The Oxytocin Receptor: From Intracellular Signaling to Behavior.

The many facets of the oxytocin (OXT) system of the brain and periphery elicited nearly 25,000 publications since 1930 (see FIGURE 1 , as listed in PubMed), which revealed central roles for OXT and its receptor (OXTR) in reproduction, and social and emotional behaviors in animal and human studies focusing on mental and physical health and disease. In this review, we discuss the mechanisms of OXT expression and release, expression and binding of the OXTR in brain and periphery, OXTR-coupled signaling cascades, and their involvement in behavioral outcomes to assemble a comprehensive picture of the central and peripheral OXT system. Traditionally known for its role in milk let-down and uterine contraction during labor, OXT also has implications in physiological, and also behavioral, aspects of reproduction, such as sexual and maternal behaviors and pair bonding, but also anxiety, trust, sociability, food intake, or even drug abuse. The many facets of OXT are, on a molecular basis, brought about by a single receptor. The OXTR, a 7-transmembrane G protein-coupled receptor capable of binding to either Gαi or Gαq proteins, activates a set of signaling cascades, such as the MAPK, PKC, PLC, or CaMK pathways, which converge on transcription factors like CREB or MEF-2. The cellular response to OXT includes regulation of neurite outgrowth, cellular viability, and increased survival. OXTergic projections in the brain represent anxiety and stress-regulating circuits connecting the paraventricular nucleus of the hypothalamus, amygdala, bed nucleus of the stria terminalis, or the medial prefrontal cortex. Which OXT-induced patterns finally alter the behavior of an animal or a human being is still poorly understood, and studying those OXTR-coupled signaling cascades is one initial step toward a better understanding of the molecular background of those behavioral effects.

Experimental Models of SARS-CoV-2 Infection: Possible Platforms to Study COVID-19 Pathogenesis and Potential Treatments.

In December 2019, a novel coronavirus crossed species barriers to infect humans and was effectively transmitted from person to person, leading to a worldwide pandemic. Development of effective clinical interventions, including vaccines and antiviral drugs that could prevent or limit theburden or transmission of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), is a global health priority. It is thus of utmost importance to assess possible therapeutic strategies against SARS-CoV-2 using experimental models that recapitulate aspects of the human disease. Here, we review available models currently being developed and used to study SARS-CoV-2 infection and highlight their application to screen potential therapeutic approaches, including repurposed antiviral drugs and vaccines. Each identified model provides a valuable insight into SARS-CoV-2 cellular tropism, replication kinetics, and cell damage that could ultimately enhance understanding of SARS-CoV-2 pathogenesis and protective immunity.

Structure-function relationships of the disease-linked A218T oxytocin receptor variant.

Various single nucleotide polymorphisms (SNPs) in the oxytocin receptor (OXTR) gene have been associated with behavioral traits, autism spectrum disorder (ASD) and other diseases. The non-synonymous SNP rs4686302 results in the OXTR variant A218T and has been linked to core characteristics of ASD, trait empathy and preterm birth. However, the molecular and intracellular mechanisms underlying those associations are still elusive. Here, we uncovered the molecular and intracellular consequences of this mutation that may affect the psychological or behavioral outcome of oxytocin (OXT)-treatment regimens in clinical studies, and provide a mechanistic explanation for an altered receptor function. We created two monoclonal HEK293 cell lines, stably expressing either the wild-type or A218T OXTR. We detected an increased OXTR protein stability, accompanied by a shift in Ca2+ dynamics and reduced MAPK pathway activation in the A218T cells. Combined whole-genome and RNA sequencing analyses in OXT-treated cells revealed 7823 differentially regulated genes in A218T compared to wild-type cells, including 429 genes being associated with ASD. Furthermore, computational modeling provided a molecular basis for the observed change in OXTR stability suggesting that the OXTR mutation affects downstream events by altering receptor activation and signaling, in agreement with our in vitro results. In summary, our study provides the cellular mechanism that links the OXTR rs4686302 SNP with genetic dysregulations associated with aspects of ASD.

Chronic oxytocin-driven alternative splicing of Crfr2α induces anxiety.

