down-regulation of SERT, although this mutation had no effect on the physical interaction between SERT and SCAMP2. Taken together, our results suggest that SCAMP2 plays an important role in the regulation of the subcellular distribution of SERT.

Integrating the monoamine, neurotrophin and cytokine hypotheses of depression--a central role for the serotonin transporter?

Monoamine, in particular serotonergic neurotransmission has long been recognized as an important factor in the aetiology of depression. The serotonin transporter (SERT) is the primary regulator of serotonin levels in the brain and a key target for widely used antidepressant drugs, such as selective serotonin reuptake inhibitors (SSRIs). In realising the limitations of current antidepressant therapy, depression research has branched out to encompass other areas such as synaptic plasticity, neurogenesis and brain structural remodelling as factors which influence mood and behaviour. More recently, the immune system has been implicated in the development of depression and various intriguing observations have inspired the cytokine hypothesis of depression. Over the past two decades evidence of in vitro and in vivo regulation of SERT function by pro-inflammatory cytokines as well as by mechanisms of synaptic plasticity has been accumulating, offering a mechanistic link between the monoamine, neurotrophin and cytokine theories of depression. This review will focus firstly on the interconnected roles of serotonin and neurotrophins in depression and antidepressant therapy, secondly on the impact of the immune system on serotonin transporter regulation and neurotrophin signalling and finally we propose a model of reciprocal regulation of serotonin and neurotrophin signalling in the context of inflammation-induced depression.

Modulation of the dopamine transporter by interaction with Secretory Carrier Membrane Protein 2.

The monoamine transporters for dopamine (DAT), norepinephrine (NET) and serotonin (SERT) facilitate the homeostatic balance of neurotransmitters in the synaptic cleft and thus, play a fundamental role in regulating neuronal activity. Despite the importance of these monoamine transporters in controlling brain function, only relatively little information is available regarding the cellular and molecular regulation of these proteins. The monoamine transporters have been found to associate with a number of different proteins that regulate the function and subcellular localization of the transporters. We recently reported a functional interaction between SERT and the Secretory Carrier Membrane Protein 2 (SCAMP2). Here, we demonstrate that SCAMP2 also plays a role in the functional regulation of DAT. DAT and SCAMP2 interaction is here verified by co-immunoprecipitation and fluorescence resonance energy transfer (FRET) microscopy. Moreover, co-expression of DAT and SCAMP2 results in a decrease in DAT-mediated dopamine uptake caused by reduced levels of DAT molecules on the cell surface. Our finding that SCAMP2 interacts with and regulates the subcellular distribution of both DAT and SERT suggests that interaction with SCAMP2 may constitute an important mechanism for coordinating cell surface expression of monoamine transporters.

Elevation of cortical serotonin transporter activity upon peripheral immune challenge is regulated independently of p38 mitogen-activated protein kinase activation and transporter phosphorylation.

The serotonin transporter (SERT) is responsible for high‐affinity serotonin (5‐HT) uptake from extracellular fluid and is a prominent pharmacological target in the treatment of depression. In recent years, depression has also been linked to immune system activation. Inflammatory conditions can cause sickness behaviour and depression‐like symptoms in both animals and humans. Since SERT has been proposed as one of the molecular targets in inflammation‐induced depression, we applied the widely used lipopolysaccharides (LPS) model to study the effects of peripheral inflammation on SERT activity in the brain. We show that 24 h after intraperitoneal LPS administration, SERT‐mediated 5‐HT uptake is significantly enhanced in the frontal cortex. Analysis of uptake kinetics revealed that the transport capacity (Vmax) of cortical SERT was increased in LPS‐injected animals, while the Km value remained unchanged. The increase in Vmax was neither due to increased SERT protein expression nor increased synaptic surface exposure. The suppression of SERT activity upon inhibition of p38 MAPK was not selective for LPS‐induced enhancement of SERT function. In addition, SERT activity changes in LPS‐treated rats are unaffected by nitric oxide synthase and protein kinase G inhibitors. Using the Phos‐Tag method, we identified five SERT‐specific protein bands representing distinct phosphorylation states of SERT. However, the enhancement of SERT activity in LPS‐treated rats was not correlated with altered transporter phosphorylation. Together with previous studies by others, our results suggest that SERT is regulated by multiple mechanisms in response to peripheral immune system activation.

