Enzyme Tryptophan Hydroxylase Research Articles

The effects of brain serotonin deficiency on the behavioral and neurogenesis-promoting effects of voluntary exercise in tryptophan hydroxylase 2 (R439H) knock-in mice

Exercise is known to reduce depression and anxiety symptoms. Although the cellular and molecular mechanisms underlying this effect remain unknown, exercise-induced increases in neurotransmitter release and hippocampal neurogenesis have been hypothesized to play key roles. One neurotransmitter that has been implicated in both antidepressant-like effects and the regulation of hippocampal neurogenesis is serotonin (5-HT). Complete loss of function of the brain 5-HT synthesis enzyme (tryptophan hydroxylase 2, Tph2) has been reported to prevent exercise-induced increases in neurogenesis and to block a subset of antidepressant-like responses to selective serotonin reuptake inhibitors (SSRIs), but whether partial loss of Tph2 function blocks the behavioral and neurogenic effects of exercise has not been established. This study used four tests that are predictive of antidepressant efficacy to determine the impact of 5-HT deficiency on responses to exercise in male and female mice. Our results demonstrate that low 5-HT impairs the behavioral effects of exercise in females in the forced swim and novelty-suppressed feeding tests. However, genetic reductions in 5-HT synthesis did not significantly impact exercise-induced alterations in cellular proliferation or immature neuron production in the hippocampus in either sex. These findings highlight the importance of brain 5-HT in mediating behavioral responses to exercise and suggest that individual differences in brain 5-HT synthesis could influence sensitivity to the mental health benefits of exercise. Furthermore, the observed disconnect between neurogenic and behavioral responses to exercise suggests that increased neurogenesis is unlikely to be the primary driver of the behavioral effects of exercise observed here.

Clarifying the Functional Role of Serotonin in Meloidogyne graminicola Host Plant Parasitism by Immunolocalization and RNA Interference.

Serotonin (5-hydroxytryptamine) is an essential neurotransmitter involved in regulating various behaviors in plant-parasitic nematodes, including locomotion, egg laying, feeding, and mating. However, the functional role of serotonin in root-knot nematode invasion of host plants and the molecular mechanisms underlying feeding behavior remain poorly understood. In this study, we tested the effects of exogenous serotonin and the pharmacological compounds fluoxetine and methiothepin on the feeding behaviors of Meloidogyne graminicola. Our results suggested that M. graminicola possesses an endogenous serotonin signaling pathway and that serotonin plays a crucial role in modulating feeding behaviors in M. graminicola second-stage juveniles. We also identified and cloned the serotonin synthesis enzyme tryptophan hydroxylase (Mg-tph-1) in M. graminicola and investigated the role of endogenous serotonin by generating RNA interference nematodes in Mg-tph-1. Silencing Mg-tph-1 substantially reduced nematode invasion, development, and reproduction. According to the immunostaining results, we speculated that these serotonin immunoreactive cells near the nerve ring in M. graminicola are likely homologous to Caenorhabditis elegans ADFs, NSMs, and RIH serotonergic neurons. Furthermore, we investigated the impact of phytoserotonin on nematode invasion and development in rice by overexpressing OsTDC-3 or supplementing rice plants with tryptamine and found that an increase in phytoserotonin increases nematode pathogenicity. Overall, our study provides insights into the essential role of serotonin in M. graminicola host plant parasitism and proposes that the serotonergic signaling pathway could be a potential target for controlling plant-parasitic nematodes.

Effect of (R)-2-Amino-6-(1R,2S)-1,2-Dihydroxypropyl)-5,6,7,8-Tetrahydropterin-4(3H)-One and Its Structural Analogues on the Temperature Stability of Tryptophan Hydroxylase 2 with the P447R Mutation.

The enzyme tryptophan hydroxylase 2 (TPH2) catalyzes the hydroxylation of L-tryptophan to L-5-hydroxytryptophan (5-HTP), the first and the key step in 5-HT synthesis in the mammalian brain. Mutations in the human Tph2 gene reducing enzyme activity increase the risk of psychopathology. Pharmacological chaperones are small molecules that can specifically bind to mutant protein molecules, restore their disturbed 3D structure to the native state, and increase their stability and functional activity. The chaperone activity of (R)-2-amino-6-(1R,2S)-1,2-dihydroxypropyl)-5,6,7,8-tetrahydropterin-4(3H)-one (BH4) is expressed by increasing the in vitro thermal stability of mutant tyrosine hydroxylase and phenylalanine hydroxylase molecules which are similar to TPH2 in their structure and characteristics. The P447R substitution in the mouse TPH2 molecule results in a 2-fold decrease in enzyme activity in their brains. We studied the effect of this mutation on the TPH2 thermal stability, as well as on the ability of BH4 and its 8 structural analogues to increase the thermal stability of the mutant TPH2 from midbrain extracts of BALB/C mice. Temperature stability was studied by the decrease in enzyme activity during its heating for 2 min at increasing temperatures and was evaluated by the T50 value that is the temperature at which the enzyme activity decreased by half. For the mutant TPH2, the T50 value was decreased compared to the wild type enzyme. BH4 and its closest structural analogue, 6-methyl-5,6,7,8-tetrahydropterin, increased the T50 value, i.e., exhibited chaperone activity. Other close BH4 analogs, 6,7-dimethyl-5,6,7,8-tetrahydropterin and folic acid, were not effective. It can be assumed that BH4 can be effective in the treatment of mental disorders caused by mutations in the Tph2 gene.

