CONSTITUTIVE PHOTOMORPHOGENIC 1 is involved in gibberellic acid-mediated germination by repressing RGA-LIKE 2 in Arabidopsis thaliana

학위논문 (석사)-- 서울대학교 대학원 농업생명과학대학 식물생산과학부, 2017. 8. 백남천.In flowering plants, germination is a sophisticated process, which is regulated by the cross-talk between endogenous signals and environmental cues such as hormones, light, water and temperature. GA hormone is a key regulator of seed germination. Many genes are involved in GA-mediated seed germination pathway. Among them, RGL2 has been considered to be the major negative regulator by repressing germination associated genes. Here, we showed that COP1 is closely involved in regulating GA-mediated seed germination. We found that the germination rate of cop1 mutants were strongly decreased by paclobutrazol (PAC) treatments, that is inhibitor of GA biosynthesis, compared with Wild-type. However, germination of COP1 overexpressed-transgenic plants is insensitive to PAC. Analysis of western blot using antibody against COP1 recombinant proteins demonstrated that GA affected on COP1 protein stability. While imbibed wild-type seeds under GA present condition significantly increased COP1 protein than that in mock (distilled water) condition, COP1 protein was decreased by PAC treatment. The genetic study of cop1-4 rgl2 double mutants provided strong evidence that COP1 act as upstream negative regulator of RGL2. Further analysis by BiFC and in vitro pull-down assay indicated that COP1 physically interacts with RGL2. RGL2 protein was degraded in COP1 overexpressed plants. These results suggested that COP1 is partially involved negatively regulates in RGL2 protein stability. COP1 regulates the transcript levels of the genes associated with germination such as GASA6, EXPA1, EXPA2, EXPA8, and XTH33. Taken together, our results suggested that COP1 regulates GA-mediated seed germination through degradation of RGL2 proteins.INTRODUCTION 1 MATERIALS AND METHODS 4 RESULTS 11 DISCUSSION 34 REFERENCES 38 ABSTRACT IN KOREAN 45Maste

Chronic stress-induced memory deficits are reversed by regular exercise via AMPK-mediated BDNF induction

Chronic stress has a detrimental effect on neurological insults, psychiatric deficits, and cognitive impairment. In the current study, chronic stress was shown to impair learning and memory functions, in addition to reducing in hippocampal Adenosine monophosphate-activated protein kinase (AMPK) activity. Similar reductions were also observed for brain-derived neurotrophic factor (BDNF), synaptophysin, and post-synaptic density-95 (PSD-95) levels, all of which was counter-regulated by a regime of regular and prolonged exercise. A 21-day restraint stress regimen (6 h/day) produced learning and memory deficits, including reduced alternation in the Y-maze and decreased memory retention in the water maze test. These effects were reversed post-administration by a 3-week regime of treadmill running (19 m/min, 1 h/day, 6 days/week). In hippocampal primary culture, phosphorylated-AMPK (phospho-AMPK) and BDNF levels were enhanced in a dose-dependent manner by 5-amimoimidazole-4-carboxamide riboside (AICAR) treatment, and AICAR-treated increase was blocked by Compound C. A 7-day period of AICAR intraperitoneal injections enhanced alternation in the Y-maze test and reduced escape latency in water maze test, along with enhanced phospho-AMPK and BDNF levels in the hippocampus. The intraperitoneal injection of Compound C every 4 days during exercise intervention diminished exercise-induced enhancement of memory improvement during the water maze test in chronically stressed mice. Also, chronic stress reduced hippocampal neurogenesis (lower Ki-67- and doublecortin-positive cells) and mRNA levels of BDNF, synaptophysin, and PSD-95. Our results suggest that regular and prolonged exercise can alleviate chronic stress-induced hippocampal-dependent memory deficits. Hippocampal AMPK-engaged BDNF induction is at least in part required for exercise-induced protection against chronic stress. © 2016 IBRO

Exercise Prevents Memory Consolidation Defects Via Enhancing Prolactin Responsiveness of CA1 Neurons in Mice Under Chronic Stress

