Many aspects of gastrointestinal physiology are under the control of the enteric nervous system (... more Many aspects of gastrointestinal physiology are under the control of the enteric nervous system (ENS), 1 which is an extensive neural network embedded within the wall of the entire gastrointestinal tract and operates, to a large extent, independently from commands arriving from the central nervous system. 2 Enteric neurons and enteric glial cells (EGCs) form the core of the ENS and are assembled into two interconnected ganglionated plexus layers. 3 While the cell bodies of enteric neurons are mainly localized within the submucosal plexus (SMP), 4 and between the circular and longitudinal muscle layers, within the myenteric plexus (MP), 1-3,5,6 EGCs can also be found in the mucosa and muscle layers. Different enteric neuron subtypes can be classified according to their morphological, electrical, neurochemical, and molecular properties. 7-9 EGC subtypes can be classified based on their morphology and location. 8,10,11 Owing to its location close to the contractile gut musculature and intimate association with other intestinal cell types, such as epithelial, immune, and stromal cells, the development of adequate methodology to study the ENS has been a major challenge to the field. 12-14 The generation of in vitro techniques has allowed the acquisition, expansion, and manipulation of ENS cells for a wide variety of research questions. They include (but are not limited to) primary ENS cell cultures in (semi-)monolayer, stem cell-derived 3D neurospheres, induced ENS
Tissue maintenance and repair depend on the integrated activity of multiple cell types 1 . Wherea... more Tissue maintenance and repair depend on the integrated activity of multiple cell types 1 . Whereas the contributions of epithelial 2,3 , immune 4,5 and stromal cells 6,7 in intestinal tissue integrity are well understood, the role of intrinsic neuroglia networks remains largely unknown. Here, we $
The presence and identity of neural progenitors in the enteric nervous system (ENS) of vertebrate... more The presence and identity of neural progenitors in the enteric nervous system (ENS) of vertebrates is a matter of intense debate. Here we demonstrate that the non-neuronal ENS cell compartment of teleosts shares molecular and morphological characteristics with mammalian enteric glia but cannot be identified by the expression of canonical glia markers. However, unlike their mammalian counterparts, which are generally quiescent and do not undergo neuronal differentiation during homeostasis, we show that a relatively high proportion of zebrafish enteric glia proliferate under physiological conditions giving rise to progeny that differentiate into enteric neurons. We also provide evidence that, similar to brain neural stem cells, the activation and neuronal differentiation of enteric glia are regulated by Notch signalling. Our experiments reveal remarkable similarities between enteric glia and brain neural stem cells in teleosts and open new possibilities for use of mammalian enteric glia as a potential source of neurons to restore the activity of intestinal neural circuits compromised by injury or disease. Results Expression of canonical glia markers is undetectable in the zebrafish ENS To pave the way for a systematic search for cells harbouring neurogenic potential in the ENS of non-amniotic vertebrates, we first set out to characterise the non-neuronal compartment of the zebrafish ENS, the most likely source of enteric neural progenitors. Initially, we combined the SAGFF234A Gal4 transcriptional activator gene trap with the UAS:GFP transgene in order to generate SAGFF234A;UAS:GFP animals in which ENS progenitors and their descendants were labelled with GFP (Heanue et al., 2016a; Kawakami et al., 2010). In 7 day post fertilisation (dpf) larvae the majority of GFP + cells (93.76% ± 2.99) co-expressed the pan-neuronal marker HuC/D (Suppl. Fig. 1A-C), suggesting that in comparison to mammals, in which EGCs outnumber enteric neurons (Gabella, 1981; Ruhl, 2005), the nonneuronal ENS cell population of zebrafish is considerably smaller. To support this supposition, we also quantified the proportion of neurons within the ENS of Tg(-4725sox10:Cre;βactin-LoxP-STOP-LoxP-hmgb1-mCherry) transgenic fish (hereafter abbreviated as Tg(sox10:Cre;Cherry)) in which sox10-driven Cre recombinase activates a nuclear Cherry reporter in early NC cells and all derivative lineages, including the ENS (Rodrigues et al., 2012; Wang et al., 2011). Consistent with the analysis of SAGFF234A;UAS:GFP animals, the majority of Cherry + cells (84.79±7.70%) in the gut of 7 dpf Tg(sox10:Cre;Cherry) larvae were positive for HuC/D (Fig. 1A, C). Similar analysis in adult (≥ 3 months old) Tg(sox10:Cre;Cherry) zebrafish showed