Involvement of the Spinal Serotonergic System in the Analgesic Effect of [6]-Shogaol in Oxaliplatin-Induced Neuropathic Pain in Mice.

Oxaliplatin is a chemotherapy drug that can induce severe acute neuropathy in patients within hours of treatment. In our previous study, 10 mg/kg [6]-shogaol (i.p.) significantly alleviated cold and mechanical allodynia induced by a 6 mg/kg oxaliplatin injection (i.p.); however, the precise serotonin-modulatory effect has not been investigated. In this study, we showed that intrathecal injections of NAN-190 (5-HT1A receptor antagonist, 1 µg) and MDL-72222 (5-HT3 receptor antagonist, 15 µg), but not ketanserin (5-HT2A receptor antagonist, 1 µg), significantly blocked the analgesic effect of [6]-shogaol (10 mg/kg, i.p.). Furthermore, the gene expression of the serotonin-synthesizing enzyme tryptophan hydroxylase 2 (TPH2) and serotonin levels in the spinal cord and serum were significantly downregulated (p < 0.0001 and p = 0.0002) and upregulated (p = 0.0298 and p = 0.0099) after oxaliplatin and [6]-shogaol administration, respectively. Moreover, both the gene and protein expression of the spinal serotonin receptors 5-HT1A and 5-HT3 significantly increased after [6]-shogaol injections (p < 0.0001). Finally, intrathecal injections of both receptor agonists (8-OH-DPAT; 5-HT1A receptor agonist, 10 µg and m-CPBG; 5-HT3 receptor agonist, 15 µg) mimicked the effects of [6]-shogaol in oxaliplatin-injected mice. Taken together, these results demonstrate that [6]-shogaol attenuates oxaliplatin-induced neuropathic pain by modulating the spinal serotoninergic system.

FRI167 Implication Of Serotonin In The Physiopathology Of Aldosterone-Producing Adenoma

Abstract Disclosure: C. Duparc: None. M. D'agostino: None. A. Lopez: None. S. Renouf: None. E. Louiset: None. H. Lefebvre: None. In the human adrenal gland, serotonin (5-HT), released by subcapsular mast cells strongly stimulates expression of CYP11B2, encoding aldosterone-synthase and aldosterone production. The effect of 5-HT on adrenocortical cells is mediated by activation of type 4 serotonin receptor (5-HT4R) positively coupled to cAMP/protein kinase A (PKA) signaling pathway and calcium influx. Consistently, administration of cisapride, a 5-HT4R agonist, to healthy volunteers induces a significant increase in plasma aldosterone levels. In aldosterone producing adenomas (APA), the density of mast cells is enhanced in comparison with normal adrenals. Moreover, molecular studies have shown overexpression of HTR4 gene, encoding 5-HT4R, in APA tissues. Administration of cisapride to patients with APA induced more robust plasma aldosterone responses than in healthy voluntaries. These results suggest that 5-HT could play a role in physiopathology of APA. The aim of our study was to examine expression of genes and proteins involved in aldosterone production and actors of the serotonergic pathway in a series of 31 adenomas removed from patients with primary aldosteronism. We have also investigated the in vitro effect of various 5-HT4R ligands on aldosterone production by explants or cultured cells derived from adenoma tissues collected at surgery. Firstly, aldosterone synthase (AS) immunostaining allowed identification of adenoma phenotypes accordingly to the new international histopathology consensus for unilateral Primary aldosteronism. Among the 31 tissues studied, 20 were classified as classical APAs, i.e. presenting with homogenous or heterogenous AS distribution, remaining being considered as 11 non-classical APA. Expression of the gene encoding the 5-HT-synthesizing enzyme tryptophan hydroxylase was similar in APA and normal adrenals. HTR4 overexpression was more robust in classical than non-classical APAs. HTR4 mRNA levels were positively correlated to those encoding CYP11B2. In addition, 5-HT4R immunostaining was widely distributed in most APA tissues, a pattern which was not exclusively noticed in AS-positive adenomas. BIMU-8, a potent 5-HT4R agonist, strongly increased aldosterone production by perifused APA explants. This effect was blocked by administration of GR-113808, a 5-HT4R antagonist. In cultured APA cells, BIMU-8 stimulated aldosterone synthesis in a dose-dependent manner with a higher efficiency than that observed in normal adrenocortical cells. Aldosterone secretion was less strongly stimulated by prucalopride, a partial 5-HT4R agonist used in the treatment of chronic constipation. These data reveal an enhancement of the 5-HT signaling pathway in APAs in comparison with normal adrenals, suggesting that 5-HT and the 5-HT4R may participate in the pathophysiology of primary aldosteronism. Presentation: Friday, June 16, 2023

Resilience to Pain-Related Depression in σ1 Receptor Knockout Mice Is Associated with the Reversal of Pain-Induced Brain Changes in Affect-Related Genes.

