Ove Wiborg

Serotonin4 (5-HT4) Receptor Agonists Are Putative Antidepressants with a Rapid Onset of Action

Current antidepressants are clinically effective only after several weeks of administration. Here, we show that serotonin(4) (5-HT(4)) agonists reduce immobility in the forced swimming test, displaying an antidepressant potential. Moreover, a 3 day regimen with such compounds modifies rat brain parameters considered to be key markers of antidepressant action, but that are observed only after 2-3 week treatments with classical molecules: desensitization of 5-HT(1A) autoreceptors, increased tonus on hippocampal postsynaptic 5-HT(1A) receptors, and enhanced phosphorylation of the CREB protein and neurogenesis in the hippocampus. In contrast, a 3 day treatment with the SSRI citalopram remains devoid of any effect on these parameters. Finally, a 3 day regimen with the 5-HT(4) agonist RS 67333 was sufficient to reduce both the hyperlocomotion induced by olfactory bulbectomy and the diminution of sucrose intake consecutive to a chronic mild stress. These findings point out 5-HT(4) receptor agonists as a putative class of antidepressants with a rapid onset of action.

The S-enantiomer of R,S-citalopram, increases inhibitor binding to the human serotonin transporter by an allosteric mechanism. Comparison with other serotonin transporter inhibitors

The interaction of the S- and R-enantiomers (escitalopram and R-citalopram) of citalopram, with high- and low-affinity binding sites in COS-1 cell membranes expressing human SERT (hSERT) were investigated. Escitalopram affinity for hSERT and its 5-HT uptake inhibitory potency was in the nanomolar range and approximately 40-fold more potent than R-citalopram. Escitalopram considerably stabilised the [3H]-escitalopram/SERT complex via an allosteric effect at a low-affinity binding site. The stereoselectivity between escitalopram and R-citalopram was approximately 3:1 for the [3H]-escitalopram/hSERT complex. The combined effect of escitalopram and R-citalopram was additive. Paroxetine and sertraline mainly stabilised the [3H]-paroxetine/hSERT complex. Fluoxetine, duloxetine and venlafaxine have only minor effects. 5-HT stabilised the [125I]-RTI-55, [3H]-MADAM, [3H]-paroxetine, [3H]-fluoxetine and [3H]-venlafaxine/SERT complex to some extent. Thus, escitalopram shows a unique interaction with the hSERT compared with other 5-HT reuptake inhibitors (SSRIs) and, in addition to its 5-HT reuptake inhibitory properties, displays a pronounced effect via an affinity-modulating allosteric site.

Functional analysis of a novel human serotonin transporter gene promoter in immortalized raphe cells

To investigate the structural basis for genetic regulation of the human serotonin transporter gene, a 1.8 kb fragment upstream to the cap site was cloned and sequenced. The promoter possesses a polymorphic repeat region with 16 and 14 repeats, respectively. Both were cloned and characterized. The promoter sequence revealed an internal 379 bp fragment not reported in previous publications. This novel fragment contains consensus sequences for several transcription factors including SpI and GATA. DNA from 48 unrelated individuals was PCR amplified, in this region, to test for allelic variations. All were found to possess the additional 379 bp fragment. The integrity of the promoter was furthermore confirmed by genomic Southern blotting. The promoter activity was analyzed by reporter gene assays in neuronal and non-neuronal serotonergic cell lines. In immortalized serotonergic raphe neurons, RN46A, three cis-acting, cell specific, activating elements and a silencer were located. One of the activators and the silencer are located in the repeat region and one activator is positioned in the novel fragment. A fourth activating element was found to be active in both RN46A cells and in a non-neuronal serotonergic cell line, JAR. A 3.5 kb fragment from intron 1 was cloned and found to possess cell specific activity in JAR cells indicating the presence of an alternative promoter in intron 1.

