Cholinergic Synapses Research Articles

Botulinum toxin in the treatment of neuralgia and neuropathic pain

Introduction: Botulinum toxin is one of the most powerful neurotoxins currently known, with high affinity to the cholinergic synapse, which sufficiently inhibits the release of acetylcholine. Its use has proved to be effective in the treatment of many diseases of the musculoskeletal system. Although most of the therapeutic effects of botulinum toxin are due to the temporary relaxation of skeletal muscles (caused by inhibition of acetylcholine release), research is ongoing into its effects on the nervous system.
 Purpose of the work: The aim of the study is to analyze the effectiveness of botulinum toxin in the treatment of neuralgia and neuropathic pain.
 Method: The relevant samples were accessed by means of an electronic search in the PubMed database. The analysis used reviews and meta-analyzes posted on the platform over the last 10 years.
 Conclusions: Botulinum toxin has great potential in the treatment of pain. It is multitasking due to its favorable safety profile and long-lasting relief from a single injection compared to other pain medications. The side effects caused by it were assessed to be mild to moderate and included a local skin reaction (swelling), injection site pain, muscle weakness, flu symptoms, nausea and vomiting.
 Keywords: botulinum toxin; neuropathic pain; pain treatment; neuralgia; BoNT / A

Brain-derived neurotrophic factor predominantly regulates the expression of synapse-related genes in the striatum: Insights from in vitro transcriptomics.

AimThe striatum, a main component of the basal ganglia, is a critical part of the motor and reward systems of the brain. It consists of GABAergic and cholinergic neurons and receives projections of dopaminergic, glutamatergic, and serotonergic neurons from other brain regions. Brain‐derived neurotrophic factor (BDNF) plays multiple roles in the central nervous system, and striatal BDNF has been suggested to be involved in psychiatric and neurodegenerative disorders. However, the transcriptomic impact of BDNF on the striatum remains largely unknown. In the present study, we performed transcriptomic profiling of striatal cells stimulated with BDNF to identify enriched gene sets (GSs) and their novel target genes in vitro.MethodsWe carried out RNA sequencing (RNA‐Seq) of messenger RNA extracted from primary dissociated cultures of rat striatum stimulated with BDNF and conducted Generally Applicable Gene‐set Enrichment (GAGE) analysis on 10599 genes. Significant differentially expressed genes (DEGs) were determined by differential expression analysis for sequence count data 2 (DESeq2).ResultsGAGE analysis identified significantly enriched GSs that included GSs related to regulation and dysregulation of synaptic functions, such as synaptic vesicle cycle and addiction to nicotine and morphine, respectively. It also detected GSs related to various types of synapses, including not only GABAergic and cholinergic synapses but also dopaminergic and glutamatergic synapses. DESeq2 revealed 72 significant DEGs, among which the highest significance was observed in the apolipoprotein L domain containing 1 (Apold1).ConclusionsThe present study indicates that BDNF predominantly regulates the expression of synaptic‐function‐related genes and that BDNF promotes synaptogenesis in various subtypes of neurons in the developing striatum. Apold1 may represent a unique target gene of BDNF in the striatum.

Effect of Rhizoma alismatis on the expression of hub genes in the treatment of gastric cancer.

BackgroundThe aim of this study was to predict the target genes and pathways of Rhizoma alismatis (RA) in the treatment of gastric cancer (GC) by an bioinformatics analysis.MethodsThe Traditional Chinese Medicine System Pharmacology database was used to obtain the chemical components and target genes of RA. GC-related genes were downloaded from GeneCard website. GO and KEGG enrichment analyses were used to detect the potential mechanisms of RA targets. Hub genes were identified by protein-protein interaction (PPI) network and then verified by quantitative real-time polymerase chain reaction (qRT-PCR) analysis.ResultsOur analyses identified 34 target genes that contribute to the development of GC. GO analysis showed that the biological functions of the target genes mainly included activation of receptors, including the nuclear receptor, steroid hormone receptor, acetylcholine receptor, G-protein coupled serotonin receptor, serotonin receptor and others. According to KEGG analysis, we found that insulin resistance, galactose metabolism, adipocytokine signaling pathway, breast cancer pathway, and cholinergic synapse were the top 5 pathways involving RA target genes. qRT-PCR and immunohistochemical analysis indicated that RA had significant effects on the expression of hub genes, including MYC, CASP3, SP1, MAPK8, PPARG, FOS, and SLC2A1.ConclusionsOur study revealed the multi-component, multi-target, and multi-mechanisms of RA on GC, which suggest novel therapeutics for GC.

