Therapy-induced secretion of spliceosomal components mediates pro-survival crosstalk between ovarian cancer cells.

Ovarian cancer often develops resistance to conventional therapies, hampering their effectiveness. Here, using ex vivo paired ovarian cancer ascites obtained before and after chemotherapy and in vitro therapy-induced secretomes, we show that molecules secreted by ovarian cancer cells upon therapy promote cisplatin resistance and enhance DNA damage repair in recipient cancer cells. Even a short-term incubation of chemonaive ovarian cancer cells with therapy-induced secretomes induces changes resembling those that are observed in chemoresistant patient-derived tumor cells after long-term therapy. Using integrative omics techniques, we find that both ex vivo and in vitro therapy-induced secretomes are enriched with spliceosomal components, which relocalize from the nucleus to the cytoplasm and subsequently into the extracellular vesicles upon treatment. We demonstrate that these molecules substantially contribute to the phenotypic effects of therapy-induced secretomes. Thus, SNU13 and SYNCRIP spliceosomal proteins promote therapy resistance, while the exogenous U12 and U6atac snRNAs stimulate tumor growth. These findings demonstrate the significance of spliceosomal network perturbation during therapy and further highlight that extracellular signaling might be a key factor contributing to the emergence of ovarian cancer therapy resistance.

Comparison of the calcium signaling alterations in GABA-ergic medium spiny neurons produced from iPSCs of different origins.

Disease models based on induced pluripotent stem cells (iPSCs) are in high demand because of their physiological adequacy and well-reproducibility of the pathological phenotype. Nowadays, the most common approach to generate iPSCs is the reprogramming of somatic cells using vectors based on lentivirus or Sendai virus. We have previously shown impairments of calcium signaling including store-operated calcium entry in Huntington's disease-specific iPSCs-based GABA-ergic medium spiny neurons. However, different approaches for iPSCs generation make it difficult to compare the models since the mechanism of reprogramming may influence the electrophysiological properties of the terminally differentiated neurons. Here, we have studied the features of calcium homeostasis in GABA-ergic medium spiny neurons differentiated from iPSCs obtained from fibroblasts of the same donor using different methods. Our data demonstrated that there were no significant differences neither in calcium influx through the store-operated channels, nor in the levels of proteins activating this type of calcium entry in neurons differentiated from iPSCs generated with lenti- and Sendai viruses-based approaches. We also found no differences in voltage-gated calcium entry for these neurons. Thus, we clearly showed that various methods of cell reprogramming result in similar deregulations in neuronal calcium signaling which substantiates the ability to combine the experimental data on functional studies of ion channels in models based on iPSCs obtained by different methods and expands the prospects for the use of biobanking.

Is a 2D Nanostructured Surface Capable of Changing the Corrosion and Magnetic Properties of an Amorphous Alloy?

In this work, an attempt was made to reveal and explain the influence of the process of formation of 2D nanostructures at the surface of an amorphous alloy (an alloy with the composition Co75Si15Fe5Cr4.5Al0.5 (in at.%) was used for this purpose) on the corrosion and magnetic properties of such an alloy. Two-dimensional nanostructures (nanocells of 100-150 nm in size, which were obtained by anodizing the initial sample in an ionic liquid) are essentially a pattern on the surface of the sample, and they cannot completely cover and block the surface from external effects. It was postulated that the presence of these nanostructures during corrosion and magnetic tests has no significant effect. However, a noticeable inhibition effect was observed during corrosion tests and a less noticeable (but still detectable) effect was observed during magnetic tests. The authors believe that the effect obtained, with a detailed study, can be used to increase the corrosion resistance and to improve the properties of traditional magnetic materials.

Cervical and Vaginal Microbiomes in Early Miscarriages and Ongoing Pregnancy with and without Dydrogesterone Usage.