that, although the fraction of non-neuronal Cherry + cells was higher relative to 7 dpf larvae, even at this stage the majority of ENS + cells (65.49±4.8%) were neurons (Fig. 1B, C). Therefore, the non-neuronal compartment in the zebrafish ENS is notably smaller relative to its mammalian counterpart. All non-neuronal cells of the mammalian ENS are identified as enteric glia expressing combinations of the canonical glia markers S100β, GFAP and BFABP (Hao et al., 2016; Young et al., 2003). To determine whether these marker proteins are also expressed in the zebrafish ENS, we used antibodies raised against them to immunostain 7 dpf larvae, a stage neuroectodermal gut tissues as it persisted in ret mutant larvae, which lack enteric neuroglia networks (Suppl. Fig. 1F-I) (Heanue et al., 2016a). Immunostaining signal for GFAP has previously reported in the ENS (Baker et al., 2019; Kelsh and Eisen, 2000), however in our experiments the expression is not apparently within the NC-derived lineages. Consistent with the immunostaining, expression of the Tg(gfap:GFP) transgene (Bernardos and Raymond, 2006) was also undetectable in the gut of 7dpf larvae (Fig. 1E). In contrast to the ENS, these immunostaining reagents identified the expected signal in the spinal cord (Suppl. Fig. J-O). To ascertain that the lack of glia marker expression was not due to delayed maturation of enteric glia, we also immunostained adult zebrafish gut for GFAP, S100β, BFABP and (in the case of gfap:GFP transgenics) GFP. Similar to 7dpf animals, no apparent ENS-specific expression of these markers or the gfap:GFP transgene detected in adult gut (Fig. 1F-G, Suppl. Fig 1P-Q). Finally, contrary to reports indicating expression of Nestin in non-neuronal cells of mammalian enteric ganglia (Kulkarni et al., 2017), no expression of the nestin:GFP transgene was detected in the ENS of adult zebrafish (Suppl. Fig. 1R). Taken together, our studies demonstrate that the non-neuronal compartment of the zebrafish ENS is considerably smaller relative to its mammalian counterpart and cannot be labelled by immunohistochemical reagents commonly used for the identification of enteric glia. Non-neuronal cells of the zebrafish ENS share with mammalian EGCs early NC cell and ENS progenitor markers To explore further the gene expression profile of the non-neuronal ENS cell compartment in zebrafish, we carried out bulk RNA sequencing of fluorescent-labelled nuclei (nRNAseq) isolated from Tg(sox10:Cre;Cherry) adult gut muscularis externa. This strategy, which we described recently (Obata et al., 2020), avoids lengthy protocols of tissue dissociation and cell isolation that are often associated with considerable cell damage. Since the available transgenic tools did not allow us to label specifically the non-neuronal ENS cell compartment, bulk nRNAseq was performed on nuclei purified by FACS (fluorescentactivated cell sorting) representing both the Cherry + (entire ENS) and Cherry-(non-ENS) muscularis externa cell populations of Tg(sox10:Cre;Cherry) zebrafish gut (Fig. 2A and Suppl. Fig. 2A; see also Materials and Methods). Principal component analysis (PCA) demonstrated a clear separation of the Cherry + and Cherrynuclear transcriptomes along PC1 (Suppl. Fig. 2B), indicating that variability along this axis is determined predominantly by the lineage origin (NC vs non-NC) of the two cell populations. As expected, genes associated with non-NC tissues, such as smooth muscle cells (mylka, myh11a, cald1a, srfa, gata6,. Tg(her4.3:EGFP)-expressing cells are integral to the ENS, we introduced the her4.3:EGFP transgene into the Tg(sox10:Cre;Cherry) genetic background and immunostained gut preparations from adult Tg(her4.3:EGFP;sox10:Cre;Cherry) animals for GFP, HuC/D and Cherry. As expected, GFP + cells were negative for HuC/D but expressed the Cherry reporter (Fig. 3C), indicating that they belong to the non-neuronal compartment of the ENS. Consistent with this idea, GFP + cells co-expressed sox2 and sox10 (Fig. 3D, E), which were identified by our transcriptomic analysis as genes expressed by the non-neuronal compartment of the zebrafish ENS. The GFP + HuC/Dcell population in Tg(her4.3:EGFP;sox10:Cre;Cherry) represented approximately a quarter (24.20 ± 5.18%) of all Cherry + ENS cells, but 12.93 ± 5.33% of Cherry + cells were negative for both GFP and HuC/D (Cherry + GFP-HuC/D-) (Fig. 3B). Therefore, the majority of non-neuronal ENS cells in adult zebrafish gut can be identified by the expression of the Notch activity reporter Tg(her4.3:EGFP). GFP + cells in the ENS of Tg(her4.3:EGFP) zebrafish have morphological characteristics of mammalian EGCs To provide evidence that Tg(her4.3:EGFP)-expressing cells in the zebrafish ENS are equivalent to mammalian EGCs, we characterised the morphology of GFP + cells in the gut of .
