Cellular and transcriptional responses of SH-SY5Y human neurocytes following in vitro exposure to Gelsemium sempervirens

Background: Gelsemium sempervirens (Gelsemium s.) is a highly toxic plant but is employed at low doses and/or high dilutions as an anxiolytic and antidepressant. Previous investigations in our laboratory [1,2] have shown a significant anxiolytic-like activity of Gelsemium s., using emotional response models in laboratory mice. Although there is some biochemical evidence of a possible role of neurosteroid metabolism [3], the cellular and molecular mechanisms involved in the effects of Gelsemium s. at the level of nervous system are largely unknown. To help determine these pathways, we used human neurocytes (SH-SY5Y cell line) treated in vitro with different dilutions of Gelsemium s. and evaluated their vitality and gene expression changes. Methods: The drugs were produced by Boiron Laboratoires, Lyon (F), starting from a whole-plant-hydroalcoholic extract of Gelsemium s. Solutions 1C, 2C, 3C, 4C, 8C and 29C (C= centesimal dilution/dynamization prepared in 30% ethanol/distilled water) were provided in 30-ml glass bottles, wrapped in aluminium foil and were stored in the dark at room temperature in a metal cupboard. Control solutions (“placebo”) were serially diluted/dynamized 30% ethanol/distilled water. Before each experiment, 0.05 ml samples of Gelsemium s. and placebo were added to 5 ml of distilled sterile and apyrogenic water in a 15 ml Falcon polystyrene plastic tube, closed and shaken in mechanical shaker DinaA for 7.5 sec (150 strokes) to obtain the final 2C, 3C, 4C, 5C, 9C and 30C succussed dilutions, with ethanol concentration of 0.3% (v/v) (final 0.03% in the assay system). Human neuroblastoma cell line SHSY5Y was grown in DMEM-F12 medium (Lonza), with 10% fetal bovine serum (FBS), penicillin (100 units/ml) and streptomycin (100 mg/ml). To assess cell viability and metabolism, 20,000 cells per well were seeded in 96 microplate wells in 200 μl of medium. After overnight incubation, 22 μl of drug or placebo were added and the plate was incubated at 37°C with 5% CO2 in a humidified atmosphere for 72 hours. Then the viability test with reagent WST-1 (Roche) was performed for 3 hours and the absorbance was detected with multiplate reader. A total of 17 experiments, each done with six replicate microwells. To make a relative measurement of protein, the cells were lysed and a Bradford assay was done directly in the plate. The Student t-test and the sign rank test for paired data were utilized for data analysis. To obtain a profile of gene expression, cells were pre preconditioned with Gelsemium s./placebo dilutions for 24 h, then RNA was isolated and analysed by microarray and RT-PCR. SHSY5Y cells were plated onto Petri dishes (day 1) and the day after the medium was replaced with the medium with 2% FBS (day 2). After 24h, 10%v/v Gelsemium s. or placebo dilutions were added to the medium (day 3) and cells were incubated for a further 24h. On day 4, cells were then harvested and the RNA extracted using the Qiagen RNAeasy Mini Kit following the manufacturer’s instructions. Microarray analysis was performed on a custom 12 x 135 k human NimbleGen microarray containing 45033 genes with 3 probes per target gene. Four biological replicates were analysed for each condition. Analysis of differentially expressed genes was performed using linear modelling and empirical Bayes methods and p-values were adjusted for multiple testing with the Benjamini and Hochberg method. A Human Neurotransmitter Receptors and Regulators RT2 Profiler PCR array (Qiagen) was performed in profiling the expression of genes involved in modulating the biological processes of neurotransmitter biosynthesis, uptake, transport and signaling through neurotransmitter receptors. Results: In viability tests, cells treated with Gelsemium s. showed slightly higher metabolic activity (3-4 %) than those treated with placebo. Overall comparison of the data for the whole sample of placebo versus that of Gelsemium s. using the Student t-test showed a small but significant difference (p < 0.001). Furthermore, a non parametric approach comparing the two treatments at the same dilution yielded a significant difference under the sign test (p < 0.01) and Wilcoxon rank test for paired data (p < 0.05), so that the values of differences were also considered. The differences between groups having the same dilution (placebo 2C versus Gelsemium s. 2C etc.) were significant in four dilutions: 2 C, 3 C, 4C (p < 0.01), 9 C (p < 0.02), while 5 C and 30 C yielded non-significant values. No changes due to Gelsemium s. were detected using protein assay, suggesting that the viability test revealed effects on metabolic activity instead of on cell proliferation. In microarray analysis, transcripts expression was analyzed and genes differentially expressed by the Gelsemium s. dilutions were selected. A gene was considered to be differentially expressed if it showed an absolute value of log-ratio greater than or equal to 0.5, an index that translates to a fold-change of 1.4 in transcript quantity. Out of a total of 45,033 transcripts, exposure to Gelsemium s. 2C promoted the selective downexpression of 49 genes (p values adj

