Inhibition of Indoleamine 2,3 Dioxygenase Does Not Improve Cancer-Related Symptoms in a Murine Model of Human Papilloma Virus-Related Head and Neck Cancer.

The expression of indoleamine 2,3 dioxygenase (IDO) by tumors can contribute to immunotolerance, and IDO induced by inflammation can also increase risk for the development of behavioral alterations. Thus, this study was initiated to determine whether IDO inhibition, intended to facilitate tumor clearance in response to treatment, attenuates behavioral alterations associated with tumor growth and treatment. We used a murine model of human papilloma virus-related head and neck cancer. We confirmed that tumor cells express IDO and expression was increased by radiotherapy. Interestingly, inhibition of IDO activation by the competitive inhibitor 1-methyl tryptophan mildly exacerbated treatment-associated burrowing deficits (burrowing is a sensitive index of sickness in tumor-bearing mice). Genetic deletion of IDO worsened tumor outcomes and had no effect on the behavioral response as by decreased burrowing or reduced voluntary wheel running. In contrast, oral administration of a specific inhibitor of IDO1 provided no apparent benefit on the tumor response to cancer therapy, yet decreased voluntary wheel-running activity independent of treatment. These results indicate that, independent of its potential effect on tumor clearance, inhibition of IDO does not improve cancer-related symptoms.

Latent toxoplasmosis and psychiatric symptoms - A role of tryptophan metabolism?

Toxoplasma gondii (TOX) is a common parasite which infects approximately one third of the human population. In recent years, it has been suggested that latent toxoplasmosis may be a risk factor for the development of mental disorders, particularly schizophrenia and anxiety. With regards to depression the results have been varied. The main objective of this study was to examine subpopulations from the Danish PRISME and GENDEP populations for TOX IgG antibodies. These consisted of: a group with symptoms of anxiety, a group suffering from burnout syndrome, as well as two different subpopulations with depression of differing severity. The secondary objective of this study was to examine whether tryptophan metabolism was altered in TOX-positive subjects within each subpopulation. Our results show that the anxiety and burnout populations were more likely to be TOX IgG seropositive. Furthermore, we find that the moderate-severe but not mild-moderate depressive subpopulation were associated with TOX seropositivety, suggesting a possible role of symptom severity. Additionally, we found that TOX positive subjects in the anxiety and burnout subpopulations had altered tryptophan metabolism. This relationship did not exist in the mild-moderate depressive subpopulation. These results suggest that TOX seropositivity may be related to anxiety, burnout and potentially to severity of depression. We furthermore show that the psychiatric symptoms could be associated with an altered tryptophan metabolism.

Upregulation of neuronal kynurenine 3-monooxygenase mediates depression-like behavior in a mouse model of neuropathic pain.

Pain and depression often co-occur, but the underlying mechanisms have not been elucidated. Here, we used the spared nerve injury (SNI) model in mice to induce both neuropathic pain and depression-like behavior. We investigated whether brain interleukin (IL)-1 signaling and activity of kynurenine 3-monoxygenase (KMO), a key enzyme for metabolism of kynurenine into the neurotoxic NMDA receptor agonist quinolinic acid, are necessary for comorbid neuropathic pain and depression-like behavior. SNI mice showed increased expression levels of Il1b and Kmo mRNA in the contralateral side of the brain. The SNI-induced increase of Kmo mRNA was associated with increased KMO protein and elevated quinolinic acid and reduced kynurenic acid in the contralateral hippocampus. The increase in KMO-protein in response to SNI mostly took place in hippocampal NeuN-positive neurons rather than microglia. Inhibition of brain IL-1 signaling by intracerebroventricular administration of IL-1 receptor antagonist after SNI prevented the increase in Kmo mRNA and depression-like behavior measured by forced swim test. However, inhibition of brain IL-1 signaling has no effect on mechanical allodynia. In addition, intracerebroventricular administration of the KMO inhibitor Ro 61-8048 abrogated depression-like behavior without affecting mechanical allodynia after SNI. We show for the first time that the development of depression-like behavior in the SNI model requires brain IL-1 signaling and activation of neuronal KMO, while pain is independent of this pathway. Inhibition of KMO may represent a promising target for treating depression.

Drugs with antidepressant properties affect tryptophan metabolites differently in rodent models with depression-like behavior.