We investigated the effects of regular exercise on chronic stress-induced memory consolidation impairment and its underlying mechanism. We focused on prolactin (PRL)-modulated calcium-permeable (CP)-α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors (AMPARs) in neurons in the CA1 stratum lacunosum-moleculare (SLM) area of the dorsal hippocampus. Regular exercise protected against memory retention defects and prevented dendritic retraction in apical distal segments of hippocampal CA1 neurons, as indicated by enhanced dendritic ramification, dendritic length, spine density, and synaptic protein levels following chronic stress. Regular exercise normalized synaptic CP-AMPAR assembly in the hippocampal CA1 SLM area, as evidenced by an enhanced ratio of GluR1 to GluR2 during chronic stress. This alteration in AMPARs was critical to memory retention, whereby memory retention was blunted by local blockage of CP-AMPARs in the SLM of naïve and exercised mice. Regular exercise improved PRL responsiveness in the hippocampal CA1 region during chronic stress, which led to increased binding of PRL to its receptor (PRLR) and PRL-dependent enhancement in phosphorylated signal transducer and activator of transcription 5 levels. The improvement in PRL responsiveness contributed to memory retention during chronic stress, as the protective action of exercise on memory persistence during stress was abolished by PRLR knockdown in the hippocampal CA1 area. Finally, in primary hippocampal cultures, repeated treatment with corticosterone led to decreased AMPAR-mediated Ca2+ influx, which was restored by PRL treatment. The above findings suggest a protective role for exercise against chronic stress-evoked defects in memory consolidation via PRL-modulated incorporation of CP-AMPARs into hippocampal CA1 synapses. © 2019, Springer Science+Business Media, LLC, part of Springer Nature

Chronic stress-induced dendritic reorganization and abundance of synaptosomal PKA-dependent CP-AMPA receptor in the basolateral amygdala in a mouse model of depression

Chronic stress is a precipitating factor for disorders including depression. The basolateral amygdala (BLA) is a critical substrate that interconnects with stress-modulated neural networks to generate emotion- and mood-related behaviors. The current study shows that 3 h per day of restraint stress for 14 days caused mice to exhibit long-term depressive behaviors, manifested by disrupted sociality and despair levels, which were rescued by fluoxetine. These behavioral changes corresponded with morphological and molecular changes in BLA neurons, including chronic stress-elicited increases in arborization, dendritic length, and spine density of BLA principal neurons. At the molecular level, calcium-permeable α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors (CP-AMPARs) within the synaptosome exhibited an increased GluR1:GluR2 subunit ratio. We also observed increased GluR1 phosphorylation at Ser 845 and enhanced cyclic AMP-dependent protein kinase (PKA) activity in the BLA. These molecular changes reverted to the basal state post-treatment with fluoxetine. The expression of synaptophysin (SYP) and postsynaptic density protein 95 (PSD-95) at BLA neuronal synapses was also enhanced by chronic stress, which was reversed post-treatment. Finally, chronic stress-provoked depressive behavior was overcome by local blockage of CP-AMPARs in the BLA via stereotaxic injection (IEM-1460). Chronic stress-elicited depressive behavior may be due to hypertrophy of BLA neuronal dendrites and increased of PKA-dependent CP-AMPAR levels in BLA neurons. Furthermore, fluoxetine can reverse chronic stress-triggered cytoarchitectural and functional changes of BLA neurons. These findings provide insights into depression-linked structural and functional changes in BLA neurons. © 2017 Elsevier Inc

Exercise exerts an anxiolytic effect against repeated restraint stress through 5-HT2A-mediated suppression of the adenosine A(2A) receptor in the basolateral amygdala

Repeated or chronic stressful stimuli induce emotion- and mood-related abnormalities, such as anxiety and depression. Conversely, regular exercise exerts protective effects. Here, we found that exercise recovered anxiety-like behaviors, as measured using the open field and elevated plus maze tests in an anxiety mouse model. In addition to behavioral improvement, exercise enhanced the synaptic density of the 5-hydroxytryptamine 2A receptor (5-HT2AR), but not the 5-HT1AR in the basolateral amygdala (BLA) region in this mouse model. Furthermore, global treatment with a selective 5-HT2AR antagonist (MDL11930) generated an anxiety phenotype. Thus, synaptic recruitment of 5-HT2AR in BLA neurons may mediate the anxiolytic effects of exercise. The exercise regimen also reduced adenosine A(2A) receptor (A(2A)R)-mediated protein kinase A (PKA) activation, and the anxiolytic effect of the exercise was blunted by local activation of A(2A)R within the BLA using CGS21680, a selective A(2A)R agonist. Particularly, A(2A)R-mediated PKA activity was shown to be dependent on 5-HT2AR signaling in the BLA. These results imply that repeated stress upregulates A(2A)R-mediated adenosine signaling to facilitate PKA activation, whereas regular exercise inhibits A(2A)R function by increasing 5-HT2AR in the BLA. Accordingly, this integrated modulation of 5-HT and adenosine signaling, via 5-HT2AR and A(2A)R respectively, may be a mechanism underlying the anxiolytic effect of regular exercise

