Periodic Reporting for period 1 - SynGut (Molecular, morphological, and functional requirements for gastrointestinal serotonin release)
Reporting period: 2020-03-01 to 2022-02-28
Signaling between the gut and the brain is important for the regulation of many different behavioral and physiological processes. In the intestine, a highly heterogenous group of sensory secretory cells named enteroendocrine cells (EECs) is found embedded within the intestinal epithelium and capable of signaling through a variety of different peptide hormones and neurotransmitters. Due to their prime physical location, EECs are exposed to the contents of the gut lumen, sense changes in this environment, and transmit this information to the body and neurons of the enteric nervous system and those signaling to the brain. EECs are therefore considered integral components of the microbiota-gut-brain-axis and EEC dysfunction or abnormal signaling has been linked to many different disorders including obesity, diabetes, and inflammatory bowel diseases. Serotonergic enterochromaffin (EC) cells form the largest EEC subclass and these cells are found in all the different regions of the gut while comprising a rather heterogeneous group themselves. In general, EC cells function as chemo- and mechanoreceptors, which means that they must integrate multimodal sensory information on the cellular level and respond in an activated state by secreting the classical neurotransmitter serotonin. Gastrointestinal serotonin mediates diverse physiological processes in the body, most notably the regulation of gut motility. While there has been a growing understanding of the molecular mechanisms and sensory stimuli by which EC cells are activated, the process(es) via which excitation-secretion coupling is achieved and the molecular mechanisms that control serotonin release from these cells remain incompletely understood. Interestingly, several studies have proposed that some EECs, including EC cells, adopt morphological features (i.e. long axon-like processes termed ‘neuropods’) and exhibit molecular properties (i.e. expression of components of the synaptic vesicle fusion machinery) that determine fast cell-to-cell signaling in neurons of the brain. It has therefore been hypothesized that EC cells may communicate with neurons via fast, synapse-like mechanisms. However, as outlined above, evidence supporting this notion has largely been circumstantial.
The overall goal of this project was therefore to answer the question whether mouse EC cells signal in a synaptic-like fashion with the ENS and sensory afferents to mediate gut-brain-communication and to determine the molecular release machinery that mediates serotonin release from these cells. To achieve this, three specific objectives were formulated:
1. To define functional properties and molecular requirements of vesicle fusion in EC cells.
2. To dissect the functional organization of EC cell release sites and EC connectivity.
3. To probe consequences of defective serotonin release.
The project results achieved so far are described in detail below. In summary, we found that despite the expression of key components of the neuronal presynaptic neurotransmitter release machinery in EC cells, cultured cells release the majority of serotonin with relatively slow kinetics from large secretory vesicles, unlike fast synaptic transmission, but similar to the signaling mode of other endocrine cell types.
The overall goal of this project was therefore to answer the question whether mouse EC cells signal in a synaptic-like fashion with the ENS and sensory afferents to mediate gut-brain-communication and to determine the molecular release machinery that mediates serotonin release from these cells. To achieve this, three specific objectives were formulated:
1. To define functional properties and molecular requirements of vesicle fusion in EC cells.
2. To dissect the functional organization of EC cell release sites and EC connectivity.
3. To probe consequences of defective serotonin release.
The project results achieved so far are described in detail below. In summary, we found that despite the expression of key components of the neuronal presynaptic neurotransmitter release machinery in EC cells, cultured cells release the majority of serotonin with relatively slow kinetics from large secretory vesicles, unlike fast synaptic transmission, but similar to the signaling mode of other endocrine cell types.
To address the three main objectives, we employed a multidisciplinary strategy that would allow us to dissect molecular, morphological, and functional requirements for serotonin release from mouse intestinal EC cells in vitro. For this, we used a combination of intestinal stem cell-derived organoid cultures and 2D epithelial monolayer cultures generated from a transgenic mouse line that expresses a fluorescent reporter specifically in EC cells. This enabled us to unequivocally identify EC cells amongst the other epithelial cells for targeted functional assays. To probe serotonin secretion on the single-cell level we used a combination of whole-cell patch clamp electrophysiology and carbon fibre amperometry for the direct electrochemical detection of serotonin released from individually fusing vesicles. We compared the kinetics of the release process to that of catecholamine released from another well-studied neuroendocrine cell type and found that cultured mouse EC cells release serotonin with similarly slow kinetics. We interpret our data as such that the vesicle fusion process in EC cells is much slower than that in neuronal synapses in the brain.
To identify potential regulators of the vesicle fusion process in EC cells, we performed RT-PCR gene expression analysis of fluorescence-activated cell sorting (FACS) purified EC cells and analysed previously published RNA sequencing data sets. Our data confirmed that EC cells express genes encoding for key components of the synaptic neurotransmitter release machinery. We then determined the subcellular localisation of proteins of interests in EC cells in cultures and gut tissue sections immunocytochemistry and immunohistochemistry, respectively. While we could confirm the expression of several key components of the synaptic neurotransmitter release machinery on the protein level, our preliminary analysis failed to reveal the presence of proteins that constitute synapse-like presynaptic specialisations (“active zones”).
To gain a better understanding of the molecular mechanisms that control the 5-HT release process from EC cells, we are in the process of generating knockout organoid lines using CRISPR/Cas9 gene editing strategies in vitro. The aim is to systematically screen for defects in 5-HT secretion using our high temporal electrophysiology and electrochemistry assays.
The first results of this project are summarized in a recent preprint (Shaaban et al., BioRxiv, 2021, doi: https://doi.org/10.1101/2021.05.28.446100).
To identify potential regulators of the vesicle fusion process in EC cells, we performed RT-PCR gene expression analysis of fluorescence-activated cell sorting (FACS) purified EC cells and analysed previously published RNA sequencing data sets. Our data confirmed that EC cells express genes encoding for key components of the synaptic neurotransmitter release machinery. We then determined the subcellular localisation of proteins of interests in EC cells in cultures and gut tissue sections immunocytochemistry and immunohistochemistry, respectively. While we could confirm the expression of several key components of the synaptic neurotransmitter release machinery on the protein level, our preliminary analysis failed to reveal the presence of proteins that constitute synapse-like presynaptic specialisations (“active zones”).
To gain a better understanding of the molecular mechanisms that control the 5-HT release process from EC cells, we are in the process of generating knockout organoid lines using CRISPR/Cas9 gene editing strategies in vitro. The aim is to systematically screen for defects in 5-HT secretion using our high temporal electrophysiology and electrochemistry assays.
The first results of this project are summarized in a recent preprint (Shaaban et al., BioRxiv, 2021, doi: https://doi.org/10.1101/2021.05.28.446100).
While the project addresses basic science questions, it is anticipated that the results have implications for human health. That said, the socio-economic impact of this work is difficult to predict. Understanding the fundamental cell biological and molecular processes that control EC cell function and consequential serotonin release may on the one hand contribute in the future to a better understanding of disease pathologies linked to altered serotonin signaling in the gut and periphery and, on the other hand, may help identify new potential avenues and molecular targets to treat these disorders using pharmacological interventions.