The metabolism of tryptophan through kynurenine and serotonin pathways is linked to depression. Here, effects of different drugs with antidepressant properties (vortioxetine, fluoxetine, and ketamine) on various tryptophan metabolites in different brain regions and plasma were examined using tandem mass spectrometry (LC-MS/MS), in Flinders Sensitive Line rats, a genetic rat model of depression, and its controls: Flinders Sensitive Line and Sprague-Dawley rats. Protein levels of kynurenine pathway enzymes were measured in the brains and livers of these rat strains. Furthermore, effects of vortioxetine on tryptophan metabolites were assessed in the cortical regions of lupus mice (MRL/MpJ-FasIpr ), a murine model of increased depression-like behavior associated with inflammation. Sustained vortioxetine or fluoxetine (at doses aimed to fully occupy serotonin transporter via food or drinking water for at least 14 days) reduced levels of the excitotoxin quinolinic acid (QUIN) in various brain regions in all rats. Furthermore, chronic vortioxetine reduced levels of QUIN in MRL/MpJ-FasIpr mice. Acute i.p. administration of fluoxetine (10 mg/kg) or vortioxetine (10 mg/kg) led to reduced brain 5-hydroxyindoleacetic acid in Sprague-Dawley rats (2, 4, 6, and 8 h) and a similar trend was evident in Flinders Sensitive Line and Flinders Sensitive Line rats after 4 h. In contrast, single or repeated administration of ketamine (15 mg/kg i.p.) did not induce significant changes in metabolite levels. In conclusion, sustained vortioxetine and fluoxetine administration decreased QUIN independent of species, while ketamine was ineffective. These results support the hypothesis that modulating tryptophan metabolism may be part of the mechanism of action for some antidepressants.

Quantitative analysis of lipids: a higher-throughput LC-MS/MS-based method and its comparison to ELISA.

AIM: Lipids such as prostaglandins, leukotrienes and thromboxanes are released as a result of an inflammatory episode in pain (central and peripheral). METHODOLOGY & RESULTS: To measure these lipids as potential mechanistic biomarkers in neuropathic pain models, we developed a higher-throughput LC-MS/MS-based method with simultaneous detection of PGE2, PGD2, PGF2α, LTB4, TXB2 and 2-arachidonoyl glycerol in brain and spinal cord tissues. We also demonstrate that the LC-MS/MS method was more sensitive and specific in differentiating PGE2 levels in CNS tissues compared with ELISA. CONCLUSION: The ability to modify the LC-MS/MS method to accommodate numerous other lipids in one analysis, demonstrates that the presented method offers a cost-effective and more sensitive alternative to ELISA method useful in drug discovery settings.

Task- and Treatment Length-Dependent Effects of Vortioxetine on Scopolamine-Induced Cognitive Dysfunction and Hippocampal Extracellular Acetylcholine in Rats.

Major depressive disorder (MDD) is a common psychiatric disorder that often features impairments in cognitive function, and these cognitive symptoms can be important determinants of functional ability. Vortioxetine is a multimodal antidepressant that may improve some aspects of cognitive function in patients with MDD, including attention, processing speed, executive function, and memory. However, the cause of these effects is unclear, and there are several competing theories on the underlying mechanism, notably including regionally-selective downstream enhancement of glutamate neurotransmission and increased acetylcholine (ACh) neurotransmission. The current work sought to evaluate the ACh hypothesis by examining vortioxetine's ability to reverse scopolamine-induced impairments in rodent tests of memory and attention. Additionally, vortioxetine's effects on hippocampal extracellular ACh levels were examined alongside studies of vortioxetine's pharmacokinetic profile. We found that acute vortioxetine reversed scopolamine-induced impairments in social and object recognition memory, but did not alter scopolamine-induced impairments in attention. Acute vortioxetine also induced a modest and short-lived increase in hippocampal ACh levels. However, this short-term effect is at variance with vortioxetine's moderately long brain half life (5.1 hours). Interestingly, subchronic vortioxetine treatment failed to reverse scopolamine-induced social recognition memory deficits and had no effects on basal hippocampal ACh levels. These data suggest that vortioxetine has some effects on memory that could be mediated through cholinergic neurotransmission, however these effects are modest and only seen under acute dosing conditions. These limitations may argue against cholinergic mechanisms being the primary mediator of vortioxetine's cognitive effects, which are observed under chronic dosing conditions in patients with MDD.

