Activation of the JAK-STAT pathway by olanzapine is necessary for desensitization of serotonin2A receptor-stimulated phospholipase C signaling in rat frontal cortex but not serotonin2A receptor-stimulated hormone release

Chronic treatment with olanzapine causes desensitization of serotonin2A receptor signaling. The purpose of the current study is to further understand the mechanisms underlying this desensitization response of serotonin2A receptor signaling in vivo. We now report that desensitization of serotonin2A receptor stimulated-phospholipase C activity in rat frontal cortex induced by olanzapine is dependent on activation of the JAK-STAT pathway. Olanzapine treatment for 7 days significantly increased the levels of the regulator of G protein signaling (RGS7) protein, RGS7 mRNA levels, and activation of JAK2 in rat frontal cortex. Pretreatment with a JAK2 inhibitor AG490, significantly attenuated the olanzapine-induced reductions in serotonin2A receptor-stimulated phospholipase C activity and prevented the olanzapine-induced increases in RGS7 mRNA and protein levels. In contrast, inhibition of the JAK-STAT pathway with AG490 did not reverse the olanzapine-induced desensitization of the serotonin2A receptor pathway in the hypothalamic paraventricular nucleus mediating increases in plasma hormone levels. AG490 dose-dependently inhibited serotonin2A receptor-stimulated oxytocin and corticosterone release. Taken together, these results suggest that the olanzapine-induced increase in RGS7 expression is mediated by activation of JAK-STAT and is necessary for olanzapine-induced desensitization of serotonin2A receptor-stimulated phospholipase C activity in the frontal cortex but not serotonin2A receptor-stimulated hormone release

Erratum: Integrin-FAK Signaling Rapidly and Potently Promotes Mitochondrial Function Through STAT3 (Cell Communication and Signaling (2016) 14 (32) DOI: 10.1186/s12964-016-0157-7)

Reference. Unfortunately, after publication of this article [1], it was noticed that the Acknowledgements and Funding sections were incomplete. The Acknowledgements section currently reads, “We are grateful for the technical support by Aruna Visavadiya, Ying Li, and Rhesa Dykes” and the Funding section currently reads, “This work was supported by NIH grant NS45734 and ETSU medical school funds”. The full, corrected sections can be seen below. Acknowledgements We are grateful for the technical support by Aruna Visavadiya, Ying Li, and Rhesa Dykes. Dr. Britta Engelhardt (Theodor Kocher institute) is thanked for providing the bEnd5 cells. Funding This work was supported by NIH grant NS45734 and in part by NIH grant C06RR0306551 and the ETSU College of Medicine. Further to this, a duplicate image in Fig. 4e was reported. The correct image is presented in this correction article. (Figure Presented)

The Anxiety- and Depression-like Behavioral Profile of Astrocytic FAK Knockout Mice

The anxiety- and depression-like behavioral profile of Astrocytic FAK knockout mice Steve Yang and Dr. Cuihong Jia, Department of Biomedical Sciences, College of Medicine, East Tennessee State University, Johnson City, TN. Ciliary neurotrophic factor (CNTF) is expressed by astrocytes where it is believed to promote neurotransmitter synthesis and yet depression-like behaviors in mice, especially female mice based on our previous study. We have also discovered that inhibition of focal adhesion kinase (FAK) in astrocytes can upregulate CNTF. However, whether FAK inhibition-induced CNTF affects anxiety- or depression-like behaviors is still unclear. We, therefore, used Cre-lox system to upregulate CNTF expression in mice brains via selective and inducible knockout FAK in astrocytes. We then used behavioral tests, such as open field, elevated T-maze, sucrose swim, object location memory, to measure target mice\u27s anxiety-like and depression-like behaviors. As a result of completing the behavior tests and by comparing to the control group, we learned that knockout of FAK in astrocytes did not affect anxiety – and depression-like behaviors in female mice, nor did it alter learning or memory abilities. Nevertheless, we found that FAK knockout in astrocytes of female mice seemed to increase their locomotor function. Further study will use the same procedures to expand on male mice, compare the results, and investigate the mechanism underlying the locomotor activities. We hope this work can be a useful tool in better understanding anxiety and mood disorders from a molecular level, especially posttraumatic stress disorder, and major depressive disorder so that more specific pharmacologic therapies can be developed to treat these illnesses

Effect of IP3R3 and NPY on Age-Related Declines in Olfactory Stem Cell Proliferation

Losing the sense of smell because of aging compromises health and quality of life. In the mouse olfactory epithelium, aging reduces the capacity for tissue homeostasis and regeneration. The microvillous cell subtype that expresses both inositol trisphosphate receptor type 3 (IP3R3) and the neuroproliferative factor neuropeptide Y (NPY) is critical for regulation of homeostasis, yet its role in aging is undefined. We hypothesized that an age-related decline in IP3R3 expression and NPY signaling underlie age-related homeostatic changes and olfactory dysfunction. We found a decrease in IP3R3+ and NPY+ microvillous cell numbers and NPY protein and a reduced sensitivity to NPY-mediated proliferation over 24months. However, in IP3R3-deficient mice, there was no further age-related reduction in cell numbers, proliferation, or olfactory function compared with wild type. The proliferative response was impaired in aged IP3R3-deficient mice when injury was caused by satratoxin G, which induces IP3R3-mediated NPY release, but not by bulbectomy, which does not evoke NPY release. These data identify IP3R3 and NPY signaling as targets for improving recovery following olfactotoxicant exposure

Mechanisms of constitutive and ATP-evoked ATP release in neonatal mouse olfactory epithelium