Serotonin Transporter Associated Protein Complexes Are Enriched in Synaptic Vesicle Proteins and Proteins Involved in Energy Metabolism and Ion Homeostasis

The serotonin transporter (SERT) mediates Na+-dependent high-affinity serotonin uptake and plays a key role in regulating extracellular serotonin concentration in the brain and periphery. To gain novel insight into SERT regulation, we conducted a comprehensive proteomics screen to identify components of SERT-associated protein complexes in the brain by employing three independent approaches. In vivo SERT complexes were purified from rat brain using an immobilized high-affinity SERT ligand, amino-methyl citalopram. This approach was combined with GST pulldown and yeast two-hybrid screens using N- and C-terminal cytoplasmic transporter domains as bait. Potential SERT associated proteins detected by at least two of the interaction methods were subjected to gene ontology analysis resulting in the identification of functional protein clusters that are enriched in SERT complexes. Prominent clusters include synaptic vesicle proteins, as well as proteins involved in energy metabolism and ion homeostasis. Using subcellular fractionation and electron microscopy we provide further evidence that SERT is indeed associated with synaptic vesicle fractions, and colocalizes with small vesicular structures in axons and axon terminals. We also show that SERT is found in close proximity to mitochondrial membranes in both, hippocampal and neocortical regions. We propose a model of the SERT interactome, in which SERT is distributed between different subcellular compartments through dynamic interactions with site-specific protein complexes. Finally, our protein interaction data suggest novel hypotheses for the regulation of SERT activity and trafficking, which ultimately impact on serotonergic neurotransmission and serotonin dependent brain functions.

Human amniocytes regulate serotonin levels by active uptake and express genes suggestive of a wider role in facilitating neurotransmitter regulation in the fetal environment.

Fetal serotonin levels, which mediate multiple developmental processes, are highly regulated. However, an incomplete picture exists on the component parts of such regulation during fetal growth. Serotonin and its metabolite 5-hydroxyindoleacetic acid (5-HIAA) are found in the amniotic fluid, also containing significant numbers of amniocytes, previously thought to be the result of cell shedding as a byproduct of growth. The aim of the present study was to examine human amniocytes as a potentially active and dynamic component of serotonin regulation in the fetal environment. Using amniocytes derived from multiple donors of amniocentesis, we found all components necessary for serotonin metabolism. We identified a strong expression of the serotonin transporter and confirmed the high-affinity serotonin transporter-mediated uptake of serotonin (5-HT), along with uptake via the norepinephrine transporter, and an evidence of 5-HT breakdown due to the expression of the degradative enzymes monoamine oxidase A and B. Additionally, wider expression analysis for biogenic amine and cholinergic metabolism suggests a capability for cholinergic synthesis and release and for catecholamine storage. Our results shed new light on amniocytes, consistent with a role in the homeostasis of neurotransmitters during fetal development. Moreover, these results may provide clinical significance for amniocytes as new targets for uptake inhibitors such as tricyclic antidepressants, selective serotonin reuptake inhibitors, and drugs of abuse such as cocaine, with implications on their regulation during pregnancy. This work shows for the first time an inherent in vivo function of amniocytes and more broadly implicates them as a new and active component of the fetal-maternal regulatory system.

TNFα-dependent anhedonia and upregulation of hippocampal serotonin transporter activity in a mouse model of collagen-induced arthritis

&NA; The serotonin transporter (SERT) facilitates high affinity reuptake of 5‐HT from the extracellular fluid and dysregulation of transporter function has been implicated in a range of mood disorders including depression. Recent studies have linked immune system activation to depression as well as to altered serotonin transporter activity. Advancing previous studies, which have mainly focussed on acute effects of immune system activation, in this study we used collagen‐induced arthritis (CIA) in mice as a model of chronic inflammatory disease, to investigate the effect of prolonged inflammation on brain SERT function and behaviour. We found that 5–6 weeks after immunisation, CIA mice display anhedonia, a core depression‐like behaviour. Behavioural symptoms are temporally correlated with a region‐specific upregulation of SERT activity in the hippocampus, which occurs at a post‐translational level and is independent of SERT trafficking. Kinetic analysis of 5‐HT uptake revealed that the elevation of transporter activity is due to an increase in 5‐HT transport capacity (Vmax) with no change in apparent Km values, suggesting that different regulatory mechanisms govern SERT modulation under chronic versus acute inflammatory conditions. Protein expression of tumour necrosis factor receptor 1 (TNFR1) was specifically upregulated in the hippocampus of CIA mice, indicating altered TNF&agr; signalling. Anti‐TNF&agr; treatment using etanercept not only diminished joint inflammation, but also prevented the development of anhedonia and the upregulation of SERT activity in the hippocampus, suggesting a key role for TNF&agr; signalling in brain function regulation in this disease model. Our study provides novel insight into molecular mechanisms underlying mood symptoms in chronic inflammatory diseases, with particular relevance to rheumatoid arthritis. HighlightsCollagen‐induced arthritis causes symptoms of anhedonia in mice.Serotonin transporter activity is upregulated in the hippocampus of CIA mice.Anti‐TNF&agr; treatment abolishes anhedonia and normalises SERT activity.This study provides novel insight into mechanisms underlying inflammation induced depression.

The pro-inflammatory cytokine TNF-α regulates the expression of the serotonin transporter (SERT) gene in primary astrocytes and the C6 glioma cell line

516 A role for the innate immune system in age-related deficits in synaptic function and cognition