Emerging evidence suggests that the reproductive tract microbiota is a key modulator of local inflammatory and immune pathways throughout pregnancy and may subsequently impact pregnancy outcomes. In this study, our objective was to analyze the cervical and vaginal microbiomes during early pregnancy among three groups: women with healthy ongoing pregnancies, women undergoing dydrogesterone treatment, and those who experienced miscarriages. The experiment involved 51 women at 8-11 weeks of gestation. The microbiome was examined using 16S rRNA sequencing on the Ion Torrent PGM platform. Across all groups, Lactobacillus iners was predominant, suggesting that the vaginal community type CST III is common among the majority of participants. Notably, our data highlighted the significant roles of Gardnerella vaginalis and Mycoplasma girerdii in the pathogenesis of early miscarriage. Conversely, L. iners and Bifidobacterium longum have a protective effect in early pregnancy. Moreover, dydrogesterone intake appeared to influence notable differences between the cervical and vaginal microbiomes. Overall, our study enhanced our understanding of the cervical and vaginal microbiome composition in the eastern European population during early pregnancy.

PAPP-A-Specific IGFBP-4 Proteolysis in Human Induced Pluripotent Stem Cell-Derived Cardiomyocytes.

The insulin-like growth factors IGF-I and IGF-II-as well as their binding proteins (IGFBPs), which regulate their bioavailability-are involved in many pathological and physiological processes in cardiac tissue. Pregnancy-associated plasma protein A (PAPP-A) is a metalloprotease that preferentially cleaves IGFBP-4, releasing IGF and activating its biological activity. Previous studies have shown that PAPP-A-specific IGFBP-4 proteolysis is involved in the pathogenesis of cardiovascular diseases, such as ischemia, heart failure, and acute coronary syndrome. However, it remains unclear whether PAPP-A-specific IGFBP-4 proteolysis participates in human normal cardiomyocytes. Here, we report PAPP-A-specific IGFBP-4 proteolysis occurring in human cardiomyocytes derived from two independent induced pluripotent cell lines (hiPSC-CMs), detected both on the cell surface and in the cell secretome. PAPP-A was measured by fluoroimmune analysis (FIA) in a conditioned medium of hiPSC-CMs and was detected in concentrations of up to 4.3 ± 1.33 ng/mL and 3.8 ± 1.1 ng/mL. The level of PAPP-A-specific IGFBP-4 proteolysis was determined as the concentration of NT-IGFBP-4 proteolytic fragments using FIA for a proteolytic neo-epitope-specific assay. We showed that PAPP-A-specific IGFBP-4 proteolysis is IGF-dependent and inhibited by EDTA and 1,10-phenanthroline. Therefore, it may be concluded that PAPP-A-specific IGFBP-4 proteolysis functions in human normal cardiomyocytes, and hiPSC-CMs contain membrane-bound and secreted forms of proteolytically active PAPP-A.

An Efficient 2D Protocol for Differentiation of iPSCs into Mature Postmitotic Dopaminergic Neurons: Application for Modeling Parkinson's Disease.

About 15% of patients with parkinsonism have a hereditary form of Parkinson's disease (PD). Studies on the early stages of PD pathogenesis are challenging due to the lack of relevant models. The most promising ones are models based on dopaminergic neurons (DAns) differentiated from induced pluripotent stem cells (iPSCs) of patients with hereditary forms of PD. This work describes a highly efficient 2D protocol for obtaining DAns from iPSCs. The protocol is rather simple, comparable in efficiency with previously published protocols, and does not require viral vectors. The resulting neurons have a similar transcriptome profile to previously published data for neurons, and have a high level of maturity marker expression. The proportion of sensitive (SOX6+) DAns in the population calculated from the level of gene expression is higher than resistant (CALB+) DAns. Electrophysiological studies of the DAns confirmed their voltage sensitivity and showed that a mutation in the PARK8 gene is associated with enhanced store-operated calcium entry. The study of high-purity DAns differentiated from the iPSCs of patients with hereditary PD using this differentiation protocol will allow for investigators to combine various research methods, from patch clamp to omics technologies, and maximize information about cell function in normal and pathological conditions.

iPSC-derived cells lack immune tolerance to autologous NK-cells due to imbalance in ligands for activating and inhibitory NK-cell receptors.