Mice lacking the σ1 receptor chaperone (σ1R-/-) are resilient to depressive-like behaviors secondary to neuropathic pain. Examining the resilience's brain mechanisms could help develop conceptually novel therapeutic strategies. We explored the diminished motivation for a natural reinforcer (white chocolate) in the partial sciatic nerve ligation (PSNL) model in wild-type (WT) and σ1R-/- mice. In the same mice, we performed a comprehensive reverse transcription quantitative PCR (qPCR) analysis across ten brain regions of seven genes implicated in pain regulation and associated affective disorders, such as anxiety and depression. PSNL induced anhedonic-like behavior in WT but not in σ1R-/- mice. In WT mice, PSNL up-regulated dopamine transporter (DAT) and its rate-limiting enzyme, tyrosine hydroxylase (Th), in the ventral tegmental area (VTA) and periaqueductal gray (PAG) as well as the serotonin transporters (SERT) and its rate-limiting enzyme tryptophan hydroxylase 2 (Tph2) in VTA. In addition, μ-opioid receptor (MOR) and σ1R were up-regulated in PAG, and MOR was also elevated in the somatosensory cortex (SS) but down-regulated in the striatum (STR). Finally, increased BDNF was found in the medial prefrontal cortex (mPFC) and hypothalamus (HPT). Sham surgery also produced PSNL-like expression changes in VTA, HPT, and STR. Genetic deletion of the σ1R in mice submitted to PSNL or sham surgery prevented changes in the expression of most of these genes. σ1R is critically involved in the supraspinal gene expression changes produced by PSNL and sham surgery. The changes in gene expression observed in WT mice may be related to pain-related depression, and the absence of these changes observed in σ1R-/- mice may be related to resilience.

Impact of the circadian nuclear receptor REV-ERBα in dorsal raphe 5-HT neurons on social interaction behavior, especially social preference

Social interaction among conspecifics is essential for maintaining adaptive, cooperative, and social behaviors, along with survival among mammals. The 5-hydroxytryptamine (5-HT) neuronal system is an important neurotransmitter system for regulating social behaviors; however, the circadian role of 5-HT in social interaction behaviors is unclear. To investigate whether the circadian nuclear receptor REV-ERBα, a transcriptional repressor of the rate-limiting enzyme tryptophan hydroxylase 2 (Tph2) gene in 5-HT biosynthesis, may affect social interaction behaviors, we generated a conditional knockout (cKO) mouse by targeting Rev-Erbα in dorsal raphe (DR) 5-HT neurons (5-HTDR-specific REV-ERBα cKO) using the CRISPR/Cas9 gene editing system and assayed social behaviors, including social preference and social recognition, with a three-chamber social interaction test at two circadian time (CT) points, i.e., at dawn (CT00) and dusk (CT12). The genetic ablation of Rev-Erbα in DR 5-HTergic neurons caused impaired social interaction behaviors, particularly social preference but not social recognition, with no difference between the two CT points. This deficit of social preference induced by Rev-Erbα in 5-HTDR-specific mice is functionally associated with real-time elevated neuron activity and 5-HT levels at dusk, as determined by fiber-photometry imaging sensors. Moreover, optogenetic inhibition of DR to nucleus accumbens (NAc) 5-HTergic circuit restored the impairment of social preference in 5-HTDR-specific REV-ERBα cKO mice. These results suggest the significance of the circadian regulation of 5-HT levels by REV-ERBα in regulating social interaction behaviors.

Efficient biosynthesis of high-value 5‑Hydroxytryptophan using a multienzyme cascade

5-Hydroxytryptophan (5-HTP) is a precursor of serotonin. The increasing demand for 5-HTP is driving the search for a more efficient and cost-effective production method. In this study, a new in vitro multi-enzyme cascade system (MECCS) was developed to convert L-tryptophan (L-trp) to 5-HTP in a single container while achieving the cycling and regeneration of cofactors and coenzymes. The rate-limiting enzyme tryptophan hydroxylase (TPH) was truncated to improve the overall efficiency of the cascade. After screening, the mutant M1 (NΔ143/CΔ26) showed excellent enzymatic performance, with a 2.3-fold increase in catalytic activity. Molecular dynamics simulations showed that the increase in active center binding force and the improvement of substrate tunnel in M1 improved the overall catalytic efficiency. In addition, the rigidity enhancement of the ligand binding pocket surrounding the region improved the thermal stability of the enzyme. Under optimized conditions, the MECCS converted 50 mM L-trp to 36.9 mM 5-HTP, with a conversion rate of 73.8%. This study provides an alternative pathway for the efficient production of 5-HTP.