Female Flinders Sensitive Line rats show estrous cycle-independent depression-like behavior and altered tryptophan metabolism.

Clinical studies suggest a link between depression and dysfunctional tryptophan (TRP) metabolism. Even though depression is twice as prevalent in women as men, the impact of the estrous cycle on TRP metabolism is not well-understood. Here we investigated 13 kynurenine and serotonin metabolites in female Flinders Sensitive Line (FSL) rats, a genetic rat model of depression. FSL rats and controls (Flinders Resistant Line rats), 12-20weeks old, were subject to the forced swim test (FST), a commonly used measure of depression-like behavior. Open field was used to evaluate locomotor ability and agoraphobia. Subsequently, plasma and hemispheres were collected and analyzed for their content of TRP metabolites using liquid chromatography-tandem mass spectrometry. Vaginal saline lavages were obtained daily for ⩾2 cycles. To estimate the effects of sex and FST we included plasma from unhandled, naïve male FSL and FRL rats. Female FSL rats showed a depression-like phenotype with increased immobility in the FST, not confounded by anxiety. In the brain, 3-hydroxykynurenine was increased whereas anthranilate and 5-hydroxytryptophan were decreased. In plasma, anthranilate and quinolinate levels were lower in FSL rats compared to the control line, independent of sex and FST. The estrous cycle neither impacted behavior nor TRP metabolite levels in the FSL rat. In conclusion, the female FSL rat is an interesting preclinical model of depression with altered TRP metabolism, independent of the estrous cycle. The status of the pathway in brain was not reflected in the plasma, which may indicate that an inherent local, cerebral regulation of TRP metabolism occurs.

Histamine may contribute to vortioxetine's procognitive effects; possibly through an orexigenic mechanism.

Vortioxetine is a novel multimodal antidepressant that acts as a serotonin (5-HT)3, 5-HT7, and 5-HT1D receptor antagonist; 5-HT1B receptor partial agonist; 5-HT1A receptor agonist; and 5-HT transporter inhibitor in vitro. In preclinical and clinical studies vortioxetine demonstrates positive effects on cognitive dysfunction. Vortioxetine's effect on cognitive function likely involves the modulation of several neurotransmitter systems. Acute and chronic administration of vortioxetine resulted in changes in histamine concentrations in microdialysates collected from the rat prefrontal cortex and ventral hippocampus. Based on these results and a literature review of the current understanding of the interaction between the histaminergic and serotonergic systems and the role of histamine on cognitive function, we hypothesize that vortioxetine through an activation of the orexinergic system stimulates the tuberomammilary nucleus and enhances histaminergic neurotransmission, which contributes to vortioxetine's positive effects on cognitive function.

Development of a Surface Plasmon Resonance Assay for the Characterization of Small-Molecule Binding Kinetics and Mechanism of Binding to Kynurenine 3-Monooxygenase.

Kynurenine 3-monooxygenase (KMO), a pivotal enzyme in the kynurenine pathway, was identified as a potential therapeutic target for treating neurodegenerative and psychiatric disorders. In this article, we describe a surface plasmon resonance (SPR) assay that delivers both kinetics and the mechanism of binding (MoB) data, enabling a detailed characterization of KMO inhibitors for the enzyme in real time. SPR assay development included optimization of the protein construct and the buffer conditions. The stability and inhibitor binding activity of the immobilized KMO were significantly improved when the experiments were performed at 10°C using a buffer containing 0.05% n-dodecyl-ß-d-maltoside (DDM) as the detergent. The KD values of the known KMO inhibitors (UPF648 and RO61-8048) from the SPR assay were in good accordance with the biochemical LC/MS/MS assay. Also, the SPR assay was able to differentiate the binding kinetics (k(a) and k(d)) of the selected unknown KMO inhibitors. For example, the inhibitors that showed comparable IC50 values in the LC/MS/MS assay displayed differences in their residence time (τ = 1/k(d)) in the SPR assay. To better define the MoB of the inhibitors to KMO, an SPR-based competition assay was developed, which demonstrated that both UPF648 and RO61-8048 bound to the substrate-binding site. These results demonstrate the potential of the SPR assay for characterizing the affinity, the kinetics, and the MoB profiles of the KMO inhibitors.