The neuropeptide oxytocin (OXT) has generated considerable interest as potential treatment for psychiatric disorders, including anxiety and autism spectrum disorders. However, the behavioral and molecular consequences associated with chronic OXT treatment and chronic receptor (OXTR) activation have scarcely been studied, despite the potential therapeutic long-term use of intranasal OXT. Here, we reveal that chronic OXT treatment over two weeks increased anxiety-like behavior in rats, with higher sensitivity in females, contrasting the well-known anxiolytic effect of acute OXT. The increase in anxiety was transient and waned 5 days after the infusion has ended. The behavioral effects of chronic OXT were paralleled by activation of an intracellular signaling pathway, which ultimately led to alternative splicing of hypothalamic corticotropin-releasing factor receptor 2α (Crfr2α), an important modulator of anxiety. In detail, chronic OXT shifted the splicing ratio from the anxiolytic membrane-bound (mCRFR2α) form of CRFR2α towards the soluble CRFR2α (sCRFR2α) form. Experimental induction of alternative splicing mimicked the anxiogenic effects of chronic OXT, while sCRFR2α-knock down reduced anxiety-related behavior of male rats. Furthermore, chronic OXT treatment triggered the release of sCRFR2α into the cerebrospinal fluid with sCRFR2α levels positively correlating with anxiety-like behavior. In summary, we revealed that the shifted splicing ratio towards expression of the anxiogenic sCRFR2α underlies the adverse effects of chronic OXT treatment on anxiety.

Co-Stimulation of Oxytocin and Arginine-Vasopressin Receptors Affect Hypothalamic Neurospheroid Size.

Oxytocin (OXT) is a neuropeptide involved in a plethora of behavioral and physiological processes. However, there is a prominent lack of 3D cell culture models that investigate the effects of OXT on a cellular/molecular level. In this study, we established a hypothalamic neuronal spheroid model to investigate the cellular response in a more realistic 3D setting. Our data indicate that the formation of spheroids itself does not alter the basic characteristics of the cell line and that markers of cellular morphology and connectivity are stably expressed. We found that both OXT and arginine vasopressin (AVP) treatment increase spheroid size (surface area and volume), as well as individual nucleus size, which serves as an indicator for cellular proliferation. The cellular response to both OXT and AVP seems mainly to be mediated by the AVP receptor 1a (V1aR); however, the OXT receptor (OXTR) contributes significantly to the observed proliferative effect. When we blocked the OXTR pharmacologically or knocked down the OXTR by siRNA, the OXT- or AVP-induced cellular proliferation decreased. In summary, we established a 3D cell culture model of the neuronal response to OXT and AVP and found that spheroids react to the treatment via their respective receptors but also via cross-talk between the two receptor types.

Substrate stiffness affects the morphology and gene expression of epidermal neural crest stem cells in a short term culture.

According to the intrinsic plasticity of stem cells, controlling their fate is a critical issue in cell-based therapies. Recently, a growing body of evidence has suggested that substrate stiffness can affect the fate decisions of various stem cells. Epidermal neural crest stem cells as one of the main neural crest cell derivatives hold great promise for cell therapies due to presenting a high level of plasticity. This study was conducted to define the influence of substrate stiffness on the lineage commitment of these cells. Here, four different polyacrylamide hydrogels with elastic modulus in the range of 0.7-30 kPa were synthesized and coated with collagen and stem cells were seeded on them for 24 hr. The obtained data showed that cells can attach faster to hydrogels compared with culture plate and cells on <1 kPa stiffness show more neuronal-like morphology as they presented several branches and extended longer neurites over time. Moreover, the transcription of actin downregulated on all hydrogels, while the expression of Nestin, Tubulin, and PDGFR-α increased on all of them and SOX-10 and doublecortin gene expression were higher only on <1 kPa. Also, it was revealed that soft hydrogels can enhance the expression of glial cell line-derived neurotrophic factor, neurotrophin-3, and vascular endothelial growth factor in these stem cells. On the basis of the results, these cells can respond to the substrate stiffness in the short term culture and soft hydrogels can alter their morphology and gene expression. These findings suggested that employing proper substrate stiffness might result in cells with more natural profiles similar to the nervous system and superior usefulness in therapeutic applications.