Network Pharmacology-Based Analysis of the Effects of Corydalis decumbens (Thunb.) Pers. in Non-Small Cell Lung Cancer.

Non-small cell lung cancer (NSCLC) is one of the most malignant tumors worldwide. The main treatment for NSCLC is based on Western medicine; however, the overall effect is unsatisfactory. This study aimed to investigate the potential therapeutic targets and pharmacological mechanisms of action of the traditional Chinese medicine Corydalis decumbens (Thunb.) Pers. in NSCLC based on network pharmacology and bioinformatics. The overlapping genes between Corydalis decumbens (Thunb.) Pers. and NSCLCs were screened using Venn analysis. Cytoscape 3.7.1 software was used to analyze the overlapping target protein-protein interaction (PPI) network. Gene ontology and pathway enrichment analysis using the Kyoto Encyclopedia of Genes and Genomics database were performed to exploring biological functions of the overlapping genes. The gene expression profiling interactive analysis dataset was used to analyze the correlation between hub gene expression and disease. This study revealed 38 nodes with 191 edges, which may be therapeutic targets for NSCLC. PPI network analysis showed that the most likely association was between the genes AR and NCOA2, NCOA2, and RXRA and ESR1 and NCOA2. These overlapping genes were mainly enriched in the estrogen signaling pathway, calcium signaling pathway, cholinergic synapse, and PI3K-Akt signaling pathway. ESR2 mRNA levels were significantly downregulated in patients with lung adenocarcinoma (LUAD) getting worse, and KDR levels were lower in lung squamous cell carcinoma (LUSC) than those in normal tissue. PTGS2 expression was correlated with the median survival time of LUAD, and ESR1 expression was correlated with the median survival time of LUSC. The application of network pharmacology revealed the potential mechanism underlying the effects of Corydalis decumbens (Thunb.) Pers. in NSCLC treatment and provided a theoretical basis for further in-depth research in this field.

A network pharmacology study with molecular docking to investigate the possibility of licorice against posttraumatic stress disorder.

Post traumatic stress disorder (PTSD) is a mental health condition that has a debilitating effect on a person's quality of life and leads to a high socioeconomic burden. Licorice has been demonstrated to have neuroprotective and antidepressant-like effects, but little is known about its effects for the treatment of PTSD. The present study aimed to explore the potential of licorice for PTSD therapy using a network pharmacology approach with molecular docking studies. The compounds of licorice were obtained from databases with screening by absorption, distribution, metabolism and excretion (ADME) evaluation. Genes associated with compounds or PTSD were obtained from public databases, and the genes overlapping between licorice compounds and PTSD were compared by Venn diagram. A network of medicine-ingredients-targets-disease was constructed, visualized, and analyzed using cytoscape software. Protein-protein interactions, gene ontology, pathway enrichment and molecular docking were performed to evaluate the effect of licorice for the treatment of PTSD. 69 potential compounds were screened after ADME evaluation. A total of 81 compound-related genes and 566 PTSD-related genes were identified in the databases with 27 overlapping genes. Licorice compounds (e.g., medicarpin, 7-methoxy-2-methyl isoflavone, shinpterocarpin, formononetin, licochalcone a) and target proteins (e.g., ESR1, PTGS2, NOS2, and ADRB2) with high degree in the network were involved in G protein-coupled receptor signaling pathways at the postsynaptic/synaptic membrane. Moreover, neuroactive ligand-receptor interactions, calcium signaling, cholinergic synapse, serotonergic synapse and adrenergic signaling in cardiomyocytes may play important roles in the treatment of PTSD by licorice. This study provides molecular evidence of the beneficial effects of licorice for the treatment of PTSD.