Behavioral Deficits Are Accompanied by Immunological and Neurochemical Changes in a Mouse Model for Neuropsychiatric Lupus (NP-SLE).

Neuropsychiatric symptoms of systemic lupus erythematosus (NP-SLE) have been understudied compared to end-organ failure and peripheral pathology. Neuropsychiatric symptoms, particularly affective and cognitive indications, may be among the earliest manifestations of SLE. Among the potential pathophysiological mechanisms responsible for NP-SLE are increased peripheral pro-inflammatory cytokines, subsequent induction of indoleamine-2,3-dioxygenase (IDO) and activation of the kynurenine pathway. In the MRL/MpJ-Faslpr (MRL/lpr) murine model of lupus, depression-like behavior and cognitive dysfunction is evident before significant levels of autoantibody titers and nephritis are present. We examined the behavioral profile of MRL/lpr mice and their congenic controls, a comprehensive plasma cytokine and chemokine profile, and brain levels of serotonin and kynurenine pathway metabolites. Consistent with previous studies, MRL/lpr mice had increased depression-like behavior and visuospatial memory impairment. Plasma levels of different inflammatory molecules (Haptoglobin, interleukin 10 (IL-10), interferon γ-inducible protein 10 (IP-10/CXCL10), lymphotactin, macrophage inhibitory protein 3ß (MIP-3ß/CCL19), monocyte chemotactic protein 1, 3 and 5 (MCP-1/CCL2, MCP-3/CCL7, MCP-5/CCL12), vascular cell adhesion molecule 1 (VCAM-1), lymphotactin and interferon γ (IFN-γ)) were increased in MRL/lpr mice. In cortex and hippocampus, MRL/lpr mice had increased levels of kynurenine pathway metabolites (kynurenine, 3-hydroxykynurenine, 3-hydroxynthranilic acid and quinolinic acid). Therefore, our study suggests that increased cytokine expression may be critical in the regulation subtle aspects of brain function in NP-SLE via induction of IDO and tryptophan/kynurenine metabolism.

Interferon-alpha treatment induces depression-like behaviour accompanied by elevated hippocampal quinolinic acid levels in rats.

Immunotherapy with the cytokine interferon-alpha (IFN-α) can induce symptoms of depression, and it is likely that the tryptophan-kynurenine pathway may be involved in this regard. In this study we investigated the effects of IFN-α on depression-like behaviour and central metabolites of the tryptophan-kynurenine pathway in rats. Secondly, we explored the modulating effects of an antidepressant (imipramine) and anti-inflammatory drug (celecoxib) on IFN-α-induced behavioural and pathophysiological changes in the brain. The following treatment groups were used: Control (saline), IFN-α (6×10(4)IU/kg s.c.), IFN-α+imipramine or IFN-α+celecoxib. Drugs were administered daily for 1 week. IFN-α treatment induced depression-like behaviour by increasing immobility in the forced swim test (FST), and decreased tryptophan levels in the brain. There was a trend for an increased kynurenine/tryptophan ratio, indicative of indoleamine 2,3-dioxygenase (IDO) activation, and increased quinolinic acid in the hippocampus. Imipramine decreased immobility in the FST, but did not reverse the IFN-α-induced changes in the tryptophan-kynurenine pathway. There was a trend for celecoxib to decrease immobility and to reverse the IFN-α-induced increase in the kynurenine/tryptophan ratio. Thus, our study provides further evidence for IFN-α-induced depression-like behaviour through central changes of the tryptophan-kynurenine pathway.

Peripheral indoleamine 2,3-dioxygenase 1 is required for comorbid depression-like behavior but does not contribute to neuropathic pain in mice.