Serotonin in the Pathogenesis of Lymphocytic Colitis.

Lymphocytic colitis (LC) is a chronic inflammatory disease associated with watery diarrhea, abdominal pain, and colonic intraepithelial lymphocytosis. Serotonin (5-hydroxytryptamine, 5-HT) is reported to increase in certain colon diseases; however, little is known regarding its metabolism in LC. In the present work, the level of 5-HT in serum and the number of enteroendocrine cells (EECs) as well as the expression of the 5-HT rate-limiting enzyme tryptophan hydroxylase 1 (TPH1) in colonic biopsies and urine 5-hydroxyindoeoacetic acid (5-HIAA) were determined in 36 LC patients that were treated with budesonide and 32 healthy controls. The 5-HT serum and 5-HIAA urine levels were measured using ELISA, the EEC number was determined immunohistochemically, and the colonic TPH1 mRNA expression was determined using RT-PCR. The levels of 5-HT and 5-HIAA and the number of EECs were higher in LC patients than in the controls, and positive correlations were observed between the 5-HT and 5-HIAA levels, 5-HT and EEC number, TPH1 mRNA and EEC number, as well as the severity of disease symptoms and 5-HIAA. Budesonide decreased the levels of 5-HT, 5-HIAA, and TPH1 expression and the number of EECs to values that did not differ from those for controls. In conclusion, the serotonin metabolism may be important for LC pathogenesis, and the urinary level of 5-HIAA may be considered as a non-invasive marker of this disease activity.

Serotonin as a Mitogen in the Gastrointestinal Tract: Revisiting a Familiar Molecule in a New Role.

Serotonin signaling is ubiquitous in the gastrointestinal (GI) system, where it acts as a neurotransmitter in the enteric nervous system (ENS) and influences intestinal motility and inflammation. Since its discovery, serotonin has been linked to cellular proliferation in several types of tissues, including vascular smooth muscle, neurons, and hepatocytes. Activation of serotonin receptors on distinct cell types has been shown to induce well-known intracellular proliferation pathways. In the GI tract, potentiation of serotonin signaling results in enhanced intestinal epithelial proliferation, and decreased injury from intestinal inflammation. Furthermore, activation of the type 4 serotonin receptor on enteric neurons leads to neurogenesis and neuroprotection in the setting of intestinal injury. It is not surprising that the mitogenic properties of serotonin are pronounced within the GI tract, where enterochromaffin cells in the intestinal epithelium produce 90% of the body’s serotonin; however, these proliferative effects are attributed to increased serotonin signaling within the ENS compartment as opposed to the intestinal mucosa, which are functionally and chemically separate by virtue of the distinct tryptophan hydroxylase enzyme isoforms involved in serotonin synthesis. The exact mechanism by which serotonergic neurons in the ENS lead to intestinal proliferation are not known, but the activation of muscarinic receptors on intestinal crypt cells indicate that cholinergic signaling is essential to this signaling pathway. Further understanding of serotonin’s role in mucosal and enteric nervous system mitogenesis may aid in harnessing serotonin signaling for therapeutic benefit in many GI diseases, including inflammatory bowel disease, malabsorptive conditions, and cancer.

Inhibition of mechanistic target of rapamycin signaling decreases levels of O-GlcNAc transferase and increases serotonin release in the human placenta.

Changes in placental function, in particular down-regulation of placental O-linked N-acetylglucosamine (O-GlcNAc) transferase (OGT) in response to maternal stress and increased placental secretion of serotonin into the fetal circulation following maternal infection, have been mechanistically linked to adverse neurodevelopment in mice. We hypothesized that mechanistic target of rapamycin (mTOR) signaling is a key regulator of trophoblast serotonin synthesis and OGT protein expression and that serotonin is secreted by the human placenta into the fetal circulation. Placental homogenates (n=46) from elective terminations at 8-22 weeks of gestation and from healthy-term women were sexed and the protein levels of OGT and enzymes involved in serotonin synthesis was determined. Primary human trophoblast (PHT) cells were isolated from normal term placenta (n=27), cultured and transfected (n=8) with siRNA targeting a scramble sequence (control), raptor (inhibits mTOR Complex 1 (mTORC1)), or rictor (inhibits mTOR Complex 2 (mTORC2)). Subsequently, conditioned media and PHT cell lysates were collected. Free serotonin concentration was measured using ELISA in cell culture media and in platelet-depleted normal term umbilical vein and artery plasma (n=38). Both mTORC1 and mTORC2 inhibition down-regulated OGT levels in PHT cells. The level of serotonin synthesis enzyme tryptophan hydroxylase (TPH-1) was higher in early gestation female placentas and at term serotonin concentration was three-fold higher in the umbilical vein than in the umbilical artery. Inhibition of mTORC2, but not mTORC1, increased cultured PHT cell serotonin secretion. Our data are consistent with the model that mTOR signaling is a key regulator of trophoblast serotonin synthesis and OGT protein expression.