Activation of Neuronal Nicotinic Receptors Inhibits Acetylcholine Release in the Neuromuscular Junction by Increasing Ca2+ Flux through Cav1 Channels.

Cholinergic neurotransmission is a key signal pathway in the peripheral nervous system and in several branches of the central nervous system. Despite the fact that it has been studied extensively for a long period of time, some aspects of its regulation still have not yet been established. One is the relationship between the nicotine-induced autoregulation of acetylcholine (ACh) release with changes in the concentration of presynaptic calcium levels. The mouse neuromuscular junction of m. Levator Auris Longus was chosen as the model of the cholinergic synapse. ACh release was assessed by electrophysiological methods. Changes in calcium transients were recorded using a calcium-sensitive dye. Nicotine hydrogen tartrate salt application (10 μM) decreased the amount of evoked ACh release, while the calcium transient increased in the motor nerve terminal. Both of these effects of nicotine were abolished by the neuronal ACh receptor antagonist dihydro-beta-erythroidine and Cav1 blockers, verapamil, and nitrendipine. These data allow us to suggest that neuronal nicotinic ACh receptor activation decreases the number of ACh quanta released by boosting calcium influx through Cav1 channels.

Apoptosis is involved in paraoxon-induced histological changes in rat cerebellum

Acute toxicity of organophosphorus compounds is primarily caused by inhibition of acetylcholinesterase (AChE) at cholinergic synapses. The current study was designed to investigate the effects of paraoxon on histological changes as well as the role of mitochondrion-dependent apoptosis in causing this damage in the rat cerebellum. Adult male Wistar rats were intraperitoneally injected with paraoxon at 0.3, 0.7, or 1 mg/kg. Control animals were injected with corn oil as a vehicle. At 14 or 28 days after intoxication, histological changes and alterations in the expression of apoptosis-related proteins, including Bax, Bcl-2, and caspase-3, were investigated in the cerebellum using cresyl violet staining and western blotting, respectively. Findings showed the decreased thickness of both molecular and granular layers and reduction in the number of Purkinje cells in animals treated with a higher convulsive dose of paraoxon (1 mg/kg). In addition, exposure of rats to 1 mg/kg of paraoxon activated apoptosis pathway confirmed by an increase in Bax and caspase-3 and a decrease in Bcl-2 protein levels. According to our results, cerebellar histological changes and alterations in the expression of apoptosis-related proteins occur following exposure to a high convulsive dose of paraoxon and persist for a long time.

Striatal Cholinergic Dysregulation after Neonatal Decrease in X-Linked Dystonia Parkinsonism-Related TAF1 Isoforms.

X-linked dystonia parkinsonism is a generalized, progressive dystonia followed by parkinsonism with onset in adulthood and accompanied by striatal neurodegeneration. Causative mutations are located in a noncoding region of the TATA-box binding protein-associated factor 1 (TAF1) gene and result in aberrant splicing. There are 2 major TAF1 isoforms that may be decreased in symptomatic patients, including the ubiquitously expressed canonical cTAF1 and the neuronal-specific nTAF1. The objective of this study was to determine the behavioral and transcriptomic effects of decreased cTAF1 and/or nTAF1 in vivo. We generated adeno-associated viral (AAV) vectors encoding microRNAs targeting Taf1 in a splice-isoform selective manner. We performed intracerebroventricular viral injections in newborn mice and rats and intrastriatal infusions in 3-week-old rats. The effects of Taf1 knockdown were assayed at 4 months of age with evaluation of motor function, histology, and RNA sequencing of the striatum, followed by its validation. We report motor deficits in all cohorts, more pronounced in animals injected at P0, in which we also identified transcriptomic alterations in multiple neuronal pathways, including the cholinergic synapse. In both species, we show a reduced number of striatal cholinergic interneurons and their marker mRNAs after Taf1 knockdown in the newborn. This study provides novel information regarding the requirement for TAF1 in the postnatal maintenance of striatal cholinergic neurons, the dysfunction of which is involved in other inherited forms of dystonia. © 2021 International Parkinson and Movement Disorder Society.