Binding of serotonin to the human serotonin transporter. Molecular modeling and experimental validation

Molecular modeling and structure-activity relationship studies were performed to propose a model for binding of the neurotransmitter serotonin (5-HT) to the human serotonin transporter (hSERT). Homology models were constructed using the crystal structure of a bacterial homologue, the leucine transporter from Aquifex aeolicus, as the template and three slightly different sequence alignments. Induced fit docking of 5-HT into hSERT homology models resulted in two different binding modes. Both show a salt bridge between Asp98 and the charged primary amine of 5-HT, and both have the 5-HT C6 position of the indole ring pointing toward Ala173. The difference between the two orientations of 5-HT is an enantiofacial discrimination of the indole ring, resulting in the 5-hydroxyl group of 5-HT being vicinal to either Ser438/Thr439 or Ala169/Ile172/Ala173. To assess the binding experimentally, binding affinities for 5-HT and 17 analogues toward wild type and 13 single point mutants of hSERT were measured using an approach termed paired mutant-ligand analogue complementation (PaMLAC). The proposed ligand-protein interaction was systematically examined by disrupting it through site-directed mutagenesis and re-establishing another interaction via a ligand analogue matching the mutated residue, thereby minimizing the risk of identifying indirect effects. The interactions between Asp98 and the primary amine of 5-HT and the interaction between the C6-position of 5-HT and hSERT position 173 was confirmed using PaMLAC. The measured binding affinities of various mutants and 5-HT analogues allowed for a distinction between the two proposed binding modes of 5-HT and biochemically support the model for 5-HT binding in hSERT where the 5-hydroxyl group is in close proximity to Thr439.

Characterization of an allosteric citalopram-binding site at the serotonin transporter

The serotonin transporter (SERT), which belongs to a family of sodium/chloride‐dependent transporters, is the major pharmacological target in the treatment of several clinical disorders, including depression and anxiety. In the present study we show that the dissociation rate, of [3H]S‐citalopram from human SERT, is retarded by the presence of serotonin, as well as by several antidepressants, when present in the dissociation buffer. Dissociation of [3H]S‐citalopram from SERT is most potently inhibited by S‐citalopram followed by R‐citalopram, sertraline, serotonin and paroxetine. EC50 values for S‐ and R‐citalopram are 3.6 ± 0.4 µm and 19.4 ± 2.3 µm, respectively. Fluoxetine, venlafaxine and duloxetine have no significant effect on the dissociation of [3H]S‐citalopram. Allosteric modulation of dissociation is independent of temperature, or the presence of Na+ in the dissociation buffer. Dissociation of [3H]S‐citalopram from a complex with the SERT double‐mutant, N208Q/N217Q, which has been suggested to be unable to self‐assemble into oligomeric complexes, is retarded to an extent similar to that found with the wild‐type, raising the possibility that the allosteric mechanism is mediated within a single subunit. A species‐scanning mutagenesis study comparing human and bovine SERT revealed that Met180, Tyr495 and Ser513 are important residues in mediating the allosteric effect, as well as contributing to high‐affinity binding at the primary site.

Species-scanning mutagenesis of the serotonin transporter reveals residues essential in selective, high-affinity recognition of antidepressants.

The serotonin transporter (SERT) is a high‐affinity sodium/chloride‐dependent neurotransmitter transporter responsible for reuptake of serotonin from the extracellular space. SERT is a selective target of several clinically important antidepressants. In a cross‐species analysis comparing human and bovine SERTs, the kinetic parameters for serotonin uptake were found to be similar, however, the pharmacological profiles of the two transporters differ. Following transient expression in COS‐1 cells, IC50 values were determined for several antidepressants and psychostimulants. The potencies of the antidepressants citalopram, fluoxetine, paroxetine and imipramine were several‐fold higher at hSERT compared with bSERT. No species selectivity was observed for the antidepressants fluvoxamine, and sertraline or for the psychostimulants cocaine, the cocaine analogue β‐carbomethoxy‐3β‐(4‐iodophenyl)tropane, or for 3,4‐methylenedioxymethamphetamine (MDMA). Analysis of six hSERT/bSERT chimeras and subsequent species‐scanning mutagenesis of each isoform revealed methionine‐180, tyrosine‐495, and phenylalanine‐513 to be responsible for the increase in citalopram and paroxetine potencies at hSERT and methionine‐180 and phenylalanine‐513 to confer species selectivity at hSERT for fluoxetine and imipramine. Results were obtained by doing the forward, bovine to human, mutations and confirmed by doing the reverse mutations. Citalopram analogues were used to define the roles of methionine‐180, tyrosine‐495, and phenylalanine‐513 and to reveal molecular interactions with individual functional groups of citalopram. We suggest that methionine‐180 interacts with the heterocyclic nucleus of citalopram or stabilizes the binding pocket and phenylalanine‐513 to be a steric blocker of antidepressant recognition.

