Search Results (12)

Resiniferatoxin (RTX), a potent capsaicin analog, is being investigated as a therapeutic agent for neurogenic conditions, particularly those affecting bladder control. However, the transcriptomic effects of RTX on the urinary bladder remain largely unexplored. This study aimed to characterize the transcriptomic changes in the porcine urinary bladder trigone region removed seven days post-treatment with intravesical RTX administration (500 nmol per animal in 60 mL of 5% aqueous solution of ethyl alcohol). High-throughput sequencing identified 126 differentially expressed genes (DEGs; 66 downregulated, 60 upregulated), 5 differentially expressed long non-coding RNAs (DELs), and 22 other RNAs, collectively involved in 175 gene ontology (GO) processes. Additionally, differential alternative splicing events (DASes) and single nucleotide variants (SNVs) were detected. RTX significantly modulated signaling pathways related to nerve growth and myelination. Changes in genes associated with synaptic plasticity and neuromodulation were observed, particularly within serotoninergic and cholinergic signaling. RTX altered the expression of immune-related genes, particularly those involved in chemokine signaling and immune regulation. Notably, altered gene expression patterns suggest a potential anti-cancer role for RTX. These findings provide new insights into RTX’s therapeutic effects beyond TRPV1 receptor interactions, filling a critical gap in our understanding of its molecular impact on bladder tissue. Full article

Continuous dialogue between the immune system and the brain plays a key homeostatic role in various immune responses to environmental cues. Several functions are under the control of the vagus nerve-based inflammatory reflex, a physiological mechanism through which nerve signals regulate immune functions. In the cholinergic anti-inflammatory pathway, the vagus nerve, its pivotal neurotransmitter acetylcholine, together with the corresponding receptors play a key role in modulating the immune response of mammals. Through communications of peripheral nerves with immune cells, it modulates proliferation and differentiation activities of various immune cell subsets. As a result, this pathway represents a potential target for treating autoimmune diseases characterized by overt inflammation and a decrease in vagal tone. Consistently, converging observations made in both animal models and clinical trials revealed that targeting the cholinergic anti-inflammatory pathway using pharmacologic approaches can provide beneficial effects. In parallel, bioelectronic medicine has recently emerged as an alternative approach to managing systemic inflammation. In several studies, nerve electrostimulation was reported to be clinically relevant in reducing chronic inflammation in autoimmune diseases, including rheumatoid arthritis and diabetes. In the future, these new approaches could represent a major therapeutic strategy for autoimmune and inflammatory diseases. Full article

Vagus nerve stimulation (VNS) is considered a potential method for anti-inflammation due to the involvement of the VN in the cholinergic anti-inflammatory pathway (CAP) formation of a connection between the central nervous system and peripheral immune cells that help relieve inflammation. However, whether a non-invasive transcutaneous auricular VNS (taVNS) modulates the inflammation levels via altering the parameter of taVNS is poorly understood. This study aimed to determine the differential inhibitory effects of taVNS on lipopolysaccharide (LPS)-induced systemic inflammation using electrical stimulation parameters such as pulse frequency and time. The taVNS-promoted CAP activity significantly recovered LPS-induced tissue injuries (lung, spleen, and intestine) and decreased inflammatory cytokine levels and tissue-infiltrated immune cells. Interestingly, the anti-inflammatory capacity of taVNS with 15 Hz was much higher than that of taVNS with 25 Hz. When a cytokine array was used to investigate the changes of inflammation and immune response-related cytokines/chemokines expression in taVNS with 15 Hz or 25 Hz treatment in LPS-induced endotoxemia in mice, most of the expression of cytokines/chemokines associated with pro-inflammation was severely decreased in taVNS with 15 Hz compared to 25 Hz. This study demonstrated that the taVNS parameter could differentially modulate the inflammation levels of animals, suggesting the importance of taVNS parameter selection for use in feasible interventions for acute inflammation treatment. Full article