Environmental signals modulate the activity of the nervous system and harmonize its output with t... more Environmental signals modulate the activity of the nervous system and harmonize its output with the outside world. Synaptic activity is crucial for integrating sensory and effector neural pathways but the role of transcriptional mechanisms as environmental sensors in the nervous system remains unclear. By combining a novel strategy for transcriptomic profiling of enteric neurons with microbiota manipulation, we demonstrate that the transcriptional programs of intestinal neural circuits depend on their anatomical and physiological context. We also identify the ligand-dependent transcription factor Aryl hydrocarbon Receptor (AhR) is an intrinsic regulator of enteric nervous system output. AhR is instated as a neuronal biosensor in response to microbiota colonization allowing resident enteric neurons to directly monitor and respond to the intestinal microenvironment. We suggest that AhR signaling integrates neuronal activity with host defence mechanisms towards gut homeostasis and heal...
5-Fluorouracil (5-FU) is an anticancer agent whose main side effects include intestinal mucositis... more 5-Fluorouracil (5-FU) is an anticancer agent whose main side effects include intestinal mucositis associated with intestinal motility alterations maybe due to an effect on the enteric nervous system (ENS), but the underlying mechanism remains unclear. In this report, we used an animal model to investigate the participation of the S100B/RAGE/NFκB pathway in intestinal mucositis and enteric neurotoxicity caused by 5-FU (450 mg/kg, IP, single dose). 5-FU induced intestinal damage observed by shortened villi, loss of crypt architecture and intense inflammatory cell infiltrate as well as increased GFAP and S100B co-expression and decreased HuC/D protein expression in the small intestine. Furthermore, 5-FU increased RAGE and NFκB NLS immunostaining in enteric neurons, associated with a significant increase in the nitrite/nitrate, IL-6 and TNF-α levels, iNOS expression and MDA accumulation in the small intestine. We provide evidence that 5-FU induces reactive gliosis and reduction of enteric neurons in a S100B/RAGE/NFκB-dependent manner, since pentamidine, a S100B inhibitor, prevented 5-FU-induced neuronal loss, enteric glia activation, intestinal inflammation, oxidative stress and histological injury. 5-Fluorouracil (5-FU) is an antimetabolite drug used to treat several types of cancer, including breast and colorectal cancer. Mucositis and diarrhea are common side effects of 5-FU-based anticancer regimens 1 , which contribute to the increased costs of hospitalization 2. Previous studies have reported that several inflammatory mediators are involved in 5-FU-related mucositis pathogenesis, including chemokine (C-X-C motif) ligand 4 (CXCL4) 3 , interleukin-4 (IL-4) 4 , interleukin-1β (IL-1β) 5 , chemokine (C-X-C motif) ligand 9 (CXCL9) 6 , TGF-β 6 , platelet activating factor (PAF) 7 , substance P and serotonin 8. Persistent GI over-contractility has also been demonstrated to persist, even after inflammation has resolved, suggesting that chemotherapy might affect gut neuronal function 9. The enteric nervous system (ENS) is composed of neurons and enteric glial cells (EGCs) that are organized into the following two main networks: the submucosal and myenteric plexuses 10. Previous studies reported an increase in the expression of glial fibrillary acidic protein (GFAP), a marker of EGCs activation, in rectal-biopsy specimens from patients with ulcerative colitis, Crohn's disease and infectious colitis (caused by Clostridioides difficile) 11. EGCs have an important function in the GI tract, regulating ion transport 12 and pathogen recognition since they express toll-like receptors (TLRs)-1-5,-7 and-9 13. These cells are important sources of chemotactic
Enteric glial cells were first described at the end of the 19th century, but they attracted more ... more Enteric glial cells were first described at the end of the 19th century, but they attracted more interest from researchers only in the last decades of the 20th. Although, they have a different embryological origin, the enteric GLIA share many characteristics with astrocytes, the main glial cell type of the central nervous system (CNS), such as in their expression of the same markers and in their functions. Here we review the construction of the enteric nervous system (ENS), with a focus on enteric glia, and also the main studies that have revealed the action of enteric glia in different aspects of gastrointestinal tract homeostasis, such as in the intestinal barrier, in communications with neurons, and in their action as progenitor cells. We also discuss recent discoveries about the roles of enteric glia in different disorders that affect the ENS, such as degenerative pathologies including Parkinson's and prion diseases, and in cases of intestinal diseases and injury.
