Browsing by Subject "MAPK"

Stress response in plants is influenced by several external and internal factors and is executed in a modular way. Environmental stimuli or stress is sensed by cellular receptors and the signal is transduced inside cell via the phospho-activation of highly conserved intracellular signaling cascades like mitogen activated protein kinase (MAPK) cascades. The signal then activates biosynthesis pathways of major stress response hormones like Salicylic acid (SA). In Arabidopsis about 90% SA is synthesized via isochorismate pathway and Isochorismate synthase 1 (ICS1) is a rate limiting enzyme in this pathway. In this study, goal was to select transgenic ICS1 (homozygous) candidate lines from parent ICS1-CFP by selective regeneration. Then, by molecular and physiological characterization of transgenic ICS1-CFP plants, the function of ICS1 phosphorylation, more specifically, impact of different photoperiods (Long day; LD and Short day; SD) and stress conditions on ICS1 activity would have resolved. However, there were no homozygous candidate line from any parent ICS1-CFP plants after several screening. Nevertheless, ozone treated stress sensitivity test was performed with heterozygous ICS1-CFP candidate plants (T2 generation). Ozone treated stress depends on stomata factor because ozone enters into plants through stomata. Therefore, stomata index analysis was performed with sid2 and WT (Col-0) phenotypes and grown in LD and SD conditions. Since, stomata number was different between LD and SD plants of both sid2 and WT phenotypes, a different method named Xanthine-Xanthine oxidase (X/XO) treatment was applied that induce oxidative stress regardless of stomata. Although, WT and sid2 had shown sensitivity to the treatment, the overall cell death percentage was very low. Lastly, our aim was to observe the impact of different photoperiods on the activation of two particular MAPKs i.e MPK3 and MPK6 under stress conditions. The phosphorylated (P-MPK3 and P-MPK6) are found abundantly in ozone treated plants as an early response. In this experiment, plants were grown in both LD and SD, stressed with both ozone and X/XO treatments, the activation of P-MPK3 and P-MPK6 was observed by protein level analysis (western blotting) in detailed time course. Although, the activation was visualized in both LD and SD plants, qualitatively the pattern was similar between day type samples and activation signal was very weak in both stress methods. In addition, anti-ICS1 antibody provided by Agrsera TM was tested for its efficiency to detect endogenous ICS1 protein in plants in two experimental set-up. Although the antibody could detect overexpressed ICS1-CFP protein in samples, it was not that efficient to detect endogenous ICS1 in any of the experiments.

While weeks of continuous treatment is required for conventional antidepressant drugs (e.g. fluoxetine) to bring their full therapeutic effects, a subanesthetic dose of ketamine alleviates the core symptoms of depression (anhedonia, depressed mood) and suicidal thinking within just few hours and the effects may last for days. Nitrous oxide (N2O, “laughing gas”), another NMDAR antagonist, has recently been shown to have similar rapid antidepressant effects in treatment-resistant depressed patients (Nagele et al. 2015). We previously found using naïve mice ketamine and N2O treatment to upregulate five mRNAs related to the MAPK pathway and synaptic plasticity, both implicated as being important in the action of rapid-acting antidepressants. In the current study, these shared mechanisms were further investigated in C57BL/6JHsd mice, using behavioral test batteries to study depressive-like behaviour and RT-qPCR for biochemical analyses. We first aimed to demonstrate behavioral differences between naïve mice and a chronic corticosterone-induced animal model of depression, and to use this model to investigate antidepressant-like effects of ketamine and N2O. According to the results, chronic corticosterone produced some behaviors typical of a depressive-like phenotype, namely significant worsening of coat state and decreased saccharin consumption in the saccharin preference test. Both ketamine and N2O exhibited antidepressant-like effects by reverting decreased saccharin preference. We then aimed to elucidate the effects of ketamine and N2O on five previously found shared mRNAs: Arc, Dusp1, Dusp5, Dusp6 and Nr4a1. N2O significantly upregulated all targets in the vmPFC, except Dusp5, two hours after beginning of N2O treatment. Neither ketamine nor sole chronic corticosterone produced any significant changes. The results of this study suggest that N2O is a potential candidate for rapid alleviation of depressive symptoms. We suggest that the action of rapid-acting antidepressants, more specifically N2O, is based on a homeostatic response of the brain to a presented challenge. Here this challenge would be cortical excitation previously been shown to be caused by N2O, which leads to activation of pathways such as MAPK and subsequent Arc, Dusp and Nr4a1 signaling. The level of expression of these markers would then depend on which phase this response is in and hence, the differences in time between treatment and brain sample dissection could be a reason for conflicting results to previous research. Future studies would benefit from detailed investigation of the chronic corticosterone-induced model due to its potential in controlling for behavioral variability, thus reducing the number of animals needed for preclinical research. Overall the preliminary findings of this study could be one of the first steps in the search for the mechanisms underlying the potential antidepressant effect of N2O, how these molecular markers are related to its action and how it differs from the action of ketamine.