Alterations of serotonin type 4 receptor levels are linked to mood disorders and cognitive deficits in several conditions. However, few studies have investigated 5-HT4R alterations in movement disorders. We wondered whether striatal 5-HT4R expression is altered in experimental parkinsonism. We used a brain bank tissue from a rat and a macaque model of Parkinson\'s disease (PD). We then investigated its in vivo PET imaging regulation in a cohort of macaques. Dopaminergic depletion increases striatal 5-HT4R in the two models, further augmented after dyskinesia-inducing L-Dopa. Pending confirmation in PD patients, the 5-HT4R might offer a therapeutic target for dampening PD\'s symptoms.

Serotonergic dysfunction is related to both motor and nonmotor symptoms in Parkinson\'s disease (PD). As a 5-HT receptor, 5-HT4 receptor (5-HT4R) is well-studied and already-used in clinical therapy of constipation, which is a typical non-motor symptom in PD. In this study, we investigated the role of 5-HT4R as a regulator of gut function in MPTP-induced acute PD mice model. Daily intraperitoneal injection of GR 125487 (5-HT4R antagonist) was administered 3 days before MPTP treatment until sacrifice. Seven days post-MPTP treatment, feces were collected and gastrointestinal transit time (GITT) was measured, 8 days post-MPTP treatment, behavioral tests were performed, and then animals were sacrificed for the further analysis. We found GR 125487 pretreatment not only increased GITT, but also aggravated MPTP-induced motor bradykinesia. In addition, GR 125487 pretreatment exacerbated the loss of dopaminergic neurons probably by suppressing JAK2/PKA/CREB signaling pathway and increased reactive glia and neuroinflammation in the striatum. 16 S rRNA sequencing of fecal microbiota showed that GR 125487 pretreatment altered the composition of gut microbiota, in which the abundance of Akkermansia muciniphila and Clostridium clostridioforme was increased, whereas that of Parabacteroides distasonis and Bacteroides fragilis was decreased, which are closely associated with inflammation condition. Taken together, we demonstrated that GR 125487 pretreatment exacerbates MPTP-induced striatal neurodegenerative processes possibly via the JAK2/PKA/CREB pathway and neuroinflammation by altering gut microbiota composition. In the microbiota-gut-brain axis of PD, 5-HT4R should be further explored and might serve as a target for PD diagnosis and treatment.

Hyperglycemia drives dysfunction of the intestinal barrier. 5-Hydroxytryptaine 4 receptor (5-HT 4R) agonists have been considered therapeutics for constipation in clnic. However, the roles of 5-HT 4R activation in mucosa should be fully realized. Here, we investigate the effects of 5-HT 4R activation on diabetes-induced disruption of the tight junction (TJ) barrier in the colon. Not surprisingly, the TJ barrier in diabetic mice with or without 5-HT 4R is tremendously destroyed, as indicated by increased serum fluorescein isothiocyanate (FITC)-dextran and decreased transepithelial electrical resistance (TER). Simultaneously, decreased expressions of TJ proteins are shown in both wild-type (WT) and 5-HT 4R knockout (KO) mice with diabetes. Notably, chronic treatment with intraperitoneal injection of a 5-HT 4R agonist in WT mice with diabetes repairs the TJ barrier and promotes TJ protein expressions, including occludin, claudin-1 and ZO-1, in the colon, whereas a 5-HT 4R agonist does not improve TJ barrier function or TJ protein expressions in 5-HT 4R KO mice with diabetes. Furthermore, stimulation of 5-HT 4R inhibits diabetes-induced upregulation of myosin light chain kinase (MLCK), Rho-associated coiled coil protein kinase 1 (ROCK1), and phosphorylated myosin light chain (p-MLC), which are key molecules that regulate TJ integrity, in the colonic mucosa of WT mice. However, such action induced by a 5-HT 4R agonist is not observed in 5-HT 4R KO mice with diabetes. These findings indicate that 5-HT 4R activation may restore TJ integrity by inhibiting the expressions of MLCK, ROCK1 and p-MLC, improving epithelial barrier function in diabetes.