BACKGROUND: Dozens of transplants generated from pluripotent stem cells are currently in clinical trials. The creation of patient-specific iPSCs makes personalized therapy possible due to their main advantage of immunotolerance. However, some reports have claimed recently that aberrant gene expression followed by proteome alterations and neoantigen formation can result in iPSCs recognition by autologous T-cells. Meanwhile, the possibility of NK-cell activation has not been previously considered. This study focused on the comparison of autologous and allogeneic immune response to iPSC-derived cells and isogeneic parental somatic cells used for reprogramming. METHODS: We established an isogeneic cell model consisting of parental dermal fibroblasts, fibroblast-like iPSC-derivatives (iPS-fibro) and iPS-fibro lacking beta-2-microglobulin (B2M). Using the cells obtained from two patients, we analyzed the activation of autologous and allogeneic T-lymphocytes and NK-cells co-cultured with target cells. RESULTS: Here we report that cells differentiated from iPSCs can be recognized by NK-cells rather than by autologous T-cells. We observed that iPS-fibro elicited a high level of NK-cell degranulation and cytotoxicity, while isogeneic parental skin fibroblasts used to obtain iPSCs barely triggered an NK-cell response. iPSC-derivatives with B2M knockout did not cause an additional increase in NK-cell activation, although they were devoid of HLA-I, the major inhibitory molecules for NK-cells. Transcriptome analysis revealed a significant imbalance of ligands for activating and inhibitory NK-cell receptors in iPS-fibro. Compared to parental fibroblasts, iPSC-derivatives had a reduced expression of HLA-I simultaneously with an increased gene expression of major activating ligands, such as MICA, NECTIN2, and PVR. The lack of inhibitory signals might be due to insufficient maturity of cells differentiated from iPSCs. In addition, we showed that pretreatment of iPS-fibro with proinflammatory cytokine IFNγ restored the ligand imbalance, thereby reducing the degranulation and cytotoxicity of NK-cells. CONCLUSION: In summary, we showed that iPSC-derived cells can be sensitive to the cytotoxic potential of autologous NK-cells regardless of HLA-I status. Thus, the balance of ligands for NK-cell receptors should be considered prior to iPSC-based cell therapies. Trial registration Not applicable.

Overexpression of Parkin in the Neuronal Progenitor Cells from a Patient with Parkinson's Disease Shifts the Transcriptome Towards the Normal State.

Parkinson's disease (PD) is a neurodegenerative pathology caused by the progressive loss of dopaminergic neurons in the substantia nigra. Juvenile PD is known to be strongly associated with mutations in the PARK2 gene encoding E3 ubiquitin ligase Parkin. Despite numerous studies, molecular mechanisms that trigger PD remain largely unknown. Here, we compared the transcriptome of the neural progenitor (NP) cell line, derived from a PD patient with PARK2 mutation resulting in Parkin loss, with the transcriptome of the same NPs but expressing transgenic Parkin. We found that Parkin overexpression led to the substantial recovery of the transcriptome of NPs to a normal state indicating that alterations of transcription in PD-derived NPs were mainly caused by PARK2 mutations. Among genes significantly dysregulated in PD-derived NPs, 106 genes unambiguously restored their expression after reestablishing of the Parkin level. Based on the selected gene sets, we revealed the enriched Gene Ontology (GO) pathways including signaling, neurotransmitter transport and metabolism, response to stimulus, and apoptosis. Strikingly, dopamine receptor D4 that was previously associated with PD appears to be involved in the maximal number of GO-enriched pathways and therefore may be considered as a potential trigger of PD progression. Our findings may help in the screening for promising targets for PD treatment.

Female reproductive tract microbiome and early miscarriages.

