The Microbiota-Gut-Brain Axis.

The importance of the gut-brain axis in maintaining homeostasis has long been appreciated. However, the past 15 yr have seen the emergence of the microbiota (the trillions of microorganisms within and on our bodies) as one of the key regulators of gut-brain function and has led to the appreciation of the importance of a distinct microbiota-gut-brain axis. This axis is gaining ever more traction in fields investigating the biological and physiological basis of psychiatric, neurodevelopmental, age-related, and neurodegenerative disorders. The microbiota and the brain communicate with each other via various routes including the immune system, tryptophan metabolism, the vagus nerve and the enteric nervous system, involving microbial metabolites such as short-chain fatty acids, branched chain amino acids, and peptidoglycans. Many factors can influence microbiota composition in early life, including infection, mode of birth delivery, use of antibiotic medications, the nature of nutritional provision, environmental stressors, and host genetics. At the other extreme of life, microbial diversity diminishes with aging. Stress, in particular, can significantly impact the microbiota-gut-brain axis at all stages of life. Much recent work has implicated the gut microbiota in many conditions including autism, anxiety, obesity, schizophrenia, Parkinson's disease, and Alzheimer's disease. Animal models have been paramount in linking the regulation of fundamental neural processes, such as neurogenesis and myelination, to microbiome activation of microglia. Moreover, translational human studies are ongoing and will greatly enhance the field. Future studies will focus on understanding the mechanisms underlying the microbiota-gut-brain axis and attempt to elucidate microbial-based intervention and therapeutic strategies for neuropsychiatric disorders.

A Dairy-Derived Ghrelinergic Hydrolysate Modulates Food Intake In Vivo.

Recent times have seen an increasing move towards harnessing the health-promoting benefits of food and dietary constituents while providing scientific evidence to substantiate their claims. In particular, the potential for bioactive protein hydrolysates and peptides to enhance health in conjunction with conventional pharmaceutical therapy is being investigated. Dairy-derived proteins have been shown to contain bioactive peptide sequences with various purported health benefits, with effects ranging from the digestive system to cardiovascular circulation, the immune system and the central nervous system. Interestingly, the ability of dairy proteins to modulate metabolism and appetite has recently been reported. The ghrelin receptor (GHSR-1a) is a G-protein coupled receptor which plays a key role in the regulation of food intake. Pharmacological manipulation of the growth hormone secretagogue receptor-type 1a (GHSR-1a) receptor has therefore received a lot of attention as a strategy to combat disorders of appetite and body weight, including age-related malnutrition and the progressive muscle wasting syndrome known as cachexia. In this study, a milk protein-derivative is shown to increase GHSR-1a-mediated intracellular calcium signalling in a concentration-dependent manner in vitro. Significant increases in calcium mobilisation were also observed in a cultured neuronal cell line heterologously expressing the GHS-R1a. In addition, both additive and synergistic effects were observed following co-exposure of GHSR-1a to both the hydrolysate and ghrelin. Subsequent in vivo studies monitored standard chow intake in healthy male and female Sprague-Dawley rats after dosing with the casein hydrolysate (CasHyd). Furthermore, the provision of gastro-protected oral delivery to the bioactive in vivo may aid in the progression of in vitro efficacy to in vivo functionality. In summary, this study reports a ghrelin-stimulating bioactive peptide mixture (CasHyd) with potent effects in vitro. It also provides novel and valuable translational data supporting the potential role of CasHyd as an appetite-enhancing bioactive. Further mechanistic studies are required in order to confirm efficacy as a ghrelinergic bioactive in susceptible population groups.

Molecular, biochemical and behavioural evidence for a novel oxytocin receptor and serotonin 2C receptor heterocomplex.