Stem cells have extensive proliferative potential and the ability to differentiate into one or more mature cell types. The mechanisms by which stem cells accomplish self-renewal provide fundamental insight into the origin and design of multicellular organisms. These pathways allow the repair of damage and extend organismal life beyond that of component cells, and they probably preceded the evolution of complex metazoans. Understanding the true nature of stem cells can only come from discovering how they are regulated. The concept that stem cells are controlled by particular microenvironments, also known as niches, has been widely accepted. Technical advances now allow characterization of the zones that maintain and control stem cell activity in several organs, including the brain, skin, and gut. Cholinergic neurons release acetylcholine (ACh) that mediates chemical transmission via ACh receptors such as nicotinic and muscarinic receptors. Although the cholinergic system is composed of organized nerve cells, the system is also involved in mammalian non-neuronal cells, including stem cells, embryonic stem cells, epithelial cells, and endothelial cells. Thus, cholinergic signaling plays a pivotal role in controlling their behaviors. Studies regarding this signal are beginning to unify our understanding of stem cell regulation at the cellular and molecular levels, and they are expected to advance efforts to control stem cells therapeutically. The present article reviews recent findings about cholinergic signaling that is essential to control stem cell function in a cholinergic niche. Full article

Functional characterization of muscarinic cholinergic receptors in myelinating glial cells has been well described both in central and peripheral nervous system. Rat Schwann cells (SCs) express different muscarinic receptor subtypes with the prevalence of the M2 subtype. The selective stimulation of this receptor subtype inhibits SC proliferation, improving their differentiation towards myelinating phenotype. In this work, we describe for the first time that human SCs are cholinoceptive as they express several muscarinic receptor subtypes and, as for rat SCs, M2 receptor is one of the most abundant. Human SCs, isolated from adult nerves, were cultured in vitro and stimulated with M2 muscarinic agonist arecaidine propargyl ester (APE). Similarly to that observed in rat, M2 receptor activation causes a decreased cell proliferation and promotes SC differentiation as suggested by increased Egr2 expression with an improved spindle-like shape cell morphology. Conversely, the non-selective stimulation of muscarinic receptors appears to promote cell proliferation with a reduction of SC average cell diameter. The data obtained demonstrate that human SCs are cholinoceptive and that human cultured SCs may represent an interesting tool to understand their physiology and increase the knowledge on how the cholinergic stimulation may contribute to address human SC development in normal and pathological conditions. Full article

TNFα is the main proinflammatory cytokine implicated in the pathogenesis of neurodegenerative disorders, but it also modulates physiological functions in both the developing and adult brain. In this study, we investigated a potential direct role of TNFα in determining phenotypic changes of a recently established cellular model of human basal forebrain cholinergic neuroblasts isolated from the nucleus basalis of Meynert (hfNBMs). Exposing hfNBMs to TNFα reduced the expression of immature markers, such as nestin and β-tubulin III, and inhibited primary cilium formation. On the contrary, TNFα increased the expression of TNFα receptor TNFR2 and the mature neuron marker MAP2, also promoting neurite elongation. Moreover, TNFα affected nerve growth factor receptor expression. We also found that TNFα induced the expression of DNA-methylation enzymes and, accordingly, downregulated genes involved in neuronal development through epigenetic mechanisms, as demonstrated by methylome analysis. In summary, TNFα showed a dual role on hfNBMs phenotypic plasticity, exerting a negative influence on neurogenesis despite a positive effect on differentiation, through mechanisms that remain to be elucidated. Our results help to clarify the complexity of TNFα effects in human neurons and suggest that manipulation of TNFα signaling could provide a potential therapeutic approach against neurodegenerative disorders. Full article

In the last decade, Nerve Growth Factor (NGF)-based clinical approaches have lacked specific and efficient Tyrosine Kinase A (TrkA) agonists for brain delivery. Nowadays, the characterization of novel small peptidomimetic is taking centre stage in preclinical studies, in order to overcome the main size-related limitation in brain delivery of NGF holoprotein for Central Nervous System (CNS) pathologies. Here we investigated the NGF mimetic properties of the human NGF 1–14 sequence (hNGF1–14) and its derivatives, by resorting to primary cholinergic and dorsal root ganglia (DRG) neurons. Briefly, we observed that: 1) hNGF1–14 peptides engage the NGF pathway through TrkA phosphorylation at tyrosine 490 (Y490), and activation of ShcC/PI3K and Plc-γ/MAPK signalling, promoting AKT-dependent survival and CREB-driven neuronal activity, as seen by levels of the immediate early gene c-Fos, of the cholinergic marker Choline Acetyltransferase (ChAT), and of Brain Derived Neurotrophic Factor (BDNF); 2) their NGF mimetic activity is lost upon selective TrkA inhibition by means of GW441756; 3) hNGF1–14 peptides are able to sustain DRG survival and differentiation in absence of NGF. Furthermore, the acetylated derivative Ac-hNGF1–14 demonstrated an optimal NGF mimetic activity in both neuronal paradigms and an electrophysiological profile similar to NGF in cholinergic neurons. Cumulatively, the findings here reported pinpoint the hNGF1–14 peptide, and in particular its acetylated derivative, as novel, specific and low molecular weight TrkA specific agonists in both CNS and PNS primary neurons. Full article