The human endogenous retroviruses type W family envelope (HERV-W env) gene is located on chromosome 7q21-22. Our previous studies show that HERV-W env is elevated in schizophrenia and HERV-W env can increase calcium influx. Additionally, the 5-HTergic system and particularly 5-hydroxytryptamine (5-HT) receptors play a prominent role in the pathogenesis and treatment of schizophrenia. 5-hydroxytryptamine receptor 4 (5-HT4R) agonist can block calcium channels. However, the underlying relationship between HERV-W env and 5-HT4R in the etiology of schizophrenia has not been revealed. Here, we used enzyme-linked immunosorbent assay to detect the concentration of HERV-W env and 5-HT4R in the plasma of patients with schizophrenia and we found that there were decreased levels of 5-HT4R and a negative correlation between 5-HT4R and HERV-W env in schizophrenia. Overexpression of HERV-W env decreased the transcription and protein levels of 5-HT4R but increased small conductance Ca2+-activated K+ type 2 channels (SK2) expression levels. Further studies revealed that HERV-W env could interact with 5-HT4R. Additionally, luciferase assay showed that an essential region (-364 to -176 from the transcription start site) in the SK2 promoter was required for HERV-W env-induced SK2 expression. Importantly, 5-HT4R participated in the regulation of SK2 expression and promoter activity. Electrophysiological recordings suggested that HERV-W env could increase SK2 channel currents and the increase of SK2 currents was inhibited by 5-HT4R. In conclusion, HERV-W env could activate SK2 channels via decreased 5-HT4R, which might exhibit a novel mechanism for HERV-W env to influence neuronal activity in schizophrenia.

Diabetes leads to intestinal barrier dysfunction. 5-Hydroxytryptamine 4 receptor (5-HT4R) is distributed in the colonic mucosa, but little is known about the role of its activation in diabetes-evoked colonic barrier dysfunction. This study investigates whether activation of 5-HT4Rs on goblet cells (GCs) protects the colon from commensal bacterial translocation in diabetic mice. Expression of 5-HT4R detected inside the colonic epithelium by RNAscope in situ hybridization was further observed within the mucin 2 (MUC2)-immunoreactive GCs. In diabetic mice, neither 5-HT4R transcription nor protein levels were altered compared with those in nondiabetic mice. Bacterial translocation was characterized by 16S rRNA RNAscope in situ hybridization and manifested in both crypts and lamina propria of the colon in diabetic mice. Mucin production and MUC2 expression were significantly decreased in diabetic mice. Furthermore, the loss of mitochondrial cristae of GCs and the down-regulation of mitofilin, the core protein maintaining mitochondrial homeostasis, were observed in diabetic mice. Long-term treatment with 5-HT4R agonist in diabetic mice not only prevented bacterial penetration of the whole colonic mucosa but also promoted mucin production and MUC2 expression. Markedly, 5-HT4R agonist also restored the mitochondrial cristae of GCs and up-regulated mitofilin. However, co-administration of 5-HT4R antagonist abolished the effects of 5-HT4R agonist on diabetic mice. These findings indicate that 5-HT4R in colonic mucosa is an effective target for the treatment of diabetes-induced colonic mucous barrier dysfunction.

Spinal cord injury (SCI) often leads to defecation dysfunction. Sacral nerve electrical stimulation (SNS) therapy could improve defecation function. The present study aimed to assess SNS therapy, with regard to the levels of serotonin (5‑HT) and its receptors (5‑HT3AR and 5‑HT4R) in the colon and sacral cord, a rat model of acute severe SCI was used. This rat model was made using the New York University Impactor device. Model rats were randomized to the SCI and SNS (electrical stimulation on the S3 nerve) groups. After 14 days of treatment, enteric transmission function was assessed. 5‑HT and 5‑HT3AR/5‑HT4R were measured by ELISA, quantitative PCR, immunohistochemistry and western blotting. In SCI rats, SNS significantly increased the quantity of feces, shortened the time to the first fecal passage, and improved fecal texture and colon histology. SNS elevated 5‑HT contents in the colon and spinal cord, and enhanced 5‑HT3AR/5‑HT4R protein expression and distribution in the colonic myenteric plexus and mucosa, sacral intermediolateral nucleus and dorsal horn. SNS upregulated the relative expression levels of 5‑HT3AR/5‑HT4R mRNA and protein in the colon and spinal cord. SNS can improve defecation and accelerate the recovery of colonic transmission functions in rat models of acute SCI. These effects involved upregulation of the 5‑HT/5‑HT3AR/5‑HT4R axes.

The latest estimates from world health organization suggest that more than 450 million people are suffering from depression and other psychiatric conditions. Of these, 50-60% have been reported to have progression of gut diseases. In the last two decades, researchers introduced incipient physiological roles for serotonin (5-HT) receptors (5-HTRs), suggesting their importance as a potential pharmacological target in various psychiatric and gut diseases. A growing body of evidence suggests that 5-HT systems affect the brain-gut axis in depressive patients, which leads to gut comorbidity. Recently, preclinical trials of 5-HT4R agonists and antagonists were promising as antipsychotic and prokinetic agents. In the current review, we address the possible pharmacological role and contribution of 5-HT4R in the pathophysiology of chronic depression and associated gut abnormalities. Physiologically, during depression episodes, centers of the sympathetic and parasympathetic nervous system couple together with neuroendocrine systems to alter the function of hypothalamic-pituitary-adrenal (HPA) axis and enteric nervous system (ENS), which in turn leads to onset of gastrointestinal tract (GIT) disorders. Consecutively, the ENS governs a broad spectrum of physiological activities of gut, such as visceral pain and motility. During the stages of emotional stress, hyperactivity of the HPA axis alters the ENS response to physiological and noxious stimuli. Consecutively, stress-induced flare, swelling, hyperalgesia and altered reflexes in gut eventually lead to GIT disorders. In summary, the current review provides prospective information about the role and mechanism of 5-HT4R-based therapeutics for the treatment of depressive disorder and possible consequences for the gut via brain-gut axis interactions. This article is part of the special issue entitled \'Serotonin Research: Crossing Scales and Boundaries\'.