Myocyte Enhancer Factor 2A (MEF2A) Defines Oxytocin-Induced Morphological Effects and Regulates Mitochondrial Function in Neurons.

The neuropeptide oxytocin (OT) is a well-described modulator of socio-emotional traits, such as anxiety, stress, social behavior, and pair bonding. However, when dysregulated, it is associated with adverse psychiatric traits, such as various aspects of autism spectrum disorder (ASD). In this study, we identify the transcription factor myocyte enhancer factor 2A (MEF2A) as the common link between OT and cellular changes symptomatic for ASD, encompassing neuronal morphology, connectivity, and mitochondrial function. We provide evidence for MEF2A as the decisive factor defining the cellular response to OT: while OT induces neurite retraction in MEF2A expressing neurons, OT causes neurite outgrowth in absence of MEF2A. A CRISPR-Cas-mediated knockout of MEF2A and retransfection of an active version or permanently inactive mutant, respectively, validated our findings. We also identified the phosphatase calcineurin as the main upstream regulator of OT-induced MEF2A signaling. Further, MEF2A signaling dampens mitochondrial functioning in neurons, as MEF2A knockout cells show increased maximal cellular respiration, spare respiratory capacity, and total cellular ATP. In summary, we reveal a central role for OT-induced MEF2A activity as major regulator of cellular morphology as well as neuronal connectivity and mitochondrial functioning, with broad implications for a potential treatment of disorders based on morphological alterations or mitochondrial dysfunction.

Oxytocin Regulates Stress-Induced Crf Gene Transcription through CREB-Regulated Transcription Coactivator 3.

The major regulator of the neuroendocrine stress response in the brain is corticotropin releasing factor (CRF), whose transcription is controlled by CREB and its cofactors CRTC2/3 (TORC2/3). Phosphorylated CRTCs are sequestered in the cytoplasm, but rapidly dephosphorylated and translocated into the nucleus following a stressful stimulus. As the stress response is attenuated by oxytocin (OT), we tested whether OT interferes with CRTC translocation and, thereby, Crf expression. OT (1 nmol, i.c.v.) delayed the stress-induced increase of nuclear CRTC3 and Crf hnRNA levels in the paraventricular nucleus of male rats and mice, but did not affect either parameter in the absence of the stressor. The increase in Crf hnRNA levels at later time points was parallel to elevated nuclear CRTC2/3 levels. A direct effect of Thr(4) Gly(7)-OT (TGOT) on CRTC3 translocation and Crf expression was found in rat primary hypothalamic neurons, amygdaloid (Ar-5), hypothalamic (H32), and human neuroblastoma (Be(2)M17) cell lines. CRTC3, but not CRCT2, knockdown using siRNA in Be(2)M17 cells prevented the effect of TGOT on Crf hnRNA levels. Chromatin-immunoprecipitation demonstrated that TGOT reduced CRTC3, but not CRTC2, binding to the Crf promoter after 10 min of forskolin stimulation. Together, the results indicate that OT modulates CRTC3 translocation, the binding of CRTC3 to the Crf promoter and, ultimately, transcription of the Crf gene. SIGNIFICANCE STATEMENT: The neuropeptide oxytocin has been proposed to reduce hypothalamic-pituitary-adrenal (HPA) axis activation during stress. The underlying mechanisms are, however, elusive. In this study we show that activation of the oxytocin receptor in the paraventricular nucleus delays transcription of the gene encoding corticotropin releasing factor (Crf), the main regulator of the stress response. It does so by sequestering the coactivator of the transcription factor CREB, CRTC3, in the cytosol, resulting in reduced binding of CRTC3 to the Crf gene promoter and subsequent Crf gene expression. This novel oxytocin receptor-mediated intracellular mechanism might provide a basis for the treatment of exaggerated stress responses in the future.

Antagonism of V1b receptors promotes maternal motivation to retrieve pups in the MPOA and impairs pup-directed behavior during maternal defense in the mpBNST of lactating rats.

