Trine N. Jørgensen

SLC6 Neurotransmitter Transporters: Structure, Function, and Regulation

The neurotransmitter transporters (NTTs) belonging to the solute carrier 6 (SLC6) gene family (also referred to as the neurotransmitter-sodium-symporter family or Na+/Cl−-dependent transporters) comprise a group of nine sodium- and chloride-dependent plasma membrane transporters for the monoamine neurotransmitters serotonin (5-hydroxytryptamine), dopamine, and norepinephrine, and the amino acid neurotransmitters GABA and glycine. The SLC6 NTTs are widely expressed in the mammalian brain and play an essential role in regulating neurotransmitter signaling and homeostasis by mediating uptake of released neurotransmitters from the extracellular space into neurons and glial cells. The transporters are targets for a wide range of therapeutic drugs used in treatment of psychiatric diseases, including major depression, anxiety disorders, attention deficit hyperactivity disorder and epilepsy. Furthermore, psychostimulants such as cocaine and amphetamines have the SLC6 NTTs as primary targets. Beginning with the determination of a high-resolution structure of a prokaryotic homolog of the mammalian SLC6 transporters in 2005, the understanding of the molecular structure, function, and pharmacology of these proteins has advanced rapidly. Furthermore, intensive efforts have been directed toward understanding the molecular and cellular mechanisms involved in regulation of the activity of this important class of transporters, leading to new methodological developments and important insights. This review provides an update of these advances and their implications for the current understanding of the SLC6 NTTs.

Regulation of dopamine transporter function by protein-protein interactions: new discoveries and methodological challenges

J. Neurochem. (2010) 10.1111/j.1471‐4159.2010.06599.x

The N Terminus of Monoamine Transporters Is a Lever Required for the Action of Amphetamines

The serotonin transporter (SERT) terminates neurotransmission by removing serotonin from the synaptic cleft. In addition, it is the site of action of antidepressants (which block the transporter) and of amphetamines (which induce substrate efflux). We explored the functional importance of the N terminus in mediating the action of amphetamines by focusing initially on the highly conserved threonine residue at position 81, a candidate site for phosphorylation by protein kinase C. Molecular dynamics simulations of the wild type SERT, compared with its mutations SERTT81A and SERTT81D, suggested structural changes in the inner vestibule indicative of an opening of the inner vestibule. Predictions from this model (e.g. the preferential accumulation of SERTT81A in the inward conformation, its reduced turnover number, and a larger distance between its N and C termini) were verified. Most importantly, SERTT81A (and the homologous mutations in noradrenaline and dopamine) failed to support amphetamine-induced efflux, and this was not remedied by aspartate at this position. Amphetamine-induced currents through SERTT81A were comparable with those through the wild type transporter. Both abundant Na+ entry and accumulation of SERTT81A in the inward facing conformation ought to favor amphetamine-induced efflux. Thus, we surmised that the N terminus must play a direct role in driving the transporter into a state that supports amphetamine-induced efflux. This hypothesis was verified by truncating the first 64 amino acids and by tethering the N terminus to an additional transmembrane helix. Either modification abolished amphetamine-induced efflux. We therefore conclude that the N terminus of monoamine transporters acts as a lever that sustains reverse transport.

Type I interferon signaling is involved in the spontaneous development of lupus-like disease in B6.Nba2 and (B6.Nba2 × NZW)F1 mice

Several studies have described a role for type I interferons (IFNαβ) in the initiation and/or prolongation of autoimmune diseases. Most pronounced has been the association of disease activity with what is now known as ‘the interferon signature’ of gene expression in peripheral blood mononuclear cells from lupus patients. In correlation, studies have shown that inhibition of IFNαβ signaling abrogates disease in various mouse models of lupus. New Zealand black (NZB) and B6.Nba2 congenic mice spontaneously develop elevated levels of serum anti-nuclear autoantibodies (ANAs). Nevertheless, neither of these strains develop fatal renal disease. The female F1 offspring of NZB or B6.Nba2 crossed with New Zealand white (NZW) mice do, however, develop kidney disease. We have previously shown that increases in endogenous IFNαβ levels in (B6.Nba2 × NZW)F1 mice leads to accelerated development of renal disease in an IFNαβ-dependent manner. We now show that B6.Nba2 and (B6.Nba2 × NZW)F1 mice deficient for the IFNαβ-receptor fail to develop ANA and renal disease, although the mice have substantial immune complex deposition in the glomeruli. Thus, endogenous IFNαβ might influence disease by affecting B-cell activation and differentiation, as well as the kidneys’ susceptibility to damage, the latter perhaps through induction of a local inflammatory milieu.