Miscarriage is one of the main causes of reproductive loss, which can lead to a number of physical and psychological complications and other long-term consequences. However, the role of vaginal and uterine microbiome in such complications is poorly understood. To review the published data on the function of the female reproductive tract microbiome in the pathogenesis of early miscarriages. The articles published over the past 20 years and deposited in PubMed, Google Academy, Scopus, Elibrary, ResearchGate, and EBSCO databases were analyzed. The review presents new data on the impact of the vaginal and uterine microbiome on the local immunity, including defense against sexually transmitted infections, and its association with other factors of miscarriages. The studies on the microbiome of non-pregnant women with recurrent miscarriages in the anamnesis, patients undergoing IVF, and pregnant women with miscarriages, as well as new directions in the microbiome research are discussed. The majority of studies have demonstrated that the dominant species of the vaginal and uterine microbiome in patients with early miscarriages are non-Lactobacillus bacteria. As many of these bacteria have not previously been detected by cultural studies and their role in obstetric complications is not well defined, further research on the female reproductive tract microbiome, including the microbiome of the cervix uteri, is needed to develop new approaches for the prognosis and prevention of miscarriages.

Lower Genital Tract Microbiome in Early Pregnancy in the Eastern European Population.

BACKGROUND: It is known that the features of the cervicovaginal microbiome can depend on ethnicity, which might be caused by genetic factors, as well as differences in diet and lifestyle. There is no research on the cervicovaginal microbiome of Eastern European women during early pregnancy. METHODS: We evaluated the cervical and cervicovaginal microbiome of women with first-trimester pregnancy (n = 22), further delivered at term, using the 16S rRNA sequencing method. RESULTS: The predominant bacterial species in both groups was Lactobacillus iners, followed by Prevotella copri, Ileibacterium valens, Gardnerella vaginalis and Muribaculum intestinale in the cervical samples, and Gardnerella vaginalis, Prevotella copri, Bifidobacterium longum, Ileibacterium valens and Muribaculum intestinale in the cervicovaginal samples. The cervical microbiome had higher alpha diversity; a higher abundance of Muribaculum intestinale, Aquabacterium parvum and Methyloversatilis universalis; and a lower abundance of Psychrobacillus psychrodurans. CONCLUSIONS: The Lactobacillus iners-dominated microbiome (CST III) was the predominant type of cervical and cervicovaginal microbiome in early pregnancy in the majority of the women. The presence of soil and animal bacteria in the cervicovaginal microbiome can be explained by the rural origin of patients.

Thermocatalytic Conversion of Plastics into Liquid Fuels over Clays.

Recycling polymer waste is a great challenge in the context of the growing use of plastics. Given the non-renewability of fossil fuels, the task of processing plastic waste into liquid fuels seems to be a promising one. Thermocatalytic conversion is one of the methods that allows obtaining liquid products of the required hydrocarbon range. Clays and clay minerals can be distinguished among possible environmentally friendly, cheap, and common catalysts. The moderate acidity and the presence of both Lewis and Brønsted acid sites on the surface of clays favor heavier hydrocarbons in liquid products of reactions occurring in their pores. Liquids produced with the use of clays are often reported as being in the gasoline and diesel range. In this review, the comprehensive information on the thermocatalytic conversion of plastics over clays obtained during the last two decades was summarized. The main experimental parameters for catalytic conversion of plastics according to the articles' analysis, were the reaction temperature, the acidity of modified catalysts, and the catalyst-to-plastic ratio. The best clay catalysts observed were the following: bentonite/spent fluid cracking catalyst for high-density polyethylene (HDPE); acid-restructured montmorillonite for medium-density polyethylene (MDPE); neat kaolin powder for low-density polyethylene (LDPE); Ni/acid-washed bentonite clay for polypropylene (PP); neat kaolin for polystyrene (PS); Fe-restructured natural clay for a mixture of polyethylene, PP, PS, polyvinyl chloride (PVC), and polyethylene terephthalate (PET). The main problem in using natural clays and clay minerals as catalysts is their heterogeneous composition, which can vary even within the same deposit. The serpentine group is of interest in studying its catalytic properties as fairly common clay minerals.

