The functional cooperation of 5−HT1A5-HT_{1A} and mGlu4R in HEK-293 cell line

Background The serotonin 5-HT1A receptor (5-HT1AR) and metabotropic glutamate receptor 4 (mGlu4) have been implicated as sites of antipsychotic drug action. 5-HT1AR belongs to the A class of G protein-coupled receptors (GPCRs); mGlu4 is a representative of class C GPCRs. Both receptors preferentially couple with Gi protein to inhibit cAMP formation. The present work aimed to examine the possibility of mGlu4 and 5-HT1A receptor cross-talk, the phenomenon that could serve as a molecular basis of the interaction of these receptor ligands observed in behavioral studies. Methods First, in vitro studies were performed to examine the pharmacological modulation of interaction of the mGlu4 and 5-HT1A receptors in the T-REx 293 cell line using SNAP- or HALO-tag and cAMP accumulation assay. Next, the colocalization of these two receptors was examined in some regions of the mouse brain by applying RNAScope dual fluorescence in situ hybridization, immunohistochemical labeling, and proximity ligation assay (PLA). Results The ex vivo and in vitro results obtained in the present work suggest the existence of interactions between mGlu4 and 5-HT1A receptors. The changes were observed in cAMP accumulation assay and were dependent on expression and activation of mGlu4R in T-REx 293cell line. Moreover, the existence of spots with proximity expression of both receptors were showed by PLA, immunofluorescence labeling and RNAscope methods. Conclusion The existence of interactions between mGlu4 and 5-HT1A receptors may represent another signaling pathway involved in the development and treatment psychiatric disorders such as schizophrenia or depression

Identification and characterization of aptameric inhibitors of human neutrophil elastase

Human neutrophil elastase (HNE) plays a pivotal role in innate immunity, inflammation and tissue remodelling. Aberrant proteolytic activity of HNE contributes to organ destruction in various chronic inflammatory diseases including emphysema, asthma, and cystic fibrosis. Therefore, elastase inhibitors could alleviate the progression of these disorders. Here, we used systematic evolution of ligands by exponential enrichment (SELEX) to develop single-stranded DNA aptamers that specifically target HNE. We determined the specificity of the designed inhibitors and their inhibitory efficacy against HNE using biochemical and in vitro methods, including an assay of neutrophil activity. Our aptamers inhibit the elastinolytic activity of HNE with nanomolar potency, and are highly specific for HNE and do not target other tested human proteases. As such, this study provides lead compounds suitable for the evaluation of their tissue-protective potential in animal models

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

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-HTR2C in 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 G alpha q-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

Screening and characterization of aptamers recognizing the periodontal pathogen Tannerella forsythia

Periodontal disease is one of the most common forms of inflammation. It is currently diagnosed by observing symptoms such as gingival bleeding and attachment loss. However, the detection of biomarkers that precede such symptoms would allow earlier diagnosis and prevention. Aptamers are short oligonucleotides or peptides that fold into three‐dimensional conformations conferring the ability to bind molecular targets with high affinity and specificity. Here we report the selection of aptamers that bind specifically to the bacterium Tannerella forsythia, a pathogen frequently associated with periodontal disease. Two aptamers with the highest affinity were examined in more detail, revealing that their binding is probably dependent on mirolysin, a surface‐associated protease secreted by the T. forsythia type‐9 secretion system. The aptamers showed minimal cross‐reactivity to other periodontopathogens and are therefore promising leads for the development of new tools to study the composition of the periodontitis‐associated dysbiotic bacteriome as well as inexpensive new diagnostic assays