Cholinergic Synapse Pathway Gene Polymorphisms Associated With Late-Phase Responses in Allergic Rhinitis.

Allergic rhinitis (AR) is characterized by an early-phase response (EPR), and in a subgroup of individuals, a late-phase response (LPR). We sought to investigate polymorphisms in cholinergic synapse pathway genes, previously associated with late-asthmatic responses, in the LPR. Twenty healthy participants and 74 participants with AR underwent allergen exposure using the Environmental Exposure Unit. Allergic participants were sub-phenotyped using self-reported nasal congestion scores; congestion is the predominant symptom experienced during the LPR. Acute congestion (AC, n = 36) participants developed only an EPR, while persistent congestion (PC, n = 38) participants developed both allergic responses. We interrogated blood samples collected before allergen exposure with genotyping and gene expression assays. Twenty-five SNPs located in ADCY3, AKT3, CACNA1S, CHRM3, CHRNB2, GNG4, and KCNQ4 had significantly different allele frequencies (P < 0.10) between PC and AC participants. PC participants had increased minor allele content (P = 0.009) in the 25 SNPs compared to AC participants. Two SNPs in AKT3 were associated with gene expression differences (FDR < 0.01) in PC participants. This study identified an association between the LPR and polymorphisms in the cholinergic synapse pathway genes, and developed a novel method to sub-phenotype AR using self-reported nasal congestion scores.

Physiology of cholinergic and adrenergic synapses. Role in pharmacology and practical significance

The transmission of signals between cholinergic neurons and from neurons to muscle cells (neuro-neuronal and neuromuscular transduction) occurs through synapses. They are formed by the membranes of two contacting cells, presynaptic and postsynaptic, which are separated by a narrow synaptic gap. The review article provides up-to-date information about the physiological processes in cholinergic and adrenergic synapses. The role of these synapses in pharmacology and their practical significance are presented. The transmission of excitation in cholinergic synapses occurs with the help of acetylcholine. The stages of synthesis, storage and release of acetylcholine are the same in all cholinergic neurons. The specific effects of acetylcholine mediated through cholinergic synapses depend mainly on the type of synaptic cholinergic receptors. In the system of efferent innervation, adrenergic synapses are formed by the endings of postganglionic sympathetic (adrenergic) fibers and cells of effector organs.

The HSPG syndecan is a core organizer of cholinergic synapses.

The extracellular matrix has emerged as an active component of chemical synapses regulating synaptic formation, maintenance, and homeostasis. The heparan sulfate proteoglycan (HSPG) syndecans are known to regulate cellular and axonal migration in the brain. They are also enriched at synapses, but their synaptic functions remain more elusive. Here, we show that SDN-1, the sole orthologue of syndecan in C. elegans, is absolutely required for the synaptic clustering of homomeric α7-like acetylcholine receptors (AChRs) and regulates the synaptic content of heteromeric AChRs. SDN-1 is concentrated at neuromuscular junctions (NMJs) by the neurally secreted synaptic organizer Ce-Punctin/MADD-4, which also activates the transmembrane netrin receptor DCC. Those cooperatively recruit the FARP and CASK orthologues that localize α7-like-AChRs at cholinergic NMJs through physical interactions. Therefore, SDN-1 stands at the core of the cholinergic synapse organization by bridging the extracellular synaptic determinants to the intracellular synaptic scaffold that controls the postsynaptic receptor content.

Candidate l-methionine target piRNA regulatory networks analysis response to cocaine-conditioned place preference in mice.