Efficient and fast degradation of 4-nitrophenol and detection of Fe(III) ions by Poria cocos extract stabilized silver nanoparticles.

In this study, a simple and environment-friendly method has been successfully applied for the production of silver nanoparticles (AgNPs) using Poria cocos extract. The reaction time of 60 min, the temperature of 90 °C, and silver ion concentration of 2.0 mM were identified as the best condition for the PC-AgNPs fabrication. The XRD analysis confirmed a highly crystalline face-centered cubic structure of the biosynthesized material. The PC-AgNPs were presented separately in a spherical shape with an average crystal size of 20 nm, as endorsed by the TEM and FE-SEM measurements. The presence and crucial role of biomolecules in stabilizing the nanoparticles were elucidated by FTIR, EDX, and DLS techniques. The prepared biogenic nanoparticles were further applied for the reduction of 4-nitrophenol (4-NP) and colorimetric detection of Fe3+ ions. The study results proved that PC-AgNPs exhibited superior catalytic activity and reusability in the conversion of 4-NP by NaBH4. The complete reduction of 4-NP could be achieved in 10 min with the pseudo-first-order rate constant of 0.466 min-1, and no significant performance loss was found when the material was reused five times. The colorimetric probe based on PC-AgNPs displayed outstanding sensitivity and selectivity towards Fe3+ ions with a detection limit of 1.5 µM in a linear range of 0-250 µM. Additionally, the applicability of the developed assay was explored for testing Fe3+ ions in tap water. PC-AgNPs have a great potential for further applications as a promising catalyst for reducing nitrophenols and biosensors for the routine monitoring of Fe3+ in water.

Is relaxation exercise therapy effective in the management of patients with severe arterial hypertension?

Arterial hypertension (AH) is a burning problem in the world. Antihypertensive pharmacological therapy combined by physical exercises is well-studied in patients with mild and moderate AH. However, studies that have investigated relaxation in patients with severe AH in addition to drug therapy are lacking. Optimization of a comprehensive treatment for patients with severe AH, by using a multicomponent rational antihypertensive pharmacotherapy (PT) with subsequent application of relaxation exercise therapy (RET). The study involved 32 male patients with severe AH. Initially, clinical-instrumental and laboratory examination, blood pressure registration and daily arterial blood pressure monitoring were carried out. Suitable PT was selected for all the patients. 3 months after starting PT the patients were divided in 2 groups. The patients of the 1st group were prescribed RET in addition to PT. The 2nd group of patients continued receiving PT alone. 3 months later, average daily blood pressure (ADBP)-syst and ADBP-diast were compared in both groups. Three months after PT both groups demonstrated a significant decrease in ADBP-syst and ADBP diast, but these indicators remained higher than normal and did not reach the target level. Three months after the inclusion of RET in the comprehensive treatment, the 1st group demonstrated a significant decrease in ADBP (systolic and diastolic), not only in comparison with the initial data, but also with the data observed three months after PT. After 6 months, ADBP-syst and ADBP-diast in the 1st group were significantly lower compared with those of patients in the 2nd group. The inclusion of RET in addition to a multicomponent antihypertensive PT is a promising treatment option for severe AH.

Electrochemical Synthesis of Unique Nanomaterials in Ionic Liquids.

The review considers the features of the processes of the electrochemical synthesis of nanostructures in ionic liquids (ILs), including the production of carbon nanomaterials, silicon and germanium nanoparticles, metallic nanoparticles, nanomaterials and surface nanostructures based on oxides. In addition, the analysis of works on the synthesis of nanoscale polymer films of conductive polymers prepared using ionic liquids by electrochemical methods is given. The purpose of the review is to dwell upon an aspect of the applicability of ILs that is usually not fully reflected in modern literature, the synthesis of nanostructures (including unique ones that cannot be obtained in other electrolytes). The current underestimation of ILs as an electrochemical medium for the synthesis of nanomaterials may limit our understanding and the scope of their potential application. Another purpose of our review is to expand their possible application and to show the relative simplicity of the experimental part of the work.