Glioblastoma (GBM) is considered incurable due to its resistance to current cancer treatments. So... more Glioblastoma (GBM) is considered incurable due to its resistance to current cancer treatments. So far, all clinically available alternatives for treating GBM are limited, evoking the development of novel treatment strategies that can more effectively manage these tumors. Extensive effort is being dedicated to characterize the molecular basis of GBM resistance to chemotherapy and to explore novel therapeutic procedures that may improve overall survival. Cytolysins are toxins that form pores in target cell membranes, modifying ion homeostasis and leading to cell death. These pore-forming toxins might be used, therefore, to enhance the efficiency of conventional chemotherapeutic drugs, facilitating their entrance into the cell. In this study, we show that a non-cytotoxic concentration of equinatoxin II (EqTx-II), a pore-forming toxin from the sea anemone Actinia equina, potentiates the cytotoxicity induced by temozolomide (TMZ), a first-line GBM treatment, and by etoposide (VP-16), a second- or third-line GBM treatment. We also suggest that this effect is selective to GBM cells and occurs via PI3K/Akt pathway inhibition. Finally, Magnetic resonance imaging (MRI) revealed that a non-cytotoxic concentration of EqTx-II potentiates the VP-16-induced inhibition of GBM growth in vivo. These combined therapies constitute a new and potentially valuable tool for GBM treatment, leading to the requirement of lower concentrations of chemotherapeutic drugs and possibly reducing, therefore, the adverse effects of chemotherapy.
How neurogenesis and gliogenesis are coordinated during development and why mature glial cells of... more How neurogenesis and gliogenesis are coordinated during development and why mature glial cells often share properties with neuroectodermal progenitors remains unclear. Here, we have used single cell RNA sequencing to map the regulatory landscape of neuronal and glial differentiation in the mammalian enteric nervous system (ENS). Our analysis indicates that neurogenic trajectories branch directly from a linear gliogenic axis defined by autonomic neural crest cells adopting sequential states as they progressively lose their strong neurogenic bias and acquire properties of adult enteric glia. We identify gene modules associated with transcriptional programs driving enteric neurogenesis and cell state transitions along the gliogenic axis. By comparing the chromatin accessibility profile of autonomic neural crest and adult enteric glia we provide evidence that the latter maintain an epigenetic memory of their neurogenic past. Finally, we demonstrate that adult enteric glia maintain neuro...
Glioblastoma (GBM) is considered incurable due to its resistance to current cancer treatments. So... more Glioblastoma (GBM) is considered incurable due to its resistance to current cancer treatments. So far, all clinically available alternatives for treating GBM are limited, evoking the development of novel treatment strategies that can more effectively manage these tumors. Extensive effort is being dedicated to characterize the molecular basis of GBM resistance to chemotherapy and to explore novel therapeutic procedures that may improve overall survival. Cytolysins are toxins that form pores in target cell membranes, modifying ion homeostasis and leading to cell death. These pore-forming toxins might be used, therefore, to enhance the efficiency of conventional chemotherapeutic drugs, facilitating their entrance into the cell. In this study, we show that a non-cytotoxic concentration of equinatoxin II (EqTx-II), a pore-forming toxin from the sea anemone Actinia equina, potentiates the cytotoxicity induced by temozolomide (TMZ), a first-line GBM treatment, and by etoposide (VP-16), a second- or third-line GBM treatment. We also suggest that this effect is selective to GBM cells and occurs via PI3K/Akt pathway inhibition. Finally, Magnetic resonance imaging (MRI) revealed that a non-cytotoxic concentration of EqTx-II potentiates the VP-16-induced inhibition of GBM growth in vivo. These combined therapies constitute a new and potentially valuable tool for GBM treatment, leading to the requirement of lower concentrations of chemotherapeutic drugs and possibly reducing, therefore, the adverse effects of chemotherapy.