BackgroundMethionine has been proven to inhibit addictive behaviors of cocaine dependence. However, the mechanism of methionine response to cocaine CPP is unknown. Recent evidence highlights piRNAs to regulate genes via a miRNA‐like mechanism. Here, next‐generation sequencing is used to study mechanism on methionine response to drug‐induced behaviors though piRNA.Methodsl‐methionine treatment cocaine CPP animal model was used to do non‐coding RNA sequencing. There were four groups to sequence: saline+saline (SS), MET+saline (MS), MET+cocaine (MC), and cocaine+saline. Combining mRNA sequencing data, the network and regulation of piRNA were analyzed with their corresponding mRNA and miRNA.ResultsAnalysis of the piRNAome reveals that piRNAs inversely regulated their target mRNA genes. KEGG analysis of DE‐piRNA target mRNA genes were enriched in Morphine addiction, GABAergic synapse and Cholinergic synapse pathway. Furthermore, four significantly differential expressed genes Cacna2d3, Epha6, Nedd4l, and Vav2 were identified and regulated by piRNAs in the process of l‐methionine inhibits cocaine CPP. Thereinto, Vav2 was regulated by multiple DE piRNAs by sharing the common sequence: GTCTCTCCAGCCACCTT. Meanwhile, it was found that piRNA positively regulates miRNA and three genes Bcl3, Il20ra, and Insrr were identified and regulated by piRNA through miRNA.ConclusionThe results showed that piRNA negatively regulated target mRNA genes and positively regulated target miRNA genes. Genes located in substance dependence, signal transduction and also nervous functions pathways were identified. When taken together, these data may explain the roles of l‐methionine in counteracting the effects of cocaine CPP via piRNAs.

In silico molecular docking analysis for potential anti-Alzheimer's compounds from the methanolic leaf extract of Erythroxylum monogynum using Gas chromatography–mass spectrometry

BackgroundAlzheimer's disease is the major cause of dementia by two main factors includes an accumulation of acetylcholinesterase enzyme at cholinergic synapse and deposition of β-amyloid protein at neurofibrillary tangles. The present investigation explores the binding affinities of phytoconstituents present in the methanolic leaf extract of Erytrhroxylum monogynum identified by gas chromatography-mass spectrometry against selected targets, i.e., β-amyloid and acetylcholinesterase. ObjectivesThe main objective of present study was to explore anti-Alzheimer's potential of phytoconstituents from methanolic leaf extract of Erytrhroxylum monogynum. MethodsErytrhroxylum monogynum leaf material was subjected to Soxhlet extraction by three different solvents, namely n-hexane, chloroform and methanol. The methanolic leaf extract was subjected to Gas chromatography–mass spectrometric analysis for identification of novel phytoconstituents and chromatogram revealed the presence of 45 novel phytoconstituents. These phytoconstituents were not reported earlier from any part of this plant material. These phytoconstituents were explored for their anti-Alzheimer’s potential by iGEMDOCK software against selected targets, namely recombinant human acetylcholinesterase (protein data bank ID: 3LII) and β-amyloid (protein data bank ID: 2LMN). ResultsFrom the results of docking analysis, the top five phytoconstituents with the highest binding affinity towards the selected targets were further subjected to ADMET prediction by in silico admetSAR web portal. ConclusionsMolecular docking analysis revealed the presence of 7 phytoconstituents are having the highest binding affinity towards the selected targets when compared to their respective standard compounds namely 9, 12-octa-decadienoic acid, methyl ester, (E, E); octadecanoic acid, methyl ester; hexadecanoic acid, 2-hydroxy-1-(hydroxymethyl) ethyl ester; 3, 6-ditigloyloxy-7-hydroxytropane; 3, 7,11, 15-tetramethyl-2-hexa-decen-1-ol; neophytadiene and phytol. The lead hits were further subjected to LigPlot analysis to understand their intermolecular interactions. Thus the result collectively obtained from the in silico studies using computational approaches may become prospective novel lead compounds against the selected targets of Alzheimer's disease.

Machine learning models to select potential inhibitors of acetylcholinesterase activity from SistematX: a natural products database.