Recent studies using V1b receptor (V1bR) knockout mice or central pharmacological manipulations in lactating rats highlighted the influence of this receptor for maternal behavior. However, its role in specific brain sites known to be important for maternal behavior has not been investigated to date. In the present study, we reveal that V1bR mRNA (qPCR) and protein levels (Western blot) within either the medial preoptic area (MPOA) or the medial-posterior part of the bed nucleus of the stria terminalis (mpBNST) did not differ between virgin and lactating rats. Furthermore, we characterized the effects of V1bR blockade via bilateral injections of the receptor subtype-specific antagonist SSR149415 within the MPOA or the mpBNST on maternal behavior (maternal care under non-stress and stress conditions, maternal motivation to retrieve pups in a novel environment, maternal aggression) and anxiety-related behavior in lactating rats. Blocking V1bR within the MPOA increased pup retrieval, whereas within the mpBNST it decreased pup-directed behavior, specifically licking/grooming the pups, during the maternal defense test. In addition, immediately after termination of the maternal defense test, V1bR antagonism in both brain regions reduced nursing, particularly arched back nursing. Anxiety-related behavior was not affected by V1bR antagonism in either brain region. In conclusion our data indicate that V1bR antagonism significantly modulates different aspects of maternal behavior in a brain region-dependent manner.

Oxytocin Receptor Expression in Hair Follicle Stem Cells: A Promising Model for Biological and Therapeutic Discovery in Neuropsychiatric Disorders.

The intricate nature of the human brain and the limitations of existing model systems to study molecular and cellular causes of neuropsychiatric disorders represent a major challenge for basic research. The promising progress in patient-derived stem cell technology and in our knowledge on the role of the brain oxytocin (OXT) system in health and disease offer new possibilities in that direction. In this study, the rat hair follicle stem cells (HFSCs) were isolated and expanded in vitro. The expression of oxytocin receptors (OXTR) was evaluated in these cells. The cellular viability was assessed 12 h post stimulation with OXT. The activation of OXTR-coupled intracellular signaling cascades, following OXT treatment was determined. Also, the influence of OXT on neurite outgrowth and cytoskeletal rearrangement were defined. The assessment of OXTR protein expression revealed this receptor is expressed abundantly in HFSCs. As evidenced by the cell viability assay, no adverse or cytotoxic effects were detected following 12 h treatment with different concentrations of OXT. Moreover, OXTR stimulation by OXT resulted in ERK1/2, CREB, and eEF2 activation, neurite length alterations, and cytoskeletal rearrangements that reveal the functionality of this receptor in HFSCs. Here, we introduced the rat HFSCs as an easy-to-obtain stem cell model that express functional OXTR. This cell-based model can contribute to our understanding of the progression and treatment of neuropsychiatric disorders with oxytocinergic system deficiency.

The beneficial effects of chick embryo extract preconditioning on hair follicle stem cells: A promising strategy to generate Schwann cells.

The beneficial effects of hair follicle stem cells in different animal models of nervous system conditions have been extensively studied. While chick embryo extract (CEE) has been used as a growth medium supplement for these stem cells, this is the first study to show the effect of CEE on them. The rat hair follicle stem cells were isolated and supplemented with 10% fetal bovine serum plus 10% CEE. The migration rate, proliferative capacity and multipotency were evaluated along with morphometric alteration and differentiation direction. The proteome analysis of CEE content identified effective factors of CEE that probably regulate fate and function of stem cells. The CEE enhances the migration rate of stem cells from explanted bulges as well as their proliferation, likely due to activation of AP-1 and translationally controlled tumour protein (TCTP) by thioredoxin found in CEE. The increased length of outgrowth may be the result of cyclic AMP response element binding protein (CREB) phosphorylation triggered by active CamKII contained in CEE. Further, CEE supplementation upregulates the expression of vascular endothelial growth factor (VEGF), brain-derived neurotrophic factor and glial cell line-derived neurotrophic factor. The elevated expression of target genes and proteins may be due to CREB, AP-1 and c-Myc activation in these stem cells. Given the increased transcript levels of neurotrophins, VEGF, and the expression of PDGFR-α, S100B, MBP and SOX-10 protein, it is possible that CEE promotes the fate of these stem cells towards Schwann cells.