Brain Organoid Generation from Induced Pluripotent Stem Cells in Home-Made Mini Bioreactors.

The iPSC-derived brain organoid is a promising technology for in vitro modeling the pathologies of the nervous system and drug screening. This technology has emerged recently. It is still in its infancy and has some limitations unsolved yet. The current protocols do not allow obtaining organoids to be consistent enough for drug discovery and preclinical studies. The maturation of organoids can take up to a year, pushing the researchers to launch multiple differentiation processes simultaneously. It imposes additional costs for the laboratory in terms of space and equipment. In addition, brain organoids often have a necrotic zone in the center, which suffers from nutrient and oxygen deficiency. Hence, most current protocols use a circulating system for culture medium to improve nutrition. Meanwhile, there are no inexpensive dynamic systems or bioreactors for organoid cultivation. This paper describes a protocol for producing brain organoids in compact and inexpensive home-made mini bioreactors. This protocol allows obtaining high quality organoids in large quantities.

Global implication of mesoproterozoic (~ 1.4 Ga) magmatism within the Sette-Daban Range (Southeast Siberia).

Mesoproterozoic period included several global tectonic events like break-up of Nuna and formation of Rodinia. However, although Siberia is a significant piece of both supercontinents, Mesoproterozoic time is marked by quiescence of magmatic and tectonic activity in it. We report here a mafic dyke (named Gornostakh dyke) in the southeastern Siberian Craton dated at 1419 ± 32 Ma by LA-ICPMS U-Pb geochronology of apatite. The dyke has tholeiitic compositions with high MgO and alkaline content, low-Ti, and arc-like trace element pattern. Due to the absence of subduction tectonics in the study area, geochemical data could be attributed to a significant contribution from metasomatically enriched subcontinental lithospheric mantle previously modified by subduction processes. That kind of composition is common for low-Ti dykes of intraplate flood basalt provinces similar to, for example, Permian-Triassic Siberian large igneous province (LIP). Paleogeographic reconstructions suggest that Siberia was connected to Laurentia and Baltica and their reconfiguration interrupts a prolonged tectonic quiescence in the Siberian Craton from ca. 1.88 Ga reflecting a transition from Nuna to Rodinia configuration. The mafic magmatism on 1419 Ma on the southeastern margin of the Siberian Craton together with coeval extensional tectonics observed in the structure of the Sette-Daban ridge proposes a hypothetical LIP which may be a direct consequence of the beginning of this transition.

Cerebral Organoids-Challenges to Establish a Brain Prototype.

The new cellular models based on neural cells differentiated from induced pluripotent stem cells have greatly enhanced our understanding of human nervous system development. Highly efficient protocols for the differentiation of iPSCs into different types of neural cells have allowed the creation of 2D models of many neurodegenerative diseases and nervous system development. However, the 2D culture of neurons is an imperfect model of the 3D brain tissue architecture represented by many functionally active cell types. The development of protocols for the differentiation of iPSCs into 3D cerebral organoids made it possible to establish a cellular model closest to native human brain tissue. Cerebral organoids are equally suitable for modeling various CNS pathologies, testing pharmacologically active substances, and utilization in regenerative medicine. Meanwhile, this technology is still at the initial stage of development.

EGCG as an anti-SARS-CoV-2 agent: Preventive versus therapeutic potential against original and mutant virus.

In the search for anti-SARS-CoV-2 drugs, much attention is given to safe and widely available native compounds. The green tea component epigallocatechin 3 gallate (EGCG) is particularly promising because it reportedly inhibits viral replication and viral entry in vitro. However, conclusive evidence for its predominant activity is needed. We tested EGCG effects on the native virus isolated from COVID-19 patients in two independent series of experiments using VERO cells and two different treatment schemes in each series. The results confirmed modest cytotoxicity of EGCG and its substantial antiviral activity. The preincubation scheme aimed at infection prevention has proven particularly beneficial. We complemented that finding with a detailed investigation of EGCG interactions with viral S-protein subunits, including S2, RBD, and the RBD mutant harboring the N501Y mutation. Molecular modeling experiments revealed N501Y-specific stacking interactions in the RBD-ACE2 complex and provided insight into EGCG interference with the complex formation. Together, these findings provide a molecular basis for the observed EGCG effects and reinforce its prospects in COVID-19 prevention therapy.