Human serotonin receptor 4 (HTR4) encodes a 5-HT4 receptor involved in learning, memory, depression, anxiety, and feeding behavior. The aim of this study was to investigate the association between the deoxyribonucleic acid (DNA) methylation of HTR4 promoter and autism spectrum disorder (ASD), a disease characterized by communication disorder and repetitive or restrictive behavior.Peripheral blood DNA was obtained from 61 ASD children and 66 healthy children, and the DNA methylation of HTR4 promoter was assessed by quantitative methylation-specific polymerase chain reaction. We used percentage of methylated reference (PMR) to represent DNA methylation level.Due to significant age differences between ASD cases and controls (3 [2, 5] years and 6 [5, 6] years, P = 3.34E-10), we used binary logistic regression analysis for adjustment. Our results showed that the DNA methylation levels of HTR4 promoter were significantly lower in children with ASD than in healthy children (median PMR: 66.23% vs 94.31%,P = .028, age-adjusted P = .034). In addition, the DNA methylation of HTR4 promoter was inversely associated with age in male ASD cases (total cases: r = -0.283, P = .027; male cases: r = -0.431, P = .002; female cases: r = -0.108, P = .752). Dual-luciferase reporter gene assay showed that the reporter gene expression in the strain with recombinant pGL3-promoter-HTR4 plasmid was significantly higher than that in the strain with pGL3-promoter plasmid (fold change = 2.01, P = .0065), indicating that the HTR4 promoter fragment may contain transcription factors to upregulate promoter activity.Our study suggested that hypomethylation of the HTR4 promoter is a potential biomarker for predicting the risk of male ASD.

Serotonin (5-HT) homeostasis is critical for the brain development which influences neurogenesis, neuronal migration, and circuit formation. Distinctive distribution patterns of serotonin receptors (5-HTRs) in the brain govern various physiological activities. Amongst the 5-HTRs, serotonin 4 receptor (5-HT4R) is widely expressed in embryonic forebrain and affects neuronal development, synaptogenesis, and behavior, but its specific role in brain development is still not completely understood. Therefore, in the present study, we addressed the roles of 5-HT4R in the growth of hippocampal neurons during the development of mice brain. We cultured hippocampal neurons of the mouse at embryonic day 18 and then treatment of 5-HT4R agonist RS67333 was employed. We found RS67333 significantly increased the axonal length, diameter and branching along with total dendritic length, number of primary dendrites and their branching. In addition, these effects were neutralized by the concomitant treatment of 5-HT4R antagonist GR125487, which confirmed the specific role of the 5-HT4R in the growth of axon and dendrites. Further, the treatment of RS67333 upregulated the mRNA expression of collapsin response mediator protein-2 (CRMP2) and non-phosphorylated CRMP2 (npCRMP2) together with neurotrophic factors (BDNF, NT-3, NGF) and TRK-A. Additionally, the current research findings reveal that the knockdown of CRMP2 inhibited RS67333-induced growth of the axons and dendrites, which indicates that CRMP2 is required for the 5-HT4R-mediated growth of the axons and dendrites. Overall, the findings of the present in vitro study enrich the understanding and provide insight roles of 5-HT4R in embryonic brain development by promoting the growth of hippocampal neurons.

The Janus kinase (JAK)/signal transducer and activator of transcription 3 (STAT3) signaling pathway plays central roles in cancer cell growth and survival. Drug repurposing strategies have provided a valuable approach for developing antitumor drugs. Zelnorm (tegaserod maleate) was originally designed as an agonist of 5-hydroxytryptamine 4 receptor (5-HT4R) and approved by the FDA for treating irritable bowel syndrome with constipation (IBS-C). Through the use of a high-throughput drug screening system, Zelnorm was identified as a JAK/STAT3 signaling inhibitor. Moreover, the inhibition of STAT3 phosphorylation by Zelnorm was independent of its original target 5-HT4R. Zelnorm could cause G1 cell cycle arrest, induce cell apoptosis and inhibit the growth of a variety of cancer cells. The present study identifies Zelnorm as a novel JAK/STAT3 signaling inhibitor and reveals a new clinical application of Zelnorm upon market reintroduction.