The ever-changing roles of serotonin.

Serotonin (5-HT) has traditional roles as a key neurotransmitter in the central nervous system and as a regulatory hormone controlling a broad range of physiological functions. Perhaps the most classically-defined functions of 5-HT are centrally in the control of mood, sleep and anxiety and peripherally in the modulation of gastrointestinal motility. A more recently appreciated role for 5-HT has emerged, however, as an important metabolic hormone contributing to glucose homeostasis and adiposity, with a causal relationship existing between circulating 5-HT levels and metabolic diseases. Almost all peripheral 5-HT is derived from specialised enteroendocrine cells, called enterochromaffin (EC) cells, located throughout the length of the lining of the gastrointestinal tract. EC cells are important luminal sensory cells that can detect and respond to an array of ingested nutrients, as well as luminal gut microbiota and their associated metabolites. Intriguingly, the interaction between gut microbiota and EC cells is dynamic in nature and has strong implications for host physiology. In this review, we discuss the traditional and modern functions of 5-HT and highlight an emerging pathway by which gut microbiota influences host health. Serotonin, also known as 5-hydroxytryptamine (5-HT), is an important neurotransmitter, growth factor and hormone that mediates a range of physiological functions. In mammals, serotonin is synthesized from the essential amino acid tryptophan by the rate-limiting enzyme tryptophan hydroxylase (TPH), for which there are two isoforms expressed in distinct cell types throughout the body. Tph1 is mainly expressed by specialized gut endocrine cells known as enterochromaffin (EC) cells and by other non-neuronal cell types such as adipocytes (Walther et al., 2003). Tph2 is primarily expressed in neurons of the raphe nuclei of the brain stem and a subset of neurons in the enteric nervous system (ENS) (Yabut et al., 2019). As 5-HT cannot readily cross the blood-brain barrier, the central and peripheral pools of 5-HT are anatomically separated and as such, act in their own distinct manners (Martin et al., 2017c). In this review we discuss the peripheral roles of serotonin, with particular focus on the interaction of gut-derived serotonin with the gut microbiota, and address emerging evidence linking this relationship with host homeostasis.

Mice lacking spinal α2GABAA receptors: Altered GABAergic neurotransmission, diminished GABAergic antihyperalgesia, and potential compensatory mechanisms preventing a hyperalgesic phenotype

Diminished synaptic inhibition in the superficial spinal dorsal horn contributes to exaggerated pain responses that accompany peripheral inflammation and neuropathy. α2GABAA receptors (α2GABAAR) constitute the most abundant GABAAR subtype at this site and are the targets of recently identified antihyperalgesic compounds. Surprisingly, hoxb8-α2−/− mice that lack α2GABAAR from the spinal cord and peripheral sensory neurons exhibit unaltered sensitivity to acute painful stimuli and develop normal inflammatory and neuropathic hyperalgesia. Here, we provide a comprehensive analysis of GABAergic neurotransmission, of behavioral phenotypes and of possible compensatory mechanisms in hoxb8-α2−/− mice. Our results confirm that hoxb8-α2−/− mice show significantly diminished GABAergic inhibitory postsynaptic currents (IPSCs) in the superficial dorsal horn but no hyperalgesic phenotype. We also confirm that the potentiation of dorsal horn GABAergic IPSCs by the α2-preferring GABAAR modulator HZ-166 is reduced in hoxb8-α2−/− mice and that hoxb8-α2−/− mice are resistant to the analgesic effects of HZ-166. Tonic GABAergic currents, glycinergic IPSCs, and sensory afferent-evoked EPSCs did not show significant changes in hoxb8-α2−/− mice rendering a compensatory up-regulation of other GABAAR subtypes or of glycine receptors unlikely. Although expression of serotonin and of the serotonin producing enzyme tryptophan hydroxylase (TPH2) was significantly increased in the dorsal horn of hoxb8-α2−/− mice, ablation of serotonergic terminals from the lumbar spinal cord failed to unmask a nociceptive phenotype. Our results are consistent with an important contribution of α2GABAAR to spinal nociceptive control but their ablation early in development appears to activate yet-to-be identified compensatory mechanisms that protect hoxb8-α2−/− mice from hyperalgesia.