Chronic pain frequently co-occurs with major depressive disorder but the mechanisms are poorly understood. We investigated the contribution of indoleamine 2,3-dioxygenase-1 (IDO1), a rate-limiting enzyme in the conversion of tryptophan to neurotoxic metabolites, to this comorbidity using the spared nerve injury (SNI) model of neuropathic pain in mice. SNI resulted in unilateral mechanical allodynia, reduced social interaction, and increased immobility in the forced swim test without changes in locomotor activity. These findings indicate SNI-induced pain and comorbid depression-like behavior. These behavioral responses were accompanied by increases in plasma kynurenine/tryptophan ratios and increased expression of Ido1 and Il1b mRNA in the liver. Interestingly, SNI did not induce detectable changes in spinal cord or brain Ido1 mRNA levels. SNI was associated with spinal cord inflammatory activity as evidenced by increased Il1b mRNA expression. The SNI-induced increase of liver Ido1and Il1b mRNA was abrogated by intrathecal administration of the IL-1 inhibitor IL-1RA. Intrathecal IL-1RA also inhibited both mechanical allodynia and depression-like behavior. We also show that Ido1 is required for the development of depression-like behavior because Ido1(-/-) mice do not develop increased immobility in the forced swim test or decreased social exploration in response to SNI. Mechanical allodynia was similar in WT and Ido1(-/-) mice. In conclusion, our findings show for the first time that neuropathic pain is associated with an increase of Ido1 in liver, but not brain, downstream of spinal cord IL-1ß signaling and that Ido1 mediates comorbid depression. Moreover, comorbidity of neuropathic pain and depression are only partially mediated by a common mechanism because mechanical hyperalgesia develops independently of Ido1.

Vortioxetine, but not escitalopram or duloxetine, reverses memory impairment induced by central 5-HT depletion in rats: evidence for direct 5-HT receptor modulation.

Depressed patients suffer from cognitive dysfunction, including memory deficits. Acute serotonin (5-HT) depletion impairs memory and mood in vulnerable patients. The investigational multimodal acting antidepressant vortioxetine is a 5-HT3, 5-HT7 and 5-HT1D receptor antagonist, 5-HT1B receptor partial agonist, 5-HT1A receptor agonist and 5-HT transporter (SERT) inhibitor that enhances memory in normal rats in novel object recognition (NOR) and conditioned fear (Mørk et al., 2013). We hypothesized that vortioxetine's 5-HT receptor mechanisms are involved in its memory effects, and therefore investigated these effects in 5-HT depleted rats. Four injections of the irreversible tryptophan hydroxylase inhibitor 4-chloro-dl-phenylalanine methyl ester hydrochloride (PCPA, 86mg/kg, s.c.) induced 5-HT depletion, as measured in hippocampal homogenate and microdialysate. The effects of acute challenge with vortioxetine or the 5-HT releaser fenfluramine on extracellular 5-HT were measured in PCPA-treated and control rats. PCPA's effects on NOR and spontaneous alternation (SA) performance were assessed along with the effects of acute treatment with 5-hydroxy-l-tryptophan (5-HTP), vortioxetine, the selective 5-HT reuptake inhibitor escitalopram, or the 5-HT norepinephrine reuptake inhibitor duloxetine. SERT occupancies were estimated by ex vivo autoradiography. PCPA depleted central 5-HT by >90% in tissue and microdialysate, and impaired NOR and SA performance. Restoring central 5-HT with 5-HTP reversed these deficits. At similar SERT occupancies (>90%) vortioxetine, but not escitalopram or duloxetine, restored memory performance. Acute fenfluramine significantly increased extracellular 5-HT in control and PCPA-treated rats, while vortioxetine did so only in control rats. Thus, vortioxetine restores 5-HT depletion impaired memory performance in rats through one or more of its receptor activities.

Age-dependent alterations of the kynurenine pathway in the YAC128 mouse model of Huntington disease.

Indoleamine 2,3 dioxygenase (Ido1), the first and rate-limiting enzyme of the kynurenine pathway (KP), is a striatally enriched gene with increased expression levels in the YAC128 mouse model of Huntington disease (HD). Our objective in this study was to delineate age-related KP alterations in this model. Three enzymes potentially catalyze the first step of the KP; Ido1 and Indoleamine 2,3 dioxygenase-2 were highly expressed in the striatum and Tryptophan 2,3 dioxygenase (Tdo2) in the cerebellum. During development, Ido1 mRNA expression is dynamically regulated and chronically up-regulated in YAC128 mice. Kynurenine (Kyn) to tryptophan (Trp) ratio, a measure of activity in the first step of the KP, was elevated in YAC128 striatum, but no change in Tdo2 mRNA levels or Kyn to Trp ratio was detected in the cerebellum. Ido1 induction was coincident with Trp depletion at 3 months and Kyn accumulation at 12 months of age in striatum. Changes in downstream KP metabolites of YAC128 mice generally followed a biphasic pattern with neurotoxic metabolites reduced at 3 months and increased at 12 months of age. Striatally specific induction of Ido1 and downstream KP alterations suggest involvement in HD pathogenesis, and should be taken into account in future therapeutic developments for HD.