Postendocytic Sorting of Constitutively Internalized Dopamine Transporter in Cell Lines and Dopaminergic Neurons

The dopamine transporter (DAT) mediates reuptake of released dopamine and is the target for psychostimulants, such as cocaine and amphetamine. DAT undergoes marked constitutive endocytosis, but little is known about the fate and sorting of the endocytosed transporter. To study DAT sorting in cells lines, we fused the one-transmembrane segment protein Tac to DAT, thereby generating a transporter (TacDAT) with an extracellular antibody epitope suited for trafficking studies. TacDAT was functional and endocytosed constitutively in HEK293 cells. According to an ELISA-based assay, TacDAT intracellular accumulation was increased by the lysosomal protease inhibitor leupeptin and by monensin, an inhibitor of lysosomal degradation and recycling. Monensin also reduced TacDAT surface expression consistent with partial recycling. In both HEK293 cells and in the dopaminergic cell line 1Rb3An27, constitutively internalized TacDAT displayed primary co-localization with the late endosomal marker Rab7, less co-localization with the “short loop” recycling marker Rab4, and little co-localization with the marker of “long loop” recycling endosomes, Rab11. Removal by mutation of N-terminal ubiquitination sites did not affect this sorting pattern. The sorting pattern was distinct from a bona fide recycling membrane protein, the β2-adrenergic receptor, that co-localized primarily with Rab11 and Rab4. Constitutively internalized wild type DAT probed with the fluorescently tagged cocaine analogue JHC 1-64, exhibited the same co-localization pattern as TacDAT in 1Rb3An27 cells and in cultured midbrain dopaminergic neurons. We conclude that DAT is constitutively internalized and sorted in a ubiquitination-independent manner to late endosomes/lysosomes and in part to a Rab4 positive short loop recycling pathway.

Identification of candidate genes that influence sex hormone-dependent disease phenotypes in mouse lupus.

Ninety percent of systemic lupus erythematosus patients are female, and gender differences in lupus susceptibility are also observed in (New Zealand Black × New Zealand White)F1 (BWF1) lupus-prone mice. We followed orchiectomized, intact male and female BWF1 mice for lupus-like disease for 1 year. A comparative gene expression analysis was then used to identify candidate genes potentially responsible for gender-dependent differences in lupus susceptibility. Seven genes encoded on the sex chromosomes and 77 probe sets, including 14 immunoglobulin genes, encoded on the autosomal chromosomes were identified as differentially expressed in male versus female BWF1 splenocytes prior to disease onset. Five genes were determined to be regulated by either estradiol or dihydrotestosterone in an in vivo system and most of them were preferentially expressed in antigen-presenting cells. Gender differences in the expression of Csf3-r, Histh1c, Serpinb2, Slc6a4 and Cd22 in BWF1 mice are the result of transcriptional modification by sex hormones and warrant further investigation. The identification of candidate genes and their expression patterns in splenocyte sub-populations provide new information regarding the mechanisms by which sex hormones influence the development of mouse lupus.

Increased expression of Ifi202, an IFN-activatable gene, in B6.Nba2 lupus susceptible mice inhibits p53-mediated apoptosis.

Increased expression of p202 protein (encoded by the Ifi202 gene) in splenocytes derived from B6.Nba2 mice (congenic for the Nba2 interval derived from the New Zealand Black mice) was correlated with defects in apoptosis of splenic B cells and increased susceptibility to develop systemic lupus erythematosus. We have now investigated the molecular mechanisms by which increased expression of p202 in B6.Nba2 cells contributes to defects in apoptosis. In this study, we report that increased expression of p202 in the B6.Nba2 splenocytes, as compared with cells derived from the parental C57BL/6 (B6) mice, was correlated with increased levels of p53 protein and inhibition of p53-mediated transcription of target genes that encode proapoptotic proteins. Conversely, knockdown of p202 expression in B6.Nba2 cells resulted in stimulation of p53-mediated transcription. We found that p202 bound to p53 in the N-terminal region (aa 44–83) comprising the proline-rich region that is important for p53-mediated apoptosis. Consistent with the binding of p202 to p53, increased expression of p202 in B6.Nba2 mouse embryonic fibroblasts inhibited UV-induced apoptosis. Taken together, our observations support the idea that increased expression of p202 in B6.Nba2 mice increases the susceptibility to develop lupus, in part, by inhibiting p53-mediated apoptosis.