Interleukin-33 Induces the Enzyme Tryptophan Hydroxylase 1 to Promote Inflammatory Group 2 Innate Lymphoid Cell-Mediated Immunity

Group 2 innate lymphoid cells (ILC2s) regulate immunity, inflammation, and tissue homeostasis. Two distinct subsets of ILC2s have been described: steady-state natural ILC2s and inflammatory ILC2s, which are elicited following helminth infection. However, how tissue-specific cues regulate these two subsets of ILC2s and their effector functions remains elusive. Here, we report that interleukin-33 (IL-33) promotes the generation of inflammatory ILC2s (ILC2INFLAM) via induction of the enzyme tryptophan hydroxylase 1 (Tph1). Tph1 expression was upregulated in ILC2s upon activation with IL-33 or following helminth infection in an IL-33-dependent manner. Conditional deletion of Tph1 in lymphocytes resulted in selective impairment of ILC2INFLAM responses andincreased susceptibility to helminth infection. Further, RNA sequencing analysis revealed altered gene expression in Tph1 deficient ILC2s including inducible Tcell co-stimulator (Icos). Collectively, these data reveal a previously unrecognized function for IL-33, Tph1, and ICOS in promoting inflammatory ILC2 responses and type 2 immunity at mucosal barriers.

Serotonin pathway in carcinoid syndrome: Clinical, diagnostic, prognostic and therapeutic implications

Carcinoid syndrome represents the most common functional syndrome that affects patients with neuroendocrine neoplasms. Its clinical presentation is really heterogeneous, ranging from mild and often misdiagnosed symptoms to severe manifestations, that significantly worsen the patient's quality of life, such as difficult-to-control diarrhoea and fibrotic complications. Serotonin pathway alteration plays a central role in the pathophysiology of carcinoid syndrome, accounting for most clinical manifestations and providing diagnostic tools. Serotonin pathway is complex, resulting in production of biologically active molecules such as serotonin and melatonin, as well as of different intermediate molecules and final metabolites. These activities require site- and tissue-specific catalytic enzymes. Variable expression and activities of these enzymes result in different clinical pictures, according to primary site of origin of the tumour. At the same time, the biochemical diagnosis of carcinoid syndrome could be difficult even in case of typical symptoms. Therefore, the accuracy of the diagnostic methods of assessment should be improved, also attenuating the impact of confounding factors and maybe considering new serotonin precursors or metabolites as diagnostic markers. Finally, the prognostic role of serotonin markers has been only evaluated for its metabolite 5-hydroxyindole acetic acid but, due to heterogeneous and biased study designs, no definitive conclusions have been achieved. The most recent progress is represented by the new therapeutic agent telotristat, an inhibitor of the enzyme tryptophan hydroxylase, which blocks the conversion of tryptophan in 5-hydroxy-tryptophan. The present review investigates the clinical significance of serotonin pathway in carcinoid syndrome, considering its role in the pathogenesis, diagnosis, prognosis and therapy.

A119 AN EX VIVO MODEL TO STUDY THE GUT SEROTONERGIC SYSTEM RESPONSE TO LIVE AND HEAT-KILLED LACTOBACILLUS RHAMNOSUS STRAIN JB-1

Abstract Background Enterochromaffin (EC) cells are one of the most abundant enteroendocrine cells in the intestinal epithelium, responsible for producing and storing the largest pool of serotonin or 5-hydroxytryptamine (5-HT) in the body. 5-HT has been shown to be important for modulating a large number of gastrointestinal reflexes in health and disease. 5-HT can stimulate extrinsic (vagal and spinal afferents) or intrinsic primary afferent neurons (IPANs) which are involved in motility, secretion and vasodilation within the intestines. Where EC cell localized enzyme tryptophan hydroxylase (TpH) isoform 1 is responsible for 5-HT synthesis, serotonin reuptake transporter (Sert) and monoamine oxidase A (Mao A) are responsible for termination by uptake and metabolism of 5-HT respectively. Our previous research has demonstrated the effects Lactobacillus rhamnosus (JB-1) on the firing frequency of spinal nerve fibres and motility. Increasing interest is being focused on potential health benefits of heat-inactivated microbes and purified bacterial components. However, the effect of these heat-killed bacteria on the intestinal epithelium cells, particularly on EC cells, is unknown. Aims Small intestinal organoids are shown to recapitulate in vivo characteristics of the small intestine epithelium. The present study aims to assess the suitability of intestinal organoids to study bacterial effects on the serotonergic system in the gut. Here we determined changes in the gene expression of key mediators in the serotonergic system [serotonin reuptake transporter (Sert), tryptophan hydroxylase-1 (Tph-1) and monoamine oxidase A (Mao A)] in response to live and heat-killed JB-1. Methods Male C57bl/6 mice aged 6–8 weeks were used for both ex vivo and in vivo experiments. Jejunal organoids were grown from whole crypts isolated using DTT-EDTA solution. Live and heat-killed JB-1 bacteria were used as treatments. Gene expression analysis was performed on jejunal organoids and jejunum tissue using qRT-PCR. Results JB-1 induced a significant increase in gene expression of Sert, Mao A and Tph-1. No significant difference was observed between the effects of live and heat-killed bacteria. In contrast the JB-1 increased expression of the peptide hormone CCK. Effects of JB-1 on gene expression in organoid culture were reflective of changes observed in in vivo experiments involving feeding of the bacteria. Conclusions Ex vivo organoid culture could be a useful tool in studying mechanisms underlying bacterial effects on serotonergic signalling. The observation that heat-killed bacteria produced comparable effects to the live organism suggests the possibility of isolating active 5-HT modulating components from these strains. Future research will focus on identifying such bacterial components and how their effects on gene expression influence serotonin availability Funding Agencies None