NMDA receptor blockade by ketamine abrogates lipopolysaccharide-induced depressive-like behavior in C57BL/6J mice.

We have previously demonstrated that lipopolysaccharide (LPS) induces depressive-like behavior by activating indoleamine 2,3 dioxygenase (IDO; O'Connor et al, 2009c). IDO degrades tryptophan along the kynurenine pathway. Using mass-spectrometry (LC-MS) analysis of kynurenine metabolites in the brain of mice injected at the periphery with 1 mg/kg LPS, we show that LPS activates the kynurenine 3-monooxygenase pathway that ultimately degrades kynurenine into quinolinic acid. As quinolinic acid acts as an N-methyl-D-aspartate (NMDA) receptor agonist, we used the NMDA receptor antagonist ketamine to assess the role of NMDA receptor activation in LPS-induced depressive-like behavior. Here, we report that a low dose of ketamine (6 mg/kg, intraperitoneally) immediately before administration of LPS (0.83 mg/kg, intraperitoneally) in C57Bl/6 J mice abrogated the development of LPS-induced depressive-like behavior, without altering LPS-induced sickness measured by body weight loss, decreased motor activity, and reduced food intake. Depressive-like behavior was measured 24 h after LPS by decreased sucrose preference and increased immobility in the forced swim test (FST). Ketamine had no effect on LPS-induced cytokine expression in the liver and brain, IDO activation, and brain-derived neurotrophic factor (BDNF) transcripts. The ability of ketamine to abrogate LPS-induced depressive-like behavior independently of a possible interference with LPS-induced inflammatory signaling was confirmed when ketamine was administered 10 h after LPS instead of immediately before LPS. In contrast, ketamine had no effect when administered 24 h before LPS. To confirm that NMDA receptor antagonism by ketamine mediates the antidepressant-like activity of this compound in LPS-treated mice, mice were pretreated with the α-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA) receptor antagonist 2,3-dihydroxy-6-nitro-7-sulfamoyl-benzo(f)quinoxaline-2,3-dione (NBQX) to block enhanced AMPA receptor glutamatergic neurotransmission after NMDA receptor antagonism by ketamine. NBQX administered at the dose of 10 mg/kg intraperitoneally 15 min before ketamine in mice treated with LPS 24 h earlier restored LPS-induced decreased sucrose preference. These findings indicate that LPS-induced depressive-like behavior is mediated by NMDA receptor activation, probably as a consequence of formation of quinolinic acid.

A rodent model of premenstrual dysphoria: progesterone withdrawal induces depression-like behavior that is differentially sensitive to classes of antidepressants.

Premenstrual dysphoric disorder (PMDD) is characterized by a range of physical and affective symptoms including anxiety, irritability, anhedonia, social withdrawal and depression. We demonstrate robust and reproducible depression-like behavior during progesterone withdrawal (PWD) protocols with different methodological variables. Comparable immobility in the forced swim test was evident with different routes of administration (i.e. injections vs. implants), with and without exogenous estrogens in addition to progesterone, and in both single and multiple withdrawal paradigms. Furthermore, withdrawal from physiological doses of progesterone resulted in modest social withdrawal in the social preference test and anhedonia in the saccharin preference test without altering general activity levels or total liquid consumption. However, progesterone withdrawal did not alter serotonin levels in the cortex or hippocampus. Furthermore tryptophan depletion did not augment immobility during PWD. Neither fluoxetine nor duloxetine reduced depression-like behavior during PWD in the forced swim test. In contrast, the tricyclic antidepressant, amitriptyline, was effective in reducing the immobility in forced swim test. These data demonstrate that progesterone withdrawal is a reproducible model of PMDD in several critical behavioral domains. Furthermore, these data do not support alterations in serotonin levels in the etiology of hormonally induced depression.