The Annexin A2/S100A10 Complex: The Mutualistic Symbiosis of Two Distinct Proteins

Mutualistic symbiosis refers to the symbiotic relationship between individuals of different species in which both individuals benefit from the association. S100A10, a member of the S100 family of Ca2+-binding proteins, exists as a tight dimer and binds two annexin A2 molecules. This association forms the annexin A2/S100A10 complex known as AIIt, and modifies the distinct functions of both proteins. Annexin A2 is a Ca2+-binding protein that binds F-actin, phospholipid, RNA, and specific polysaccharides such as heparin. S100A10 does not bind Ca2+, but binds tPA, plasminogen, certain plasma membrane ion channels, neurotransmitter receptors, and the structural scaffold protein, AHNAK. S100A10 relies on annexin A2 for its intracellular survival: in the absence of annexin A2, it is rapidly destroyed by ubiquitin-dependent and independent proteasomal degradation. Annexin A2 requires S100A10 to increase its affinity for Ca2+, facilitating its participation in Ca2+-dependent processes such as membrane binding. S100A10 binds tissue plasminogen activator and plasminogen, and promotes plasminogen activation to plasmin, which is a process stimulated by annexin A2. In contrast, annexin A2 acts as a plasmin reductase and facilitates the autoproteolytic destruction of plasmin. This review examines the relationship between annexin A2 and S100A10, and how their mutualistic symbiosis affects the function of both proteins.

Correspondence between gene expression and neurotransmitter receptor and transporter density in the human brain

Neurotransmitter receptors modulate the signaling between neurons. Thus, neurotransmitter receptors and transporters play a key role in shaping brain function. Due to the lack of comprehensive neurotransmitter receptor/transporter density datasets, microarray gene expression is often used as a proxy for receptor densities. In the present report, we comprehensively test the expression-density association for a total of 27 neurotransmitter receptors, receptor binding-sites, and transporters across 9 different neurotransmitter systems, using both PET and autoradiography imaging modalities. We find poor spatial correspondences between gene expression and density for all neurotransmitter receptors and transporters except four single-protein metabotropic receptors (5-HT1A, D2, CB1, and MOR). These expression-density associations are related to population variance and change across different classes of laminar differentiation. Altogether, we recommend using direct measures of receptor and transporter density when relating neurotransmitter systems to brain structure and function.