From Hair to the Brain: The Short-Term Therapeutic Potential of Human Hair Follicle-Derived Stem Cells and Their Conditioned Medium in a Rat Model of Stroke.

The short-term therapeutic impacts of stem cells and their derivatives were frequently reported in preclinical investigations of ischemic stroke (IS); however, several drawbacks including accessibility, abundancy, and ethical concerns limited their clinical application. We describe here for the first time the therapeutic potential of human hair follicle-derived stem cells (hHFSCs) and their conditioned medium (CM) in a rat model of IS. Furthermore, we hypothesized that a combination of cell therapy with repeated CM administration might enhance the restorative efficiency of this approach compared to each treatment alone. Middle cerebral artery occlusion was performed for 30 min to induce IS. Immediately after reperfusion, hHFSCs were transplanted through the intra-arterial route and/or hHFSC-CM administered intranasally. The neurological outcomes, short-term spatial working memory, and infarct size were evaluated. Furthermore, relative expression of seven target genes in three categories of neuronal markers, synaptic markers, and angiogenic markers was assessed. The hHFSCs and hHFSC-CM treatments improved neurological impairments and reduced infarct size in the IS rats. Moreover, molecular data elucidated that IS was accompanied by attenuation in the expression of neuronal and synaptic markers in the evaluated brain regions and the interventions rescued these expression changes. Although there was no considerable difference between hHFSCs and hHFSC-CM treatments in the improvement of neurological function and decrement of infarct size, combination therapy was more effective to reduce infarction and elevation of target gene expression especially in the hippocampus. These findings highlight the curative potential of hHFSCs and their CM in a rat model of IS.

Oxytocin accelerates tight junction formation and impairs cellular migration in 3D spheroids: evidence from Gapmer-induced exon skipping.

Oxytocin (OXT) is a neuropeptide that has been associated with neurological diseases like autism, a strong regulating activity on anxiety and stress-related behavior, physiological effects during pregnancy and parenting, and various cellular effects in neoplastic tissue. In this study, we aimed to unravel the underlying mechanism that OXT employs to regulate cell-cell contacts, spheroid formation, and cellular migration in a 3D culture model of human MLS-402 cells. We have generated a labeled OXT receptor (OXTR) overexpressing cell line cultivated in spheroids that were treated with the OXTR agonists OXT, Atosiban, and Thr4-Gly7-oxytocin (TGOT); with or without a pre-treatment of antisense oligos (Gapmers) that induce exon skipping in the human OXTR gene. This exon skipping leads to the exclusion of exon 4 and therefore a receptor that lost its intracellular G-protein-binding domain. Sensitive digital PCR (dPCR) provided us with the means to differentiate between wild type and truncated OXTR in our cellular model. OXTR truncation differentially activated intracellular signaling cascades related to cell-cell attachment and proliferation like Akt, ERK1/2-RSK1/2, HSP27, STAT1/5, and CREB, as assessed by a Kinase Profiler Assay. Digital and transmission electron microscopy revealed increased tight junction formation and well-organized cellular protrusions into an enlarged extracellular space after OXT treatment, resulting in increased cellular survival. In summary, OXT decreases cellular migration but increases cell-cell contacts and therefore improves nutrient supply. These data reveal a novel cellular effect of OXT that might have implications for degenerating CNS diseases and tumor formation in various tissues.

Intranasal application of stem cells and their derivatives as a new hope in the treatment of cerebral hypoxia/ischemia: a review.

Intranasal delivery of stem cells and conditioned medium to target the brain has attracted major interest in the field of regenerative medicine. In pre-clinical investigations during the last ten years, several research groups focused on this strategy to treat cerebral hypoxia/ischemia in neonates as well as adults. In this review, we discuss the curative potential of stem cells, stem cell derivatives, and their delivery route via intranasal application to the hypoxic/ischemic brain. After intranasal application, stem cells migrate from the nasal cavity to the injured area and exert therapeutic effects by reducing brain tissue loss, enhancing endogenous neurogenesis, and modulating cerebral inflammation that leads to functional improvements. However, application of this administration route for delivering stem cells and/or therapeutic substances to the damaged sites requires further optimization to translate the findings of animal experiments to clinical trials.