Alzheimer's disease is the most common form of dementia, representing 60-70% of dementia cases. The enzyme acetylcholinesterase (AChE) cleaves the ester bonds in acetylcholine and plays an important role in the termination of acetylcholine activity at cholinergic synapses in various regions of the nervous system. The inhibition of acetylcholinesterase is frequently used to treat Alzheimer's disease. In this study, a merged BindingDB and ChEMBL dataset containing molecules with reported half-maximal inhibitory concentration (IC50) values for AChE (7032 molecules) was used to build machine learning classification models for selecting potential AChE inhibitors from the SistematX dataset (8593 secondary metabolites). A total of seven fivefold models with accuracy above 80% after cross-validation were obtained using three types of molecular descriptors (VolSurf, DRAGON 5.0, and bit-based fingerprints). A total of 521 secondary metabolites (6.1%) were classified as active in this stage. Subsequently, virtual screening was performed, and 25 secondary metabolites were identified as potential inhibitors of AChE. Separately, the crystal structure of AChE in complex with (-)-galantamine was used to perform molecular docking calculations with the entire SistematX dataset. Consensus analysis of both methodologies was performed. Only eight structures achieved combined probability values above 0.5. Finally, two sesquiterpene lactones, structures 15 and 24, were predicted to be able to cross the blood-brain barrier, which was confirmed in the VolSurf+ quantitative model, revealing these two structures as the most promising secondary metabolites for AChE inhibition among the 8593 molecules tested. A consensus analysis of classification models and molecular docking calculations identified four potential inhibitors of acetylcholinesterase from the SistematX dataset (8593 structures).

Mechanism of volatile oil from Alpinia oxyphylla in treating Alzheimer's disease based on GC-MS and network pharmacology

To study the material basis and mechanism of volatile oil from Alpinia oxyphylla in treating Alzheimer's disease(AD) based on GC-MS and network pharmacology. Ingredients of volatile oil from A.oxyphylla were analyzed by GC-MS. Targets of those ingredients were obtained through Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform(TCMSP). Relevant targets of AD were obtained through such databases as DrugBank, STITCH, OMIM. Intersection targets of ingredients and diseases were obtained by Online Venny map, and PPI network was established by STRING to screen out core targets. Gene ontology(GO) functional enrichment analysis and KEGG pathway enrichment analysis were performed by DAVID. The &quot;ingredients-target-pathway&quot; network was constructed by software Cytoscape 3.8.1 to screen out potential active ingredients of volatile oil from A.oxyphylla in the treatment of AD. The results showed that a total of 6 active ingredients were screened from the volatile oil of A.oxyphylla by GC-MS, 17 targets corresponding to 6 active ingredients were found in TCMSP database, and 3 448 AD targets were found in DrugBank database. &quot;Ingredients-target-pathway&quot; network and PPI network showed there were 4 potential active ingredients in the treatment of AD and 4 core targets. GO analysis and KEGG analysis showed 34(P&lt;0.05) and 5(P&lt;0.05) pathways, respectively, including nerve ligand receptor interaction, calcium signaling pathway, cholinergic synapse and 5-hydroxytryptaminergic synapse. This suggested that volatile oil from A.oxyphylla could synergistically treat AD by regulating calcium balance, cholinergic balance and phosphorylation. This study provided reference and guidance for further study of volatile oil from A.oxyphylla in the treatment of AD.

Study on potential effective components and mechanism of Zhishe Tongluo Capsules in treatment of ischemic stroke