NQO1 mediates the anti-inflammatory effects of nootkatone in lipopolysaccharide-induced neuroinflammation by modulating the AMPK signaling pathway

Neuroinflammation and oxidative stress play key roles in the progression of neurodegenerative diseases. Thus, the use of potent anti-inflammatory/antioxidant agents has been suggested as a promising therapeutic strategy for neurodegenerative diseases. In the present study, we investigated the anti-inflammatory and antioxidant effects of nootkatone (NKT), a sesquiterpenoid compound isolated from grapefruit, in in vitro and in vivo models of neuroinflammation. In lipopolysaccharide (LPS)-stimulated BV2 microglial cells, NKT inhibited the expression of iNOS, COX-2, and pro-inflammatory cytokines, and increased the expression of the anti-inflammatory cytokine, IL-10. In addition, NKT inhibited reactive oxygen species (ROS) production and upregulated the expression of antioxidant enzymes, such as NQO1 and HO-1. Molecular mechanistic studies showed that NKT inhibited Akt, p38 MAPK, and NF-kappa B activities, while increasing AMPK, PKA/CREB, and Nrf2/ARE signaling in LPS-stimulated BV2 cells. Since NKT dramatically increased NQO1 expression, we investigated the role of this enzyme using pharmacological inhibition or knockdown experiments. Treatment of BV2 cells with the NQO1-specific inhibitor, dicoumarol, or with NQO1 siRNA significantly blocked NKT-mediated inhibition of NO, ROS, TNF-alpha, IL-1 beta, and upregulation of IL-10. Furthermore, NQO1 inhibition reversed the effects of NKT on pm- and anti-inflammatory signaling molecules. Intriguingly, we found that the AMPK inhibitor, compound C, mimicked the effects of dicoumarol, suggesting the presence of a crosstalk between NQO1 and AMPK. Finally, we demonstrated that NKT inhibited microglial activation, lipid peroxidation, and the expression of pro-inflammatory markers in the brains of LPS-injected mice, which was also reversed by dicoumarol. These data collectively suggest that NQO1 plays a critical role in mediating the anti-inflammatory and antioxidant effects of NKT in LPS-induced neuroinflammation by modulating AMPK and its downstream signaling pathways

Administration of Alpha(s1)-Casein Hydrolysate Increases Sleep and Modulates GABA(A) Receptor Subunit Expression

Sleep is the most basic and essential physiological requirement for mental health, and sleep disorders pose potential risks of metabolic and neurodegenerative diseases. Tryptic hydrolysate of alpha(S1)-casein (alpha(S1)-CH) has been shown to possess stress relieving and sleep promoting effects. However, the differential effects of alpha(S1)-CH on electroencephalographic wave patterns and its effects on the protein levels of gamma-aminobutyric acid A (GABA(A)) receptor subtypes in hypothalamic neurons are not well understood. We found alpha(S1)-CH (120, 240 mg/kg) increased sleep duration in mice and reduced sleep-wake cycle numbers in rats. While alpha(S1)-CH (300 mg/kg) increased total sleeping time in rats, it significantly decreased wakefulness. In addition, electroencephalographic theta (theta) power densities were increased whereas alpha (alpha) power densities were decreased by alpha(S1)-CH (300 mg/kg) during sleep-wake cycles. Furthermore, protein expressions of GABA(A) receptor beta(1) subtypes were elevated in rat hypothalamus by alpha(S1)-CH. These results suggest alpha(S1)-CH, through GABA(A) receptor modulation, might be useful for treating sleep disorders

Neuroprotective effect of β-lapachone in MPTP-induced parkinson’s disease mouse model: Involvement of astroglial p-AMPK/Nrf2/HO-1 signaling pathways