Bim and Bcl-2 Mutually Affect the Expression of the Other in T Cells

The life and death of T cells is controlled to a large extent by the relative amounts of Bcl-2-related proteins they contain. The antiapoptotic protein Bcl-2 and the proapoptotic protein Bim are particularly important in this process with the amount of Bcl-2 per cell dropping by about one-half when T cells prepare to die. In this study we show that Bcl-2 and Bim each control the expression of the other. Absence of Bim leads to a drop in the amount of intracellular Bcl-2 protein, while having no effect on the amounts of mRNA for Bcl-2. Conversely, high amounts of Bcl-2 per cell allow high amounts of Bim, although in this case the effect involves increases in Bim mRNA. These mutual effects occur even if Bcl-2 is induced acutely. Thus these two proteins control the expression of the other, at either the protein or mRNA level.

Brain-derived neurotrophic factor (BDNF) enhances GABA transport by modulating the trafficking of GABA transporter-1 (GAT-1) from the plasma membrane of rat cortical astrocytes

Background: Transport of GABA into astrocytes is crucial for excitability control. Results: The neurotrophin BDNF, through TrkB-t receptor activation, enhances GABA transport into astrocytes, which requires adenosine A2A receptor signaling. Conclusion: BDNF plays an active role in the synaptic clearance of GABA. Significance: This new regulatory role for TrkB-t receptors discloses their relevance for excitability control at the tripartite synapse. The γ-aminobutyric acid (GABA) transporters (GATs) are located in the plasma membrane of neurons and astrocytes and are responsible for termination of GABAergic transmission. It has previously been shown that brain derived neurotrophic factor (BDNF) modulates GAT-1-mediated GABA transport in nerve terminals and neuronal cultures. We now report that BDNF enhances GAT-1-mediated GABA transport in cultured astrocytes, an effect mostly due to an increase in the Vmax kinetic constant. This action involves the truncated form of the TrkB receptor (TrkB-t) coupled to a non-classic PLC-γ/PKC-δ and ERK/MAPK pathway and requires active adenosine A2A receptors. Transport through GAT-3 is not affected by BDNF. To elucidate if BDNF affects trafficking of GAT-1 in astrocytes, we generated and infected astrocytes with a functional mutant of the rat GAT-1 (rGAT-1) in which the hemagglutinin (HA) epitope was incorporated into the second extracellular loop. An increase in plasma membrane of HA-rGAT-1 as well as of rGAT-1 was observed when both HA-GAT-1-transduced astrocytes and rGAT-1-overexpressing astrocytes were treated with BDNF. The effect of BDNF results from inhibition of dynamin/clathrin-dependent constitutive internalization of GAT-1 rather than from facilitation of the monensin-sensitive recycling of GAT-1 molecules back to the plasma membrane. We therefore conclude that BDNF enhances the time span of GAT-1 molecules at the plasma membrane of astrocytes. BDNF may thus play an active role in the clearance of GABA from synaptic and extrasynaptic sites and in this way influence neuronal excitability.

Links Between Type I Interferons and the Genetic Basis of Disease in Mouse Lupus

Systemic lupus erythematosus (SLE), like other autoimmune diseases, is a complex genetic trait with contributions from both major histocompatibility complex (MHC) genes and multiple non-MHC genes. Most of the contributing genes have yet to be identified. Studies of mouse models of lupus have provided important insight into the immunopathogenesis of lupus-like IgG autoantibody production and lupus nephritis, and genetic analyses of these mice are helping to unravel the complex and heterogeneous genetic basis of disease. Recent studies in both human SLE and mouse models of lupus have emphasized a potential role of type I interferons (IFN-α/β) in the initiation and perpetuation of disease. There is now increasing interest in genes that affect IFN-α/β expression–activity and IFN-regulated target genes that may be involved in the disease process. One example is interferon-inducible gene 202 (Ifi202), which has been identified as a major candidate susceptibility gene in the New Zealand hybrid model of lupus. Studies suggest that increased expression of this transcription factor leads to lupus through inhibition of lymphocyte apoptosis, although its effects on immune function are extremely complex and have yet to be fully defined. This review will focus on the genetic basis of disease in mouse lupus with a special emphasis on those genetic contributions that may affect IFN-α/β activity and those that may be target genes of IFN-α/β action.