Comparative Expression Analysis of Genes Encoding Neurotransmitter Receptors Between Entrepreneurs and Non-Entrepreneurs

Introduction and Purpose: Recent studies revealed that different behavioral and physiological processes are closely related to different receptor levels in humans. On the other hand, there weren’t any studies that investigated neurotransmitter activity and entrepreneurship relationships. Little is known about how genes expressed in the blood are associated with entrepreneurship. In this study, we measured the expression of 84 genes encoding neurotransmitter receptors between the entrepreneurs and non-entrepreneurs (n=25) using RT-PCR arrays to monitor differentially expressed genes for exploring molecular mechanism behind the entrepreneurship mindset. Materials and Methods: After determining whether participants are entrepreneurs or not, blood samples were collected. Blood samples were collected in Vacutainer® EDTA tubes as 10 ml and RNA isolation was performed at the Erzurum Technical University, Cell Culture Laboratory. Trizol® solution for RNA isolation (Thermo®, USA) was applied according to the manufacturer's instructions. Concentrations of RNA samples were measured at 260 nm using a spectrophotometer (Epoch®, Biotek). Then, total RNA was reverse transcribed into cDNA by using High-Capacity cDNA Reverse Transcription Kit (Thermo®). Total 84 genes were analyzed via RT² Profiler™ Human Neurotransmitter Receptors PCR Array (Qiagen®, USA) in Rotor-Gene Q real-time PCR cycler (Qiagen®, USA). Findings: The gene expression results obtained from RT-PCR were compared between entrepreneurs and non-entrepreneurs, and presented as Fold Changes (FC). According to our results, positive FC values indicated an increase in the expression of the genes and negative FC values indicated decrease in gene expression levels in entrepreneurs. Results: These 84 different genes regulate neurotransmitter biosynthesis, uptake, transport, and signaling via neurotransmitter receptors. According to gene expression analyses, gene expressions that could be related to the entrepreneur behavior might be connected to not only undesired psychological outcomes like various addictions and also neurological cases such as schizophrenia and depressive disorder. Our results firstly indicated that entrepreneurship was not only associated with neurotransmitter release but also with receptor levels. Keywords: Entrepreneurship, Entrepreneurs, Genes, Neurotransmitter Receptors, RT-PCR Array

Excitatory and inhibitory receptors utilize distinct post- and trans-synaptic mechanisms in vivo

Ionotropic neurotransmitter receptors at postsynapses mediate fast synaptic transmission upon binding of the neurotransmitter. Post- and trans-synaptic mechanisms through cytosolic, membrane, and secreted proteins have been proposed to localize neurotransmitter receptors at postsynapses. However, it remains unknown which mechanism is crucial to maintain neurotransmitter receptors at postsynapses. In this study, we ablated excitatory or inhibitory neurons in adult mouse brains in a cell-autonomous manner. Unexpectedly, we found that excitatory AMPA receptors remain at the postsynaptic density upon ablation of excitatory presynaptic terminals. In contrast, inhibitory GABAA receptors required inhibitory presynaptic terminals for their postsynaptic localization. Consistent with this finding, ectopic expression at excitatory presynapses of neurexin 3alpha, a putative trans-synaptic interactor with the native GABAA receptor complex, could recruit GABAA receptors to contacted postsynaptic sites. These results establish distinct mechanisms for the maintenance of excitatory and inhibitory postsynaptic receptors in the mature mammalian brain.

Comparative Expression Analysis of Genes Encoding Neurotransmitter Receptors Between Entrepreneurs and Non-Entrepreneurs