Macamides are very important secondary metabolites produced by Lepidium meyenii Walp, which possess multiple bioactivities, especially in the neuronal system. In a previous study, we observed that macamides exhibited excellent effects in the recovery of injured nerves after 1-methyl-4-phenylpyridinium (MPP⁺)-induced dopaminergic neuronal damage in zebrafish. However, the mechanism underlying this effect remains unclear. In the present study, we observed that N-benzylhexadecanamide (XA), which is a typical constituent of macamides, improved the survival rate of neurons in vitro. We determined the concentration of neurotransmitters in MN9D cells and used it in conjunction with an integrated proteomics and lipidomics approach to investigate the mechanism underlying the neuroprotective effects of XA in an MPP⁺-induced neurodegeneration cell model using QqQ MS, Q-TOF MS, and Orbitrap MS. The statistical analysis of the results led to the identification of differentially-expressed biomarkers, including 11 proteins and 22 lipids, which may be responsible for the neuron-related activities of XA. All these potential biomarkers were closely related to the pathogenesis of neurodegenerative diseases, and their levels approached those in the normal group after treatment with XA. Furthermore, seven lipids, including five phosphatidylcholines, one lysophosphatidylcholine, and one phosphatidylethanolamine, were verified by a relative quantitative approach. Moreover, four proteins (Scarb2, Csnk2a2, Vti1b, and Bnip2) were validated by ELISA. The neurotransmitters taurine and norepinephrine, and the cholinergic constituents, correlated closely with the neuroprotective effects of XA. Finally, the protein–lipid interaction network was analyzed. Based on our results, the regulation of sphingolipid metabolism and mitochondrial function were determined to be the main mechanisms underlying the neuroprotective effect of XA. The present study should help us to better understand the multiple effects of macamides and their use in neurodegenerative diseases. Full article

Haploinsufficiency of the T-box transcription factor TBX1 is responsible for many features of 22q11.2 deletion syndrome. Tbx1 is expressed dynamically in the pharyngeal apparatus during mouse development and Tbx1 homozygous mutants display numerous severe defects including abnormal cranial ganglion formation and neural crest cell defects. These abnormalities prompted us to investigate whether parasympathetic (vagal) innervation of the heart was affected in Tbx1 mutant embryos. In this report, we used an allelic series of Tbx1 mouse mutants, embryo tissue explants and cardiac electrophysiology to characterise, in detail, the function of Tbx1 in vagal innervation of the heart. We found that total nerve branch length was significantly reduced in Tbx1^+/− and Tbx1^neo2/− mutant hearts expressing 50% and 15% levels of Tbx1. We also found that neural crest cells migrated normally to the heart of Tbx1^+/−, but not in Tbx1^neo2 mutant embryos. In addition, we showed that cranial ganglia IX^th and X^th were fused in Tbx1^neo2/− but neuronal differentiation appeared intact. Finally, we used telemetry to monitor heart response to carbachol, a cholinergic receptor agonist, and found that heart rate recovered more quickly in Tbx1^+/− animals versus controls. We speculate that this condition of decreased parasympathetic drive could result in a pro-arrhythmic substrate in some 22q11.2DS patients. Full article