The Beneficial Potential of Genetically Modified Stem Cells in the Treatment of Stroke: a Review.

The last two decades have witnessed a surge in investigations proposing stem cells as a promising strategy to treat stroke. Since growth factor release is considered as one of the most important aspects of cell-based therapy, stem cells over-expressing growth factors are hypothesized to yield higher levels of therapeutic efficiency. In pre-clinical studies of the last 15 years that were investigating the efficiency of stem cell therapy for stroke, a variety of stem cell types were genetically modified to over-express various factors. In this review we summarize the current knowledge on the therapeutic efficiency of stem cell-derived growth factors, encompassing techniques employed and time points to evaluate. In addition, we discuss several types of stem cells, including the recently developed model of epidermal neural crest stem cells, and genetically modified stem cells over-expressing specific factors, which could elevate the restorative potential of naive stem cells. The restorative potential is based on enhanced survival/differentiation potential of transplanted cells, apoptosis inhibition, infarct volume reduction, neovascularization or functional improvement. Since the majority of studies have focused on the short-term curative effects of genetically engineered stem cells, we emphasize the need to address their long-term impact.

The Implementation of Preconditioned Epidermal Neural Crest Stem Cells to Combat Ischemic Stroke. Comment on Othman, F.A.; Tan, S.C. Preconditioning Strategies to Enhance Neural Stem Cell-Based Therapy for Ischemic Stroke. Brain Sci. 2020, 10, 893.

In the recent review published in Brain Sciences, Othman and Tan suggested several preconditioning strategies to improve stem cell therapy after ischemic brain injury [...].

Reconditioning the Neurogenic Niche of Adult Non-human Primates by Antisense Oligonucleotide-Mediated Attenuation of TGFß Signaling.

Adult neurogenesis is a target for brain rejuvenation as well as regeneration in aging and disease. Numerous approaches showed efficacy to elevate neurogenesis in rodents, yet translation into therapies has not been achieved. Here, we introduce a novel human TGFß-RII (Transforming Growth Factor-Receptor Type II) specific LNA-antisense oligonucleotide ("locked nucleotide acid"-"NVP-13"), which reduces TGFß-RII expression and downstream receptor signaling in human neuronal precursor cells (ReNcell CX® cells) in vitro. After we injected cynomolgus non-human primates repeatedly i.th. with NVP-13 in a preclinical regulatory 13-week GLP-toxicity program, we could specifically downregulate TGFß-RII mRNA and protein in vivo. Subsequently, we observed a dose-dependent upregulation of the neurogenic niche activity within the hippocampus and subventricular zone: human neural progenitor cells showed significantly (up to threefold over control) enhanced differentiation and cell numbers. NVP-13 treatment modulated canonical and non-canonical TGFß pathways, such as MAPK and PI3K, as well as key transcription factors and epigenetic factors involved in stem cell maintenance, such as MEF2A and pFoxO3. The latter are also dysregulated in clinical neurodegeneration, such as amyotrophic lateral sclerosis. Here, we provide for the first time in vitro and in vivo evidence for a novel translatable approach to treat neurodegenerative disorders by modulating neurogenesis.

Anxiolytic and Anxiogenic? How the Transcription Factor MEF2 Might Explain the Manifold Behavioral Effects of Oxytocin.

The neuromodulator oxytocin, since its first synthesis by du Vigneaud in 1953, has mainly been associated with beneficial physiological effects, as well as positive social and emotional behaviors. This overall positive picture of oxytocin as the "love-, cuddle-, or bonding-hormone" has repeatedly been challenged since then. Oxytocin-induced effects that would be perceived as negative by the individual, such as increased anxiety or potentiation of stress-induced ACTH release, as well as the regulation of negative approach-related emotions, such as envy and schadenfreude (gloating) have been described. The general consent is that oxytocin, instead of acting unidirectional, induces changes in the salience network to shift the emphasis of emotional contexts, and therefore can, e.g., produce both anxiolytic as well as anxiogenic behavioral outcomes. However, the underlying mechanisms leading to alterations in the salience network are still unclear. With the aim to understand the manifold effects of oxytocin on a cellular/molecular level, a set of oxytocin receptor-coupled signaling cascades and downstream effectors regulating transcription and translation has been identified. Those oxytocin-driven effectors, such as MEF2 and CREB, are known modulators of the neuronal and glial cytoarchitecture. We hypothesize that, by determining cellular morphology and connectivity, MEF2 is one of the key factors that might contribute to the diverse behavioral effects of oxytocin.