Modulation of brain serotonin by benzyl butyl phthalate in Fundulus heteroclitus (mummichog).

Endocrine-disrupting chemicals have been known to alter important animal behaviors by modulating serotonin (5-hydroxytryptamine, 5-HT) and dopamine. F. heteroclitus (mummichog) brain serotonin and dopamine levels were quantified by enzyme-linked immunosorbent assay (ELISA) following a 28-day exposure regimen involving daily doses of either 0.1 mg l-1 benzyl butyl phthalate (BBP) dissolved in acetone or acetone alone (0.1 mg l-1). No differences in mean brain mass or total protein homogenate were induced by exposure to the acetone vehicle or BBP in acetone. The acetone vehicle had no effect on dopamine, serotonin, or tyrosine hydroxylase levels, but acetone did decrease tryptophan hydroxylase levels (p = 0.011). Exposure to BBP in acetone decreased dopamine (p = 0.024), increased serotonin (p < 0.001), reduced tryptophan hydroxylase as compared to the acetone vehicle alone (p < 0.001), and had no significant effect on tyrosine hydroxylase levels. This study is the first to report modulation of F. heteroclitus brain serotonin and its enzyme tryptophan hydroxylase following sub-lethal exposure to BBP in an acetone vehicle. In addition, modulation of brain dopamine in F. heteroclitus, sans simultaneous modulation of tyrosine hydroxylase, was also observed. These findings support the use of F. heteroclitus for assessing sub-lethal BBP exposure.

Exploratory study of the effect of one week of orally administered CNSA-001 (sepiapterin) on CNS levels of tetrahydrobiopterin, dihydrobiopterin and monoamine neurotransmitter metabolites in healthy volunteers

Tetrahydrobiopterin (BH4) is a cofactor for the enzymes tyrosine hydroxylase and tryptophan hydroxylase, the rate-limiting enzymes in the production of the neurotransmitters, dopamine and serotonin, respectively, in the central nervous system (CNS). Administration of BH4 is used clinically within the management of persons with genetic BH4 deficiencies, but the BH4 molecule does not cross the blood-brain barrier sufficiently. CNSA-001 is a pharmaceutical preparation of sepiapterin, a natural precursor of BH4 that induced larger increases in plasma BH4 compared with administration of the same doses of BH4 itself in healthy volunteers in a randomized trial. Here, we report the effects of 7 days of once-daily treatment with CNSA-001 60 mg/kg (n = 6) or placebo (n = 2) on metabolites of the BH4 synthetic pathway and on biomarkers of the serotonin (5-hydroxyindoleacetic acid [5-HIAA]) and dopamine (homovanillic acid [HVA]) pathways in cerebrospinal fluid (CSF) in subjects from this trial. There were no notable changes in any metabolite in placebo-treated subjects. Administration of CNSA-001 increased mean BH4 from 18.1 (SD 3.0) to 35.1 (10.0) nmol/L, and of dihydrobiopterin (BH2) from 2.1 (0.3) to 7.9 (1.5) nmol/L. Overall, administration of CNSA-001 had little effect on mean levels (pre- vs. post-treatment) of 5-HIAA (76.1 [SD 29.8] vs. 70.1 [23.1] nmol/L) or HVA (177.2 [66.5] vs. 184.8 [35.3]) nmol/L. One subject with low 5-HIAA and HVA at baseline responded with approximately three-fold increases in CNS levels of these metabolites after CNSA-001 treatment, with post-treatment levels within the range of those seen in other subjects. Administration of CNSA-001 60 mg/kg markedly increased levels of BH4 in the CNS of healthy volunteers, with apparently little overall effect in CNS levels of already normal key neurotransmitter metabolites.

Illicit Upregulation of Serotonin Signaling Pathway in Adrenals of Patients With High Plasma or Intra-Adrenal ACTH Levels.