Many aspects of gastrointestinal physiology are under the control of the enteric nervous system (... more Many aspects of gastrointestinal physiology are under the control of the enteric nervous system (ENS), 1 which is an extensive neural network embedded within the wall of the entire gastrointestinal tract and operates, to a large extent, independently from commands arriving from the central nervous system. 2 Enteric neurons and enteric glial cells (EGCs) form the core of the ENS and are assembled into two interconnected ganglionated plexus layers. 3 While the cell bodies of enteric neurons are mainly localized within the submucosal plexus (SMP), 4 and between the circular and longitudinal muscle layers, within the myenteric plexus (MP), 1-3,5,6 EGCs can also be found in the mucosa and muscle layers. Different enteric neuron subtypes can be classified according to their morphological, electrical, neurochemical, and molecular properties. 7-9 EGC subtypes can be classified based on their morphology and location. 8,10,11 Owing to its location close to the contractile gut musculature and intimate association with other intestinal cell types, such as epithelial, immune, and stromal cells, the development of adequate methodology to study the ENS has been a major challenge to the field. 12-14 The generation of in vitro techniques has allowed the acquisition, expansion, and manipulation of ENS cells for a wide variety of research questions. They include (but are not limited to) primary ENS cell cultures in (semi-)monolayer, stem cell-derived 3D neurospheres, induced ENS
Tissue maintenance and repair depend on the integrated activity of multiple cell types 1 . Wherea... more Tissue maintenance and repair depend on the integrated activity of multiple cell types 1 . Whereas the contributions of epithelial 2,3 , immune 4,5 and stromal cells 6,7 in intestinal tissue integrity are well understood, the role of intrinsic neuroglia networks remains largely unknown. Here, we $
The presence and identity of neural progenitors in the enteric nervous system (ENS) of vertebrate... more The presence and identity of neural progenitors in the enteric nervous system (ENS) of vertebrates is a matter of intense debate. Here we demonstrate that the non-neuronal ENS cell compartment of teleosts shares molecular and morphological characteristics with mammalian enteric glia but cannot be identified by the expression of canonical glia markers. However, unlike their mammalian counterparts, which are generally quiescent and do not undergo neuronal differentiation during homeostasis, we show that a relatively high proportion of zebrafish enteric glia proliferate under physiological conditions giving rise to progeny that differentiate into enteric neurons. We also provide evidence that, similar to brain neural stem cells, the activation and neuronal differentiation of enteric glia are regulated by Notch signalling. Our experiments reveal remarkable similarities between enteric glia and brain neural stem cells in teleosts and open new possibilities for use of mammalian enteric glia as a potential source of neurons to restore the activity of intestinal neural circuits compromised by injury or disease. Results Expression of canonical glia markers is undetectable in the zebrafish ENS To pave the way for a systematic search for cells harbouring neurogenic potential in the ENS of non-amniotic vertebrates, we first set out to characterise the non-neuronal compartment of the zebrafish ENS, the most likely source of enteric neural progenitors. Initially, we combined the SAGFF234A Gal4 transcriptional activator gene trap with the UAS:GFP transgene in order to generate SAGFF234A;UAS:GFP animals in which ENS progenitors and their descendants were labelled with GFP (Heanue et al., 2016a; Kawakami et al., 2010). In 7 day post fertilisation (dpf) larvae the majority of GFP + cells (93.76% ± 2.99) co-expressed the pan-neuronal marker HuC/D (Suppl. Fig. 1A-C), suggesting that in comparison to mammals, in which EGCs outnumber enteric neurons (Gabella, 1981; Ruhl, 2005), the nonneuronal ENS cell population of zebrafish is considerably smaller. To support this supposition, we also quantified the proportion of neurons within the ENS of Tg(-4725sox10:Cre;βactin-LoxP-STOP-LoxP-hmgb1-mCherry) transgenic fish (hereafter abbreviated as Tg(sox10:Cre;Cherry)) in which sox10-driven Cre recombinase activates a nuclear Cherry reporter in early NC cells and all derivative lineages, including the ENS (Rodrigues et al., 2012; Wang et al., 2011). Consistent with the analysis of SAGFF234A;UAS:GFP animals, the majority of Cherry + cells (84.79±7.70%) in the gut of 7 dpf Tg(sox10:Cre;Cherry) larvae were positive for HuC/D (Fig. 1A, C). Similar analysis in adult (≥ 3 months old) Tg(sox10:Cre;Cherry) zebrafish showed