Epidermal neural crest stem cell transplantation as a promising therapeutic strategy for ischemic stroke.

INTRODUCTION: Cell-based therapy is considered as promising strategy to cure stroke. However, employing appropriate type of stem cell to fulfill many therapeutic needs of cerebral ischemia is still challenging. In this regard, the current study was designed to elucidate therapeutic potential of epidermal neural crest stem cells (EPI-NCSCs) compared to bone marrow mesenchymal stem cells (BM-MSCs) in rat model of ischemic stroke. METHODS: Ischemic stroke was induced by middle cerebral artery occlusion (MCAO) for 45 minutes. Immediately after reperfusion, EPI-NCSCs or BM-MSCs were transplanted via intra-arterial or intravenous route. A test for neurological function was performed before ischemia and 1, 3, and 7 days after MCAO. Also, infarct volume ratio and relative expression of 15 selected target genes were evaluated 7 days after transplantation. RESULTS: EPI-NCSCs transplantation (both intra-arterial and intravenous) and BM-MSCs transplantation (only intra-arterial) tended to result in a better functional outcome, compared to the MCAO group; however, this difference was not statistically significant. The infarct volume ratio significantly decreased in NCSC-intra-arterial, NCSC-intravenous and MSC-intra-arterial groups compared to the control. EPI-NCSCs interventions led to higher expression levels of Bdnf, nestin, Sox10, doublecortin, ß-III tubulin, Gfap, and interleukin-6, whereas neurotrophin-3 and interleukin-10 were decreased. On the other hand, BM-MSCs therapy resulted in upregulation of Gdnf, ß-III tubulin, and Gfap and down-regulation of neurotrophin-3, interleukin-1, and interleukin-10. CONCLUSION: These findings highlight the therapeutic effects of EPI-NCSCs transplantation, probably through simultaneous induction of neuronal and glial formation, as well as Bdnf over-expression in a rat model of ischemic stroke.

De Novo Protein Synthesis Mediated by the Eukaryotic Elongation Factor 2 Is Required for the Anxiolytic Effect of Oxytocin.

BACKGROUND: The neuropeptide oxytocin (OXT) mediates its actions, including anxiolysis, via its G protein-coupled OXT receptor. Within the paraventricular nucleus of the hypothalamus (PVN), OXT-induced anxiolysis is mediated, at least in part, via activation of the mitogen-activated protein kinase pathway following calcium influx through transient receptor potential cation channel subfamily V member 2 channels. In the periphery, OXT activates eukaryotic elongation factor 2 (eEF2), an essential mediator of protein synthesis. METHODS: In order to study whether OXT activates eEF2 also in neurons to exert its anxiolytic properties in the PVN, we performed in vivo and cell culture experiments. RESULTS: We demonstrate that OXT, in a protein kinase C-dependent manner, activates eEF2 both in a hypothalamic cell line and in vivo within the PVN. Next, we reveal that OXT stimulates de novo protein synthesis, while inhibition of protein synthesis within the PVN prevents the anxiolytic effect of OXT in male rats. Moreover, activation of eEF2 within the PVN conveyed an anxiolytic effect supporting a role of OXT-induced eEF2 activation and protein synthesis for its anxiolysis. Finally, we show that one of the proteins that is upregulated by OXT is the neuropeptide Y receptor 5. Infusion of a specific neuropeptide Y receptor 5 agonist into the PVN consequently led to decreased anxiety-related behavior, while pretreatment with a neuropeptide Y receptor 5 antagonist prevented the anxiolytic effect of OXT. CONCLUSIONS: Taken together, these results show that OXT recruits several intracellular signaling cascades to induce protein synthesis, which mediates the anxiolytic effects of OXT within the PVN and suggests that eEF2 represents a novel target for anxiety-related disorders.