Abstract Background ATP is an extracellular signaling molecule with many ascribed functions in sensory systems, including the olfactory epithelium. The mechanism(s) by which ATP is released in the olfactory epithelium has not been investigated. Quantitative luciferin-luciferase assays were used to monitor ATP release, and confocal imaging of the fluorescent ATP marker quinacrine was used to monitor ATP release via exocytosis in Swiss Webster mouse neonatal olfactory epithelial slices. Results Under control conditions, constitutive release of ATP occurs via exocytosis, hemichannels and ABC transporters and is inhibited by vesicular fusion inhibitor Clostridium difficile toxin A and hemichannel and ABC transporter inhibitor probenecid. Constitutive ATP release is negatively regulated by the ATP breakdown product ADP through activation of P2Y receptors, likely via the cAMP/PKA pathway. In vivo studies indicate that constitutive ATP may play a role in neuronal homeostasis as inhibition of exocytosis inhibited normal proliferation in the OE. ATP-evoked ATP release is also present in mouse neonatal OE, triggered by several ionotropic P2X purinergic receptor agonists (ATP, αβMeATP and Bz-ATP) and a G protein-coupled P2Y receptor agonist (UTP). Calcium imaging of P2X2-transfected HEK293 “biosensor” cells confirmed the presence of evoked ATP release. Following purinergic receptor stimulation, ATP is released via calcium-dependent exocytosis, activated P2X1,7 receptors, activated P2X7 receptors that form a complex with pannexin channels, or ABC transporters. The ATP-evoked ATP release is inhibited by the purinergic receptor inhibitor PPADS, Clostridium difficile toxin A and two inhibitors of pannexin channels: probenecid and carbenoxolone. Conclusions The constitutive release of ATP might be involved in normal cell turn-over or modulation of odorant sensitivity in physiological conditions. Given the growth-promoting effects of ATP, ATP-evoked ATP release following injury could lead to progenitor cell proliferation, differentiation and regeneration. Thus, understanding mechanisms of ATP release is of paramount importance to improve our knowledge about tissue homeostasis and post-injury neuroregeneration. It will lead to development of treatments to restore loss of smell and, when transposed to the central nervous system, improve recovery following central nervous system injury.</p

The Effects of Chronic Stress on CNTF/UCN3 in the pBNST and Hypothalamic PVN in Mice

Post-traumatic stress disorder (PTSD) is characterized by fear extinction deficit; chronic stress worsens this deficit. Using a Chronic Unpredictable Stress (CUS) model, we previously found that CUS increased fear extinction deficit in female mice and knockout of Ciliary Neurotrophic Factor (CNTF) attenuated it. The amygdala, specifically the medial amygdala, is strongly associated with fear conditioning and extinction. CUS increased CNTF and reduced Urocortin 3 (UCN3) in the medial amygdala, suggesting CNTF-mediated UCN3 inhibition may be involved in CUS-induced deficit of fear extinction. The medial amygdala connects to the hypothalamic paraventricular nucleus (PVN) via posterior bed nucleus of stria terminalis (pBNST) and mediates the stress response (Fig. 1). The objective of this project is to determine whether CUS affects CNTF, UCN3, and CNTF-related cytokine leukemia inhibitory factor (LIF) and interleukin-6 (IL-6) in the pBNST and hypothalamic PVN. Hippocampal CNTF expression was also examined as a brain region outside of the medial amygdala-pBNST-hypothalamic PVN circuitry. 4 groups (5 mice/group) of CNTF+/+ and CNTF-/- mice were treated with 4 weeks of CUS or control handling. At the end, fresh brain samples were collected. The hypothalamic PVN, pBNST and hippocampus were punched out from 600-700 um cryostat frozen sections. CUS was applied for 4 weeks. The control mice were handled daily for 4 weeks. RNA was extracted from tissue using QIANGEN Rneasy mini kit. BCA assay was performed to analyze protein concentration, then 10% SDS gel was used to run the protein samples. Statistical analysis included one-way ANOVA followed by Bonferroni multiple comparison or 2-tailed T test. p \u3c0.05 was defined as significant difference. In the pBNST, CUS did not affect CNTF and UCN3 mRNA expression. However, UCN3 protein was upregulated by CUS in CNTF+/+ but not CNTF-/- mice, suggesting CNTF inhibits UCN3 expression, possibly through post-transcriptional mechanism. CUS did not alter LIF and IL-3 in the pBNST. CUS did not alter CNTF mRNA expression in the PVN and further study will measure UCN3 mRNA and protein in the PVN. Finally, there was no CUS effect on CNTF, LIF and IL-6 mRNA in the hippocampus. These results and further studies are useful in development of therapeutic medications and drug targets in the case of chronic stress

Constitutive and evoked release of ATP in adult mouse olfactory epithelium

In adult olfactory epithelium (OE), ATP plays a role in constant cell turnover and post-injury neuroregeneration. We previously demonstrated that constitutive and ATP-evoked ATP release are present in neonatal mouse OE and underlie continuous cell turn-over and post-injury neuroregeneration, and that activation of purinergic P2X7 receptors is involved in the evoked release. We hypothesized that both releases are present in adult mouse OE. To study the putative contribution of olfactory sensory neurons to ATP release, we used olfactory sensory neuronal-like OP6 cells derived from the embryonic olfactory placode cells. Calcium imaging showed that OP6 cells and primary adult OE cell cultures express functional purinergic receptors. We monitored ATP release from OP6 cells and whole adult OE turbinates using HEK cells as biosensors and luciferin–luciferase assays. Constitutive ATP release occurs in OP6 cells and whole adult mouse OE turbinates, and P2X7 receptors mediated evoked ATP release occurs only in turbinates. The mechanisms of ATP release described in the present study might underlie the constant cell turn-over and post-injury neuroregeneration present in adult OE and thus, further studies of these mechanisms are warranted as it will improve our knowledge of OE tissue homeostasis and post-injury regeneration