that, although the fraction of non-neuronal Cherry + cells was higher relative to 7 dpf larvae, even at this stage the majority of ENS + cells (65.49±4.8%) were neurons (Fig. 1B, C). Therefore, the non-neuronal compartment in the zebrafish ENS is notably smaller relative to its mammalian counterpart. All non-neuronal cells of the mammalian ENS are identified as enteric glia expressing combinations of the canonical glia markers S100β, GFAP and BFABP (Hao et al., 2016; Young et al., 2003). To determine whether these marker proteins are also expressed in the zebrafish ENS, we used antibodies raised against them to immunostain 7 dpf larvae, a stage neuroectodermal gut tissues as it persisted in ret mutant larvae, which lack enteric neuroglia networks (Suppl. Fig. 1F-I) (Heanue et al., 2016a). Immunostaining signal for GFAP has previously reported in the ENS (Baker et al., 2019; Kelsh and Eisen, 2000), however in our experiments the expression is not apparently within the NC-derived lineages. Consistent with the immunostaining, expression of the Tg(gfap:GFP) transgene (Bernardos and Raymond, 2006) was also undetectable in the gut of 7dpf larvae (Fig. 1E). In contrast to the ENS, these immunostaining reagents identified the expected signal in the spinal cord (Suppl. Fig. J-O). To ascertain that the lack of glia marker expression was not due to delayed maturation of enteric glia, we also immunostained adult zebrafish gut for GFAP, S100β, BFABP and (in the case of gfap:GFP transgenics) GFP. Similar to 7dpf animals, no apparent ENS-specific expression of these markers or the gfap:GFP transgene detected in adult gut (Fig. 1F-G, Suppl. Fig 1P-Q). Finally, contrary to reports indicating expression of Nestin in non-neuronal cells of mammalian enteric ganglia (Kulkarni et al., 2017), no expression of the nestin:GFP transgene was detected in the ENS of adult zebrafish (Suppl. Fig. 1R). Taken together, our studies demonstrate that the non-neuronal compartment of the zebrafish ENS is considerably smaller relative to its mammalian counterpart and cannot be labelled by immunohistochemical reagents commonly used for the identification of enteric glia. Non-neuronal cells of the zebrafish ENS share with mammalian EGCs early NC cell and ENS progenitor markers To explore further the gene expression profile of the non-neuronal ENS cell compartment in zebrafish, we carried out bulk RNA sequencing of fluorescent-labelled nuclei (nRNAseq) isolated from Tg(sox10:Cre;Cherry) adult gut muscularis externa. This strategy, which we described recently (Obata et al., 2020), avoids lengthy protocols of tissue dissociation and cell isolation that are often associated with considerable cell damage. Since the available transgenic tools did not allow us to label specifically the non-neuronal ENS cell compartment, bulk nRNAseq was performed on nuclei purified by FACS (fluorescentactivated cell sorting) representing both the Cherry + (entire ENS) and Cherry-(non-ENS) muscularis externa cell populations of Tg(sox10:Cre;Cherry) zebrafish gut (Fig. 2A and Suppl. Fig. 2A; see also Materials and Methods). Principal component analysis (PCA) demonstrated a clear separation of the Cherry + and Cherrynuclear transcriptomes along PC1 (Suppl. Fig. 2B), indicating that variability along this axis is determined predominantly by the lineage origin (NC vs non-NC) of the two cell populations. As expected, genes associated with non-NC tissues, such as smooth muscle cells (mylka, myh11a, cald1a, srfa, gata6,. Tg(her4.3:EGFP)-expressing cells are integral to the ENS, we introduced the her4.3:EGFP transgene into the Tg(sox10:Cre;Cherry) genetic background and immunostained gut preparations from adult Tg(her4.3:EGFP;sox10:Cre;Cherry) animals for GFP, HuC/D and Cherry. As expected, GFP + cells were negative for HuC/D but expressed the Cherry reporter (Fig. 3C), indicating that they belong to the non-neuronal compartment of the ENS. Consistent with this idea, GFP + cells co-expressed sox2 and sox10 (Fig. 3D, E), which were identified by our transcriptomic analysis as genes expressed by the non-neuronal compartment of the zebrafish ENS. The GFP + HuC/Dcell population in Tg(her4.3:EGFP;sox10:Cre;Cherry) represented approximately a quarter (24.20 ± 5.18%) of all Cherry + ENS cells, but 12.93 ± 5.33% of Cherry + cells were negative for both GFP and HuC/D (Cherry + GFP-HuC/D-) (Fig. 3B). Therefore, the majority of non-neuronal ENS cells in adult zebrafish gut can be identified by the expression of the Notch activity reporter Tg(her4.3:EGFP). GFP + cells in the ENS of Tg(her4.3:EGFP) zebrafish have morphological characteristics of mammalian EGCs To provide evidence that Tg(her4.3:EGFP)-expressing cells in the zebrafish ENS are equivalent to mammalian EGCs, we characterised the morphology of GFP + cells in the gut of .