Animal models of major depression and their clinical implications.

Major depressive disorder is a common, complex, and potentially life-threatening mental disorder that imposes a severe social and economic burden worldwide. Over the years, numerous animal models have been established to elucidate pathophysiology that underlies depression and to test novel antidepressant treatment strategies. Despite these substantial efforts, the animal models available currently are of limited utility for these purposes, probably because none of the models mimics this complex disorder fully. It is presumable that psychiatric illnesses, such as affective disorders, are related to the complexity of the human brain. Here, we summarize the animal models that are used most commonly for depression, and discuss their advantages and limitations. We discuss genetic models, including the recently developed optogenetic tools and the stress models, such as the social stress, chronic mild stress, learned helplessness, and early-life stress paradigms. Moreover, we summarize briefly the olfactory bulbectomy model, as well as models that are based on pharmacological manipulations and disruption of the circadian rhythm. Finally, we highlight common misinterpretations and often-neglected important issues in this field.

Chronic mild stress for modeling anhedonia

Major depressive disorder is a complex disease implicating many brain circuitries. The clinical symptomatology is inconsistent and heterogenous and the pathogenesis is a complicated interplay of genetic and environmental factors. The episodic and recurrent nature of the disease, as well as the fact that several symptoms are only verbally expressed, make it challenging to establish valid and legitimate animal models of this disease. The purpose of this review is to provide some background knowledge and overview of valid rodent models of depression with an emphasis on our own experience with a chronic mild stress model in modeling of anhedonia and cognitive impairments associated with depression. In a final concluding remark, a ‘dying-forward’ hypothesis, for development of depression, is suggested on the basis of mainly our own data on a hippocampal pathology.

Binding and Orientation of Tricyclic Antidepressants within the Central Substrate Site of the Human Serotonin Transporter

Tricyclic antidepressants (TCAs) have been used for decades, but their orientation within and molecular interactions with their primary target is yet unsettled. The recent finding of a TCA binding site in the extracellular vestibule of the bacterial leucine transporter 11 Å above the central site has prompted debate about whether this vestibular site in the bacterial transporter is applicable to binding of antidepressants to their relevant physiological target, the human serotonin transporter (hSERT). We present an experimentally validated structural model of imipramine and analogous TCAs in the central substrate binding site of hSERT. Two possible binding modes were observed from induced fit docking calculations. We experimentally validated a single binding mode by combining mutagenesis of hSERT with uptake inhibition studies of different TCA analogs according to the paired mutation ligand analog complementation paradigm. Using this experimental method, we identify a salt bridge between the tertiary aliphatic amine and Asp98. Furthermore, the 7-position of the imipramine ring is found vicinal to Phe335, and the pocket lined by Ala173 and Thr439 is utilized by 3-substituents. These protein-ligand contact points unambiguously orient the TCA within the central binding site and reveal differences between substrate binding and inhibitor binding, giving important clues to the inhibition mechanism. Consonant with the well established competitive inhibition of uptake by TCAs, the resulting binding site for TCAs in hSERT is fully overlapping with the serotonin binding site in hSERT and dissimilar to the low affinity noncompetitive TCA site reported in the leucine transporter (LeuT).

Candidate Hippocampal Biomarkers of Susceptibility and Resilience to Stress in a Rat Model of Depression

Susceptibility to stress plays a crucial role in the development of psychiatric disorders such as unipolar depression and post-traumatic stress disorder. In the present study the chronic mild stress rat model of depression was used to reveal stress-susceptible and stress-resilient rats. Large-scale proteomics was used to map hippocampal protein alterations in different stress states. Membrane proteins were successfully captured by two-phase separation and peptide based proteomics. Using iTRAQ labeling coupled with mass spectrometry, more than 2000 proteins were quantified and 73 proteins were found to be differentially expressed. Stress susceptibility was associated with increased expression of a sodium-channel protein (SCN9A) currently investigated as a potential antidepressant target. Differential protein profiling also indicated stress susceptibility to be associated with deficits in synaptic vesicle release involving SNCA, SYN-1, and AP-3. Our results indicate that increased oxidative phosphorylation (COX5A, NDUFB7, NDUFS8, COX5B, and UQCRB) within the hippocampal CA regions is part of a stress-protection mechanism.