To explore the potential effective components and mechanism of Zhishe Tongluo Capsules in the treatment of ischemic stroke via network pharmacology, molecular docking and cellular experiment. The chemical constituents of Zhishe Tongluo Capsules were found by TCMSP, BATMAN-TCM and literatures. The constituents-target network was predicted by BATMAN-TCM database. Key words such as cerebral stroke, ischemic stroke and cerebral ischemic stroke were used to search ischemic stroke related targets, and then Venny Map was constructed based on the targets of traditional Chinese medicine and the targets of ischemic stroke. The overlapping targets were imported into STRING database to establish the interaction network. Furthermore, the core targets were screened out by Cytoscape software. Go and KEGG enrichment analysis were performed through DVIAD database. The results showed a total of 193 potential chemical constituents, 985 drug targets and 6 035 disease targets. There were 631 potential targets, 44 core targets and 55 potential active components for treating ischemic stroke through Venny mapping. GO enrichment analysis mainly involved response to hypoxia and positive regulation of ERK1/ERK2. KEGG pathway enrichment analysis mainly involved cholinergic synapse, cAMP signaling pathway, and calcium signaling pathway. Molecular docking data revealed that TP53, EGFR, IL6, INS, TNF and SRC had a good capability to bind with their corresponding active components. To ensure the protective effect Zhishe Tongluo Capsules on the inflammation reaction, an in vitro model of lipopolysaccharide(LPS)-induced RAW264.7 cells was built. The contents of IL-1α, IL-1β, IL-6 and TNF-α in the supernatant were significantly decreased by enzyme linked immunosorbent assay(ELISA). The findings suggested that Zhishe Tongluo Capsules could prevent the injury of ischemic stroke by inhibiting the inflammation.

Temporal regulation of nicotinic acetylcholine receptor subunits supports central cholinergic synapse development in Drosophila

The construction and maturation of the postsynaptic apparatus are crucial for synapse and dendrite development. The fundamental mechanisms underlying these processes are most often studied in glutamatergic central synapses in vertebrates. Whether the same principles apply to excitatory cholinergic synapses, such as those found in the insect central nervous system, is not known. To address this question, we investigated a group of projection neurons in the Drosophila larval visual system, the ventral lateral neurons (LNvs), and identified nAchRα1 (Dα1) and nAchRα6 (Dα6) as the main functional nicotinic acetylcholine receptor (nAchR) subunits in the larval LNvs. Using morphological analyses and calcium imaging studies, we demonstrated critical roles of these two subunits in supporting dendrite morphogenesis and synaptic transmission. Furthermore, our RNA sequencing analyses and endogenous tagging approach identified distinct transcriptional controls over the two subunits in the LNvs, which led to the up-regulation of Dα1 and down-regulation of Dα6 during larval development as well as to an activity-dependent suppression of Dα1 Additional functional analyses of synapse formation and dendrite dynamics further revealed a close association between the temporal regulation of individual nAchR subunits and their sequential requirements during the cholinergic synapse maturation. Together, our findings support transcriptional control of nAchR subunits as a core element of developmental and activity-dependent regulation of central cholinergic synapses.

Transcriptome Analysis of Choroid and Retina From Tree Shrew With Choroidal Neovascularization Reveals Key Signaling Moieties.

Pathological neovascularization in choroid, a leading cause of blindness, is a characteristic of many fundus diseases, such as diabetic retinopathy and age-related macular degeneration. The present study aimed to elucidate the key signaling pathways in choroidal neovascularization (CNV) by analyzing the mRNA profiles of choroid and retina in tree shrews with CNV. We induced choroidal angiogenesis by laser photocoagulation in 15 tree shrews and obtained mRNA profiles of their choroids and retinas by high-throughput transcriptome sequencing. Hierarchical cluster analysis, weighted gene co-expression network analysis (WGCNA), protein-protein interaction (PPI) network analysis, hematoxylin and eosin (HE) staining, CD31 immunohistochemistry (IHC), and reverse transcription quantitative PCR (RT-qPCR) were performed. After laser photocoagulation, we obtained a total of 350 differentially expressed genes (DEGs) in the choroid, including 59 genes in Module-FASN (“ME-FASN”) module and 28 genes in Module-RPL (“ME-RPL”) module. A total of 69 DEGs in retina, including 20 genes in Module-SLC (“ME-SLC”) module. Bioinformatics analysis demonstrated that DEGs in choroid were mainly involved in membrane transport; DEGs in “ME-RPL” were prominent in pathways associated with IgA production, antigen presentation, and cell adhesion molecules (CAMs) signaling. DEGs in “ME-FASN” were involved in fatty acid metabolism and PPAR signaling pathway, while DEGs in “ME-SLC” were involved in GABAergic synapse, neuroactive life receptor interaction, cholinergic synapse, and retrograde endocannabinoid signaling pathway. PPI network analysis demonstrated that the ribosomal protein family genes (RPL31, RPL7, RPL26L1, and RPL19) are key factors of “ME-RPL,” acyl-CoA superfamily genes (ACACA, ACAT1, ACAA2, and ACACB) and FASN are key factors of “ME-FASN” and superfamily of solid carrier genes (SLC17A6, SLC32A1, SLC12A5, and SLC6A1) and complement genes (C4A, C3, and C2) are key factors of “ME-SLC.” In conclusion, the present study discovered the important signal transductions (fatty acid metabolic pathway and CAMs signaling) and genes (ribosomal protein family and the complement system) in tree shrew CNV. We consider that our findings hold implications in unraveling molecular mechanisms that underlie occurrence and development of CNV.