Parkinson’s disease is a neurodegenerative disease characterized by the progressive loss of dopaminergic neurons within the substantia nigra pars compacta. In the present study, we investigated whether β-Lapachone (β-LAP), a natural naphthoquinone compound isolated from the lapacho tree (Tabebuia avellanedae), elicits neuroprotective effects in a 1-methyl-4-phenyl-1,2,3,6- tetrahydropyridine (MPTP)-induced Parkinson’s disease mouse model. β-LAP reduced the tyrosine hydroxylase (TH)-immunoreactive fiber loss induced by MPTP in the dorsolateral striatum, and alleviated motor dysfunction as determined by the rotarod test. In addition, β-LAP protected against MPTP-induced loss of TH positive neurons, and upregulated B-cell lymphoma 2 protein (Bcl-2) expression in the substantia nigra. Based on previous reports on the neuroprotective role of nuclear factor-E2-related factor- 2 (Nrf2) in neurodegenerative diseases, we investigated whether β-LAP induces upregulation of the Nrf2-hemeoxygenae-1 (HO-1) signaling pathway molecules in MPTP-injected mouse brains. Western blot and immunohistochemical analyses indicated that β-LAP increased HO-1 expression in glial fibrillary acidic protein-positive astrocytes. Moreover, β-LAP increased the nuclear translocation and DNA binding activity of Nrf2, and the phosphorylation of upstream adenosine monophosphate-activated protein kinase (AMPK). β-LAP also increased the localization of p-AMPK and Nrf2 in astrocytes. Collectively, our data suggest that β-LAP exerts neuroprotective effect in MPTP-injected mice by upregulating the p-AMPK/Nrf2/HO-1 signaling pathways in astrocytes. © 2019 The Korean Society of Applied Pharmacology

SK-PC-B70M alleviates neurologic symptoms in G93A-SOD1 amyotrophic lateral sclerosis mice

SK-PC-B70M, an oleanolic-glycoside saponins fraction extracted from the root of Pulsatilla koreana, carries active ingredient(s) that protects the cytotoxicity induced by Aβ(1-42) in SK-N-SH cells. It was recently demonstrated that SK-PC-B70M improved scopolamine-induced deficits of memory consolidation and spatial working memory in rats, and reduced Aβ levels and plaque deposition in the brains of the Tg2576 mouse model of Alzheimer disease. In the present study, we investigated whether SK-PC-B70M produces helpful effects on the pathology of the G93A-SOD1 transgenic mouse model of amyotrophic lateral sclerosis (ALS). Administration of SK-PC-B70M (100 or 400 mg/kg/day) from 8 weeks to 16 weeks of age attenuated neurological deficits of G93A-SOD1 mice in several motor-function-related behavioral tests. SK-PC-B70M treatment significantly suppressed the accumulation of the by-products of lipid peroxidation, malonedialdehyde (MDA) and 4-hydroxy-2-nonenal (HNE), in the spinal cord of G93A-SOD1 mice. Moreover, histologic analysis stained with cresyl violet or anti-choline acetyltransferase (ChAT) revealed that SK-PC-B70M suppressed neuronal loss in the ventral horn of the spinal cords of G93A-SOD1 mice. These results suggest that SK-PC-B70M affords a beneficial effect on neurologic deficits of G93A-SOD1 ALS mice. © 2010 Elsevier B.V. All rights reserved

Treadmill exercise restores high fat diet-induced disturbance of hippocampal neurogenesis through beta 2-adrenergic receptor-dependent induction of thioredoxin-1 and brain-derived neurotrophic factor

A high-fat diet (HFD) is known to induce metabolic disturbances that may lead to cognitive impairment. In the present study, we investigated whether a regular treadmill exercise program would improve HFD-induced hippocampal-dependent memory deficits in C57BL/6 mice. Weight gain and hepatic triglyceride levels were profoundly elevated following administration of a 60% HFD for 23 weeks, and this change was attenuated by 23-weeks of treadmill running. The exercise regimen attenuated impairments in memory function of HFD-fed mice in a water maze test and recovered HFD-induced anti-neurogenic effects as shown by immunohistochemistry data with Ki-67 and doublecortin (DCX) antibodies. Moreover, the treadmill exercise resulted in anti-inflammatory, antioxidant, and neuroprotective effects in the HFD-fed brain. The exercise inhibited HFD-induced microglial activation, expression of proinflammatory cytokines (tumor necrosis factor-alpha and interleukin-1 beta), and NF-kappa B activity in the dentate gyrus (DG) of the hippocampus. In addition, the exercise reduced malondialdehyde levels elevated by HFD and recovered antioxidant superoxide dismutase and glutathione levels in hippocampal DG of HFD-mice. The exercise also reduced the number of apoptotic cells induced by HFD, as shown by TUNEL staining in the DG region. Finally, we demonstrated that the thioredoxin-1 (TRX-1) and brain-derived neurotrophic factor (BDNF) levels were recovered by exercise, which was demonstrated to act via beta 2-adrenergic receptor enriched in synaptosomes of the DG. Therefore, our data collectively suggests that regular exercise may be a promising approach to preventing HFD-induced memory impairments via anti-inflammatory, antioxidant and neuroprotective mechanisms in the hippocampal DG region