The reduction of choline acetyltransferase, caused by the loss of cholinergic neurons, leads to the absence of acetylcholine (Ach), which is related to motor nerve degeneration. The aims of the present study were to evaluate the in vitro cholinergic nerve differentiation potential of mesenchymal stem cells from cryopreserved human dental pulp (hDPSCs-cryo) and to analyze the scale of in vivo motor nerve regeneration. The hDPSCs-cryo were isolated and cultured from cryopreserved dental pulp tissues, and thereafter differentiated into cholinergic neurons using tricyclodecane-9-yl-xanthogenate (D609). Differentiated cholinergic neurons (DF-chN) were transplanted into rats to address sciatic nerve defects, and the scale of in vivo motor nerve regeneration was analyzed. During in vitro differentiation, the cells showed neuron-like morphological changes including axonal fibers and neuron body development, and revealed high expression of cholinergic neuron-specific markers at both the messenger RNA (mRNA) and protein levels. Importantly, DF-chN showed significant Ach secretion ability. At eight weeks after DF-chN transplantation in rats with sciatic nerve defects, notably increased behavioral activities were detected with an open-field test, with enhanced low-affinity nerve growth factor receptor (p75NGFR) expression detected using immunohistochemistry. These results demonstrate that stem cells from cryopreserved dental pulp can successfully differentiate into cholinergic neurons in vitro and enhance motor nerve regeneration when transplanted in vivo. Additionally, this study suggests that long-term preservation of dental pulp tissue is worthwhile for use as an autologous cell resource in the field of nerve regeneration, including cholinergic nerves. Full article

Cholinergic signaling, which modulates cell activities via nicotinic and muscarinic acetylcholine receptors (n- and mAChRs) in response to internal or external stimuli, has been demonstrated in mammalian non-neuronal cells that synthesize acetylcholine (ACh). One of the major pathways of excitatory transmission in the enteric nervous system (ENS) is mediated by cholinergic transmission, with the transmitter ACh producing excitatory potentials in postsynaptic effector cells. In addition to ACh-synthesizing and ACh-metabolizing elements in the ENS, the presence of non-neuronal ACh machinery has been reported in epithelial cells of the small and large intestines of rats and humans. However, little is known about how non-neuronal ACh controls physiological function in the intestine. Here, experiments using crypt–villus organoids that lack nerve and immune cells in culture suggest that endogenous ACh is synthesized in the intestinal epithelium to drive organoid growth and differentiation through activation of nAChRs. Treatment of organoids with nicotine enhanced cell growth and the expression of marker genes for stem and epithelial cells. On the other hand, the nAChR antagonist mecamylamine strongly inhibited the growth and differentiation of organoids, suggesting the involvement of nAChRs in the regulation of proliferation and differentiation of Lgr5-positive stem cells. More specifically, RNA sequencing analysis revealed that Wnt5a expression was dramatically upregulated after nicotine treatment, and Wnt5a rescued organoid growth and differentiation in response to mecamylamine. Taken together, our results indicate that coordinated activities of nAChR and Wnt signaling maintain Lgr5-positive stem cell activity and balanced differentiation. Furthermore, we could clearly separate the two groups, neuronal ACh in the ENS and non-neuronal ACh in the intestinal epithelium. Dysfunction of the non-neuronal cholinergic system is involved in the pathogenesis of disease. The data will increase our understanding of the cholinergic properties of non-neuronal cells and lead to optimization of drug therapy. Full article

Hypoxic-ischemic brain injuries (HIBI) in childhood are frequently associated with poor clinical and neurological outcome. Unfortunately, there is currently no effective therapy to restore neuronal loss and to determine substantial clinical improvement. Several neurotrophins, such as Nerve Growth Factor (NGF), Brain-Derived Neurotrophic Factor (BDNF), and Glial Derived Neurotrophic Factor (GDNF), play a key role in the development, differentiation, and survival of the neurons of the peripheral and central nervous system. Experimental animal studies demonstrated their neuroprotective role in HIBI, while only a few studies examined the neuroprotective mechanisms in patients with severe HIBI. We report two cases of children with HIBI and prolonged comatose state who showed a significant improvement after intraventricular NGF administration characterized by amelioration of electroencephalogram (EEG) and cerebral perfusion at single-photon emission computed tomography (SPECT). The improvement in motor and cognitive functions of these children could be related to the neuroprotective role exerted by NGF in residual viable cholinergic neurons, leading to the restoration of neuronal networks in the damaged brain. Full article