Epigenetic Programming Reverses Cardiometabolic Dysfunctions and Modulates Hypothalamic Genes Involved in Oxidative Stress and Inflammation in Angiotensin II-Treated Male Mice

Cardiometabolic disease is a global health issue that affects millions of people worldwide. Environmental perinatal exposure affects the health outcomes of the offspring and determines their disease susceptibility later in life. Angiotensin-II (Ang-II) is a peptide known to cause vasoconstriction, elevated blood glucose levels and inflammation. Previously, we reported that perinatal exposure to a hypercaloric diet (HD) results in elevated blood pressure (BP), weight gain, fasting hyperglycemia and glucose intolerance only in male mice. In addition, subcutaneous infusion of a sub-pressor dose of Ang-II was associated with a normalization in fasting blood glucose levels and a reversal of glucose intolerance only in programmed male mice. We hypothesize that epigenetic programming blocks the deleterious effects of Ang-II by altering its inflammatory signaling pathway. C57BL6/J dams were fed HD or regular diet (RD) for 1 month before mating with RD-fed males. After weaning, offspring of HD dams (programmed) and of RD dams (controls) were maintained on RD until 3 months of age. Mice then underwent 24 h BP recording (telemetry) and were implanted with Ang-II osmotic pumps (200 ng/kg/min/2 weeks). BP (24 h) was recorded weekly for 2 weeks. Mice were then sacrificed and hypothalami were harvested for mRNA sequencing (Illumina NextSeq). Programmed mice had lower 24 h systolic BP levels compared to control males (area under the curve: 41844 ±263.2 vs. 44522 ±275.6; p<0.0001). For RNAseq analysis, data showed 62 differentially expressed genes (DEG) in programmed males compared to controls. Using iPathway analysis, we found that some of the DEG are correlated to cholinergic synapse pathway (p=0.005) and neuroactive ligand-receptor interaction pathway (p=0.003). Nicotinic acetylcholine alpha-7 receptor (Chrna7) gene, known for its anti-inflammatory and hypoglycemic effects was upregulated in programmed males (p=0.024). On the other hand, genes involved in metabolic pathways and oxidative stress were differentially expressed as well. Phospholipase A2 group 3 (Pla2g3) gene, known to be overexpressed in oxidative stress was downregulated in programmed males (p=0.04). Moreover, Thiosulfate sulfurtransferase (Tst) gene, an antioxidant enzyme and used as a marker for enhanced insulin sensitivity was upregulated (p=0.023) in programmed males. Interestingly, female mice did not show any changes in BP or gene expression between the two groups. In conclusion, perinatal exposure to HD alters the cardiovascular response to Ang-II possibly through the modulation of gene expression of Chrna7 gene and genes involved in oxidative stress. Future experiments will be investigating the signaling pathways used in epigenetic programming to affect inflammation and oxidative stress in male mice.

The Effect of Acute Unilateral Denervation Injury on Purinergic Signaling in the Cholinergic Synapse

The Effect of Acute Unilateral Denervation Injury on Purinergic Signaling in the Cholinergic Synapse