The complexity of oxytocin-mediated functions is strongly associated with its modulatory effects on other neurotransmission systems, including the serotonin (5-hydroxytryptamine, 5-HT) system. Signalling between oxytocin (OT) and 5-HT has been demonstrated during neurodevelopment and in the regulation of specific emotion-based behaviours. It is suggested that crosstalk between neurotransmitters is driven by interaction between their specific receptors, particularly the oxytocin receptor (OTR) and the 5-hydroxytryptamine 2C receptor (5-HTR2C), but evidence for this and the downstream signalling consequences that follow are lacking. Considering the overlapping central expression profiles and shared involvement of OTR and 5-HTR2C in certain endocrine functions and behaviours, including eating behaviour, social interaction and locomotor activity, we investigated the existence of functionally active OTR/5-HTR2C heterocomplexes. Here, we demonstrate evidence for a potential physical interaction between OTR and 5-HTR2Cin vitro in a cellular expression system using flow cytometry-based FRET (fcFRET). We could recapitulate this finding under endogenous expression levels of both receptors via in silico analysis of single cell transcriptomic data and ex vivo proximity ligation assay (PLA). Next, we show that co-expression of the OTR/5-HTR2C pair resulted in a significant depletion of OTR-mediated Gαq-signalling and significant changes in receptor trafficking. Of note, attenuation of OTR-mediated downstream signalling was restored following pharmacological blockade of the 5-HTR2C. Finally, we demonstrated a functional relevance of this novel heterocomplex, in vivo, as 5-HTR2C antagonism increased OT-mediated hypoactivity in mice. Overall, we provide compelling evidence for the formation of functionally active OTR/5-HTR2C heterocomplexes, adding another level of complexity to OTR and 5-HTR2C signalling functionality. This article is part of the special issue on Neuropeptides.

A ghrelin receptor and oxytocin receptor heterocomplex impairs oxytocin mediated signalling.

Oxytocin mediates its behavioural effects via the centrally expressed oxytocin receptor (OTR). Oxytocin signalling has been implicated in multiple disorders involving centrally regulated pathways, including obesity, autism, schizophrenia and depression. The OTR has been described to have a complex downstream signalling pathway and an increased understanding of oxytocinergic signalling is needed for the development of novel and better treatments for centrally regulated disorders. The ghrelin receptor (GHSR), known primarily for its role in centrally regulated energy balance and food intake, has in more recent years also been shown to play a role in mood disorders, including anxiety and depression. Although there have been suggestions of crosstalk between both signalling systems, these have largely been unexplored to date. Here we show, to our knowledge for the first-time, compelling evidence for the formation of an OTR and GHSR heterocomplex, resulting in significant modulation of OTR downstream signalling. Co-localized expression of the OTR and GHSR is shown in a heterologous cellular expression system and in primary cultures of the hypothalamus and hippocampus. A physical interaction between the OTR and GHSR is confirmed using flow-cytometry based fluorescence resonance energy transfer (fcFRET). Interestingly, co-expression of the GHSR results in a significant attenuation of OTR-mediated Gαq signalling and changes in receptor trafficking within the cell. Together, these data demonstrate a potential functional relevance of an OTR/GHSR heterocomplex and its ability to alter OTR signalling, which is poised to have important implications for future therapeutic strategies, involving oxytocinergic signalling. This article is part of the Special Issue entitled 'Receptor heteromers and their allosteric receptor-receptor interactions'.

Attenuation of Oxytocin and Serotonin 2A Receptor Signaling through Novel Heteroreceptor Formation.

The oxytocin receptor (OTR) and the 5-hydroxytryptamine 2A receptor (5-HTR2A) are expressed in similar brain regions modulating central pathways critical for social and cognition-related behaviors. Signaling crosstalk between their endogenous ligands, oxytocin (OT) and serotonin (5-hydroxytryptamine, 5-HT), highlights the complex interplay between these two neurotransmitter systems and may be indicative of the formation of heteroreceptor complexes with subsequent downstream signaling changes. In this study, we assess the possible formation of OTR-5HTR2A heteromers in living cells and the functional downstream consequences of this receptor-receptor interaction. First, we demonstrated the existence of a physical interaction between the OTR and 5-HTR2Ain vitro, using a flow cytometry-based FRET approach and confocal microscopy. Furthermore, we investigated the formation of this specific heteroreceptor complex ex vivo in the brain sections using the Proximity Ligation Assay (PLA). The OTR-5HTR2A heteroreceptor complexes were identified in limbic regions (including hippocampus, cingulate cortex, and nucleus accumbens), key regions associated with cognition and social-related behaviors. Next, functional cellular-based assays to assess the OTR-5HTR2A downstream signaling crosstalk showed a reduction in potency and efficacy of OT and OTR synthetic agonists, carbetocin and WAY267464, on OTR-mediated Gαq signaling. Similarly, the activation of 5-HTR2A by the endogenous agonist, 5-HT, also revealed attenuation in Gαq-mediated signaling. Finally, altered receptor trafficking within the cell was demonstrated, indicative of cotrafficking of the OTR/5-HTR2A pair. Overall, these results constitute a novel mechanism of specific interaction between the OT and 5-HT neurotransmitters via OTR-5HTR2A heteroreceptor formation and provide potential new therapeutic strategies in the treatment of social and cognition-related diseases.