Environmental signals modulate the activity of the nervous system and harmonize its output with t... more Environmental signals modulate the activity of the nervous system and harmonize its output with the outside world. Synaptic activity is crucial for integrating sensory and effector neural pathways but the role of transcriptional mechanisms as environmental sensors in the nervous system remains unclear. By combining a novel strategy for transcriptomic profiling of enteric neurons with microbiota manipulation, we demonstrate that the transcriptional programs of intestinal neural circuits depend on their anatomical and physiological context. We also identify the ligand-dependent transcription factor Aryl hydrocarbon Receptor (AhR) is an intrinsic regulator of enteric nervous system output. AhR is instated as a neuronal biosensor in response to microbiota colonization allowing resident enteric neurons to directly monitor and respond to the intestinal microenvironment. We suggest that AhR signaling integrates neuronal activity with host defence mechanisms towards gut homeostasis and heal...
5-Fluorouracil (5-FU) is an anticancer agent whose main side effects include intestinal mucositis... more 5-Fluorouracil (5-FU) is an anticancer agent whose main side effects include intestinal mucositis associated with intestinal motility alterations maybe due to an effect on the enteric nervous system (ENS), but the underlying mechanism remains unclear. In this report, we used an animal model to investigate the participation of the S100B/RAGE/NFκB pathway in intestinal mucositis and enteric neurotoxicity caused by 5-FU (450 mg/kg, IP, single dose). 5-FU induced intestinal damage observed by shortened villi, loss of crypt architecture and intense inflammatory cell infiltrate as well as increased GFAP and S100B co-expression and decreased HuC/D protein expression in the small intestine. Furthermore, 5-FU increased RAGE and NFκB NLS immunostaining in enteric neurons, associated with a significant increase in the nitrite/nitrate, IL-6 and TNF-α levels, iNOS expression and MDA accumulation in the small intestine. We provide evidence that 5-FU induces reactive gliosis and reduction of enteric neurons in a S100B/RAGE/NFκB-dependent manner, since pentamidine, a S100B inhibitor, prevented 5-FU-induced neuronal loss, enteric glia activation, intestinal inflammation, oxidative stress and histological injury. 5-Fluorouracil (5-FU) is an antimetabolite drug used to treat several types of cancer, including breast and colorectal cancer. Mucositis and diarrhea are common side effects of 5-FU-based anticancer regimens 1 , which contribute to the increased costs of hospitalization 2. Previous studies have reported that several inflammatory mediators are involved in 5-FU-related mucositis pathogenesis, including chemokine (C-X-C motif) ligand 4 (CXCL4) 3 , interleukin-4 (IL-4) 4 , interleukin-1β (IL-1β) 5 , chemokine (C-X-C motif) ligand 9 (CXCL9) 6 , TGF-β 6 , platelet activating factor (PAF) 7 , substance P and serotonin 8. Persistent GI over-contractility has also been demonstrated to persist, even after inflammation has resolved, suggesting that chemotherapy might affect gut neuronal function 9. The enteric nervous system (ENS) is composed of neurons and enteric glial cells (EGCs) that are organized into the following two main networks: the submucosal and myenteric plexuses 10. Previous studies reported an increase in the expression of glial fibrillary acidic protein (GFAP), a marker of EGCs activation, in rectal-biopsy specimens from patients with ulcerative colitis, Crohn's disease and infectious colitis (caused by Clostridioides difficile) 11. EGCs have an important function in the GI tract, regulating ion transport 12 and pathogen recognition since they express toll-like receptors (TLRs)-1-5,-7 and-9 13. These cells are important sources of chemotactic
Enteric glial cells were first described at the end of the 19th century, but they attracted more ... more Enteric glial cells were first described at the end of the 19th century, but they attracted more interest from researchers only in the last decades of the 20th. Although, they have a different embryological origin, the enteric GLIA share many characteristics with astrocytes, the main glial cell type of the central nervous system (CNS), such as in their expression of the same markers and in their functions. Here we review the construction of the enteric nervous system (ENS), with a focus on enteric glia, and also the main studies that have revealed the action of enteric glia in different aspects of gastrointestinal tract homeostasis, such as in the intestinal barrier, in communications with neurons, and in their action as progenitor cells. We also discuss recent discoveries about the roles of enteric glia in different disorders that affect the ENS, such as degenerative pathologies including Parkinson's and prion diseases, and in cases of intestinal diseases and injury.