STIM2 Mediates Excessive Store-Operated Calcium Entry in Patient-Specific iPSC-Derived Neurons Modeling a Juvenile Form of Huntington's Disease.

Huntington's disease (HD) is a severe autosomal-dominant neurodegenerative disorder caused by a mutation within a gene, encoding huntingtin protein. Here we have used the induced pluripotent stem cell technology to produce patient-specific terminally differentiated GABA-ergic medium spiny neurons modeling a juvenile form of HD (HD76). We have shown that calcium signaling is dramatically disturbed in HD76 neurons, specifically demonstrating higher levels of store-operated and voltage-gated calcium uptakes. However, comparing the HD76 neurons with the previously described low-repeat HD models, we have demonstrated that the severity of calcium signaling alterations does not depend on the length of the polyglutamine tract of the mutant huntingtin. Here we have also observed greater expression of huntingtin and an activator of store-operated calcium channels STIM2 in HD76 neurons. Since shRNA-mediated suppression of STIM2 decreased store-operated calcium uptake, we have speculated that high expression of STIM2 underlies the excessive entry through store-operated calcium channels in HD pathology. Moreover, a previously described potential anti-HD drug EVP4593 has been found to attenuate high levels of both huntingtin and STIM2 that may contribute to its neuroprotective effect. Our results are fully supportive in favor of the crucial role of calcium signaling deregulation in the HD pathogenesis and indicate that the cornerstone of excessive calcium uptake in HD-specific neurons is a calcium sensor and store-operated calcium channels activator STIM2, which should become a molecular target for medical treatment and novel neuroprotective drug development.

Ouabain-Induced Gene Expression Changes in Human iPSC-Derived Neuron Culture Expressing Dopamine and cAMP-Regulated Phosphoprotein 32 and GABA Receptors.

Cardiotonic steroids (CTS) are specific inhibitors and endogenous ligands of a key enzyme in the CNS-the Na+, K+-ATPase, which maintains and creates an ion gradient on the plasma membrane of neurons. CTS cause the activation of various signaling cascades and changes in gene expression in neurons and other cell types. It is known that intracerebroventricular injection of cardiotonic steroid ouabain causes mania-like behavior in rodents, in part due to activation of dopamine-related signaling cascades in the dopamine and cAMP-regulated phosphoprotein 32 (DARPP-32) expressing medium spiny neurons in the striatum. Dopaminergic projections in the striatum innervate these GABAergic medium spiny neurons. The objective of this study was to assess changes in the expression of all genes in human iPSC-derived expressing DARPP-32 and GABA receptors neurons under the influence of ouabain. We noted a large number of statistically significant upregulated and downregulated genes after a 16-h incubation with non-toxic concentration (30 nM) of ouabain. These changes in the transcriptional activity were accomplished with activation of MAP-kinase ERK1/2 and transcriptional factor cAMP response element-binding protein (CREB). Thus, it can be concluded that 30 nM ouabain incubated for 16 h with human iPSC-derived expressing DARPP-32 and GABA receptors neurons activates genes associated with neuronal maturation and synapse formation, by increasing the expression of genes associated with translation, vesicular transport, and increased electron transport chain function. At the same time, the expression of genes associated with proliferation, migration, and early development of neurons decreases. These data indicate that non-toxic concentrations of ouabain may induce neuronal maturation, neurite growth, and increased synaptogenesis in dopamine-receptive GABAergic neurons, suggesting formation of plasticity and the establishment of new neuronal junctions.