In the human adrenal, serotonin (5-HT), released by mast cells stimulates corticosteroid secretion through activation of type 4 serotonin receptors (5-HT4R). In primary pigmented nodular adrenocortical disease cells, activation of the cAMP/protein kinase A (PKA) pathway by PRKAR1A mutations triggers upregulation of the 5-HT synthesizing enzyme tryptophan hydroxylase (TPH) and the 5-HT4, 5-HT6, and 5-HT7 receptors. Because ACTH stimulates cortisol secretion through activation of PKA, adrenocortical tissues exposed to sustained stimulation by ACTH may harbor increased expression of TPH and 5-HT4/6/7 receptors. To investigate the effects of long-term ACTH stimulation on the serotonergic pathway in adrenals of patients with high plasma or intra-adrenal ACTH levels. Adrenal tissues were obtained from patients with Cushing disease, ectopic secretion of ACTH [paraneoplastic Cushing syndrome; (paraCS)], 21-hydroxylase deficiency (21-OHD), primary bilateral macronodular adrenal hyperplasia with intra-adrenal ACTH presence, or cortisol-producing adenomas. TPH and 5-HT4/6/7 receptor expression was investigated using RT-PCR and immunochemistry in comparison with normal adrenals. Primary cultured adrenocortical cells originating from a patient with paraCS were incubated with 5-HT and 5-HTR agonists/antagonists. TPH and/or 5-HT4/6/7 receptors were overexpressed in the different types of tissues. In paraCS cultured cells, the cortisol response to 5-HT was exaggerated compared with normal adrenal cells and the stimulatory action of 5-HT was reduced by 5-HT4R antagonist. Our results indicate that prolonged activation of the cAMP/PKA pathway by ACTH induces an aberrant serotonergic stimulatory loop in the adrenal cortex that likely participates in the pathogenesis of corticosteroid hypersecretion.

SUN-356 The Effects of Selective Serotonin Reuptake Inhibitors on Urinary Free Cortisol in Patients with Carney Complex and Primary Pigmented Nodular Adrenocortical Disease

PPNAD is a rare cause of ACTH-independent Cushing syndrome mainly associated with Carney complex (CNC). It is diagnosed by histologic examination or a 6-day Liddle test (LT) showing a paradoxical increase of >50% in 24-h urinary free cortisol (UFC) on the 2nd day of high-dose dexamethasone administration (Day 6 [D6]). PPNAD tissue has neuroendocrine features and can overexpress the serotonin (5-HT) synthesizing enzyme tryptophan hydroxylase type 2 and the 5-HT receptors types 4, 6, and 7, with formation of an illicit stimulatory serotonergic loop whose pharmacological inhibition in vitro decreases cortisol production [1]. SSRIs inhibit the reuptake of 5-HT and are used as 1st-line antidepressants. This is a retrospective cohort study of patients with CNC and PPNAD that underwent a LT with the objective to evaluate the effect of SSRIs on UFCs in these patients. Of the 34 patients (4-65 y) with CNC and PPNAD that had a LT at our institution between 2004 and 2018, 4 patients took an SSRI during testing. No statistically significant differences were demonstrated between the SSRI (S) group and the non-SSRI (NS) group in baseline UFCs and urine 17-hydroxycorticosteroids (17OHS) and the percent increase in UFC and 17OHS on D6. Specifically, the median (IQR) baseline UFC in the S group was 36 (13-252) mcg/24h (nl 4-56) vs 35 (13-98) mcg/24h in the NS group (P=0.95). Baseline 17OHS were 11 (8-18) mg/mg creatinine (Cr) (nl ≤6) in the S group vs 8 (5-11) mg/mgCr in the NS group (P=0.27). The percent change in UFC was 208 (93-683)% in the S group and 185 (28-364)% in the NS group (P=0.89), while the percent change in 17OHS was 56 (40-87)% in the S group and 67 (16-92)% in the NS group (P=0.91). On D6, the S group appeared to have higher UFC [118 (63-635) mcg/24h vs 112 (58-201) mcg/24h; P=0.64] and 17OHS [16 (10-39) mg/mgCr vs 13 (7-17) mg/mgCr; P=0.25] than the NS group, but these were not statistically significant. Though these data do not confirm an effect of SSRIs on UFC and ultimately cortisol production in PPNAD, and the percent change in UFC during the LT was similar between both groups, the higher D6 UFC in the S group, when the baseline UFCs between the 2 groups were similar may suggest a degree of increased cortisol production in the S group. The data are limited by the small number of patients in the S group (n=4). In addition, 50% of patients in the S group vs 77% in the NS group underwent adrenalectomy, which is indicative of more severe disease in the NS group. More data are needed from this cohort to determine if SSRI use affects cortisol production in vivo, as this may have significant diagnostic and therapeutic implications for patients with CNC and PPNAD. Reference: 1. Bram, Z., et al., PKA regulatory subunit 1A inactivating mutation induces serotonin signaling in primary pigmented nodular adrenal disease. JCI Insight, 2016. 1(15): p. e87958.