Introduction and Purpose: Recent studies revealed that different behavioral and physiological processes are closely related to different receptor levels in humans. On the other hand, there weren’t any studies that investigated neurotransmitter activity and entrepreneurship relationships. Little is known about how genes expressed in the blood are associated with entrepreneurship. In this study, we measured the expression of 84 genes encoding neurotransmitter receptors between the entrepreneurs and non-entrepreneurs (n=25) using RT-PCR arrays to monitor differentially expressed genes for exploring molecular mechanism behind the entrepreneurship mindset. Materials and Methods: After determining whether participants are entrepreneurs or not, blood samples were collected. Blood samples were collected in Vacutainer® EDTA tubes as 10 ml and RNA isolation was performed at the Erzurum Technical University, Cell Culture Laboratory. Trizol® solution for RNA isolation (Thermo®, USA) was applied according to the manufacturer's instructions. Concentrations of RNA samples were measured at 260 nm using a spectrophotometer (Epoch®, Biotek). Then, total RNA was reverse transcribed into cDNA by using High-Capacity cDNA Reverse Transcription Kit (Thermo®). Total 84 genes were analyzed via RT² Profiler™ Human Neurotransmitter Receptors PCR Array (Qiagen®, USA) in Rotor-Gene Q real-time PCR cycler (Qiagen®, USA). Findings: The gene expression results obtained from RT-PCR were compared between entrepreneurs and non-entrepreneurs, and presented as Fold Changes (FC). According to our results, positive FC values indicated an increase in the expression of the genes and negative FC values indicated decrease in gene expression levels in entrepreneurs. Results: These 84 different genes regulate neurotransmitter biosynthesis, uptake, transport, and signaling via neurotransmitter receptors. According to gene expression analyses, gene expressions that could be related to the entrepreneur behavior might be connected to not only undesired psychological outcomes like various addictions and also neurological cases such as schizophrenia and depressive disorder. Our results firstly indicated that entrepreneurship was not only associated with neurotransmitter release but also with receptor levels. Keywords: Entrepreneurship, Entrepreneurs, Genes, Neurotransmitter Receptors, RT-PCR Array

Phenothiazines as dual inhibitors of SARS-CoV-2 main protease and COVID-19 inflammation

COVID-19, caused by the severe acute respiratory coronavirus 2 (SARS-CoV-2) currently has no treatment for acute infection. The main protease (Mpro) of SARS-CoV-2 is an essential enzyme for viral replication and an attractive target for disease intervention. The phenothiazine moiety has demonstrated drug versatility for biological systems, including inhibition of butyrylcholinesterase, a property important in the cholinesterase anti-inflammatory cascade. Nineteen phenothiazine drugs were investigated using in silico modelling techniques to predict binding energies and inhibition constants (Ki values) with SARS-CoV-2 Mpro. Since most side-effects of phenothiazines are due to interactions with various neurotransmitter receptors and transporters, phenothiazines with few such interactions were also investigated. All compounds were found to bind to the active site of SARS-CoV-2 Mpro and showed Ki values ranging from 1.30 to 52.4 µM. Nine phenothiazines showed inhibition constants <10 µM. The compounds with limited interactions with neurotransmitter receptors and transporters showed micromolar (µM) Ki values. Docking results were compared with remdesivir and showed similar interactions with key residues Glu-166 and Gln-189 in the active site. This work has identified several phenothiazines with limited neurotransmitter receptor and transporter interactions and that may provide the dual action of inhibiting SARS-CoV-2 Mpro to prevent viral replication and promote the release of anti-inflammatory cytokines to curb viral-induced inflammation. These compounds are promising candidates for further investigation against SARS-CoV-2.

NMDARs, Coincidence Detectors of Astrocytic and Neuronal Activities

Synaptic plasticity is an extensively studied cellular correlate of learning and memory in which NMDARs play a starring role. One of the most interesting features of NMDARs is their ability to act as a co-incident detector. It is unique amongst neurotransmitter receptors in this respect. Co-incident detection is possible because the opening of NMDARs requires membrane depolarisation and the binding of glutamate. Opening of NMDARs also requires a co-agonist. Although the dynamic regulation of glutamate and membrane depolarization have been well studied in coincident detection, the role of the co-agonist site is unexplored. It turns out that non-neuronal glial cells, astrocytes, regulate co-agonist availability, giving them the ability to influence synaptic plasticity. The unique morphology and spatial arrangement of astrocytes at the synaptic level affords them the capacity to sample and integrate information originating from unrelated synapses, regardless of any pre-synaptic and post-synaptic commonality. As astrocytes are classically considered slow responders, their influence at the synapse is widely recognized as modulatory. The aim herein is to reconsider the potential of astrocytes to participate directly in ongoing synaptic NMDAR activity and co-incident detection.