Glioblastoma (GBM) is considered incurable due to its resistance to current cancer treatments. So... more Glioblastoma (GBM) is considered incurable due to its resistance to current cancer treatments. So far, all clinically available alternatives for treating GBM are limited, evoking the development of novel treatment strategies that can more effectively manage these tumors. Extensive effort is being dedicated to characterize the molecular basis of GBM resistance to chemotherapy and to explore novel therapeutic procedures that may improve overall survival. Cytolysins are toxins that form pores in target cell membranes, modifying ion homeostasis and leading to cell death. These pore-forming toxins might be used, therefore, to enhance the efficiency of conventional chemotherapeutic drugs, facilitating their entrance into the cell. In this study, we show that a non-cytotoxic concentration of equinatoxin II (EqTx-II), a pore-forming toxin from the sea anemone Actinia equina, potentiates the cytotoxicity induced by temozolomide (TMZ), a first-line GBM treatment, and by etoposide (VP-16), a second- or third-line GBM treatment. We also suggest that this effect is selective to GBM cells and occurs via PI3K/Akt pathway inhibition. Finally, Magnetic resonance imaging (MRI) revealed that a non-cytotoxic concentration of EqTx-II potentiates the VP-16-induced inhibition of GBM growth in vivo. These combined therapies constitute a new and potentially valuable tool for GBM treatment, leading to the requirement of lower concentrations of chemotherapeutic drugs and possibly reducing, therefore, the adverse effects of chemotherapy.
How neurogenesis and gliogenesis are coordinated during development and why mature glial cells of... more How neurogenesis and gliogenesis are coordinated during development and why mature glial cells often share properties with neuroectodermal progenitors remains unclear. Here, we have used single cell RNA sequencing to map the regulatory landscape of neuronal and glial differentiation in the mammalian enteric nervous system (ENS). Our analysis indicates that neurogenic trajectories branch directly from a linear gliogenic axis defined by autonomic neural crest cells adopting sequential states as they progressively lose their strong neurogenic bias and acquire properties of adult enteric glia. We identify gene modules associated with transcriptional programs driving enteric neurogenesis and cell state transitions along the gliogenic axis. By comparing the chromatin accessibility profile of autonomic neural crest and adult enteric glia we provide evidence that the latter maintain an epigenetic memory of their neurogenic past. Finally, we demonstrate that adult enteric glia maintain neuro...
Glioblastoma (GBM) is considered incurable due to its resistance to current cancer treatments. So... more Glioblastoma (GBM) is considered incurable due to its resistance to current cancer treatments. So far, all clinically available alternatives for treating GBM are limited, evoking the development of novel treatment strategies that can more effectively manage these tumors. Extensive effort is being dedicated to characterize the molecular basis of GBM resistance to chemotherapy and to explore novel therapeutic procedures that may improve overall survival. Cytolysins are toxins that form pores in target cell membranes, modifying ion homeostasis and leading to cell death. These pore-forming toxins might be used, therefore, to enhance the efficiency of conventional chemotherapeutic drugs, facilitating their entrance into the cell. In this study, we show that a non-cytotoxic concentration of equinatoxin II (EqTx-II), a pore-forming toxin from the sea anemone Actinia equina, potentiates the cytotoxicity induced by temozolomide (TMZ), a first-line GBM treatment, and by etoposide (VP-16), a second- or third-line GBM treatment. We also suggest that this effect is selective to GBM cells and occurs via PI3K/Akt pathway inhibition. Finally, Magnetic resonance imaging (MRI) revealed that a non-cytotoxic concentration of EqTx-II potentiates the VP-16-induced inhibition of GBM growth in vivo. These combined therapies constitute a new and potentially valuable tool for GBM treatment, leading to the requirement of lower concentrations of chemotherapeutic drugs and possibly reducing, therefore, the adverse effects of chemotherapy.
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Papers by Ana Frauches