||Acute stress induced depressive-like phenotypes via epigenetic regulation of BDNF expression in hippocampus
||Department of Pharmacology
Major depressive disorder (MDD)
Major depression has been the most affected neuropsychiatric disorders in the world. We focused on the epigenetic mechanisms for developing potential antidepressants in the future. The reduction of BDNF expression in the pathophysiology of depressive-like behaviors has been extensively studied, but the role of epigenetic regulation in major depression remained elusive. We used learned helplessness animal model as the acute stressor and evaluated the depressive-like behaviors through different paradigms. We compared learned-helplessness (LH) mice with learned-helplessness-resilient (LHR) groups in a series of experiments. Firstly, we observed increased expression of HDAC2 and HDAC5 along with decreased BDNF in depressive state of rodents. Sodium valproate administrated in mice improved the depressive-like behaviors through enhancing BDNF. Secondly, there was significant increase of methylation on exon I, III , and IV, which negatively correlated with decreased BDNF. Then, we further surveyed the key methylation factors- DNA methyltransferase 1 , 3a ,and 3b(DNMT1,3a,3b). In LH mice, mRNA and protein level of DNMT1 and DNMT3a was elevated accompanied by the reduction of BDNF. Therefore, the potent DNMT inhibitor 5-azacytidine was used to evaluate the reversal of depressive-like behaviors and reduced BDNF expression. Thirdly, the modulation of BDNF 3’ UTR by miR-206 was carried out in vitro. The data indicated that miR-206-3p preferentially bind to the vectors bearing mBDNF #3, suggesting a targeted position for interfering BDNF expression. Cotransfection of mimics and inhibitors in vectors carrying mBDNF #3 displayed dose-dependent effects both in reporter assay and in BDNF expression. In vivo experiments, Cy3-AM206 was administrated in mice to verify the distribution of Cy3 fluorescence and beneficial effects on BDNF. Both intranasal and intra-hippocampal injection of AM206 in mice exhibited a therapeutic effect through behavior and molecular modulation, which was determined by decreased immobility time and enhanced BDNF. Morphological alteration of dendritic spines and colocalization of synaptic protein markers was also used to support the favorable effects of AM206.Taken together; BDNF expression is dynamically regulated by epigenetics and has been referred to a potential therapeutic agent in treating psychiatric disorders in preclinical study.
Certificate for qualification
Abstract (in Chinese) I
Abstract (in English) IV
Tables of context IX
Figures and tables XII
Chapter 1: Introduction 1
1-1 Ideology 2
1-2 Current treatment for major depressive disorders 2
1-3 Hippocampus 2
1-4 Hippocampus and depression 2
1-5 BDNF genes 3
1-6 Structure of BDNF genes 3
1-7 BDNF expression in hippocampus 3
1-8 BDNF and depression 4
1-9 Epigenetics 4
1-10 Histone modifications 5
1-11 Histone modification in depression 5
1-12 Histone deacetylase inhibitors (HDACi) 5
1-13 DNA hyper-methylation 6
1-14 DNA hyper-methylation in depression 6
1-15 DNA hyper-methylation inhibitors and depression 7
1-16 MicroRNAs (miRNAs) 7
1-17 MicroRNAs (miRNAs) in depression 7
Chapter 2: Material and methods 12
2-1 Animals 13
2-2 Drugs 13
2-3 Learned-helplessness test (LHT) 13
2-4 Forced-swim test (FST) 14
2-5 Tail suspension test (TST) 14
2-6 Western blotting (WB) 14
2-7 Total RNA isolation 15
2-8 Real-time PCR of mRNA 16
2-9 DNA methylation assay 16
2-10 miRNA targets 17
2-11 Real-time PCR of miRNA 21
2-12 Dual luciferase assay 21
2-13 Treatment LH/LHR mice with antagomir 24
2-13-1Intra-hippocampal administration 24
2-13-2Intra-nasal administration 24
2-14 Analysis of dendritic spine density 25
2-15 Lentiviral transduction 25
2-16 Immunofluorescence 27
2-17 Statistical analyses 28
Chapter 3: Results 29
3-1 Depressive-like phenotypes are mediated by
epigenetic regulation of BDNF 30
3-2 Sod valproate reversed depressive-like phenotypes
by modulation of BDNF 31
3-3 The association of BDNF expression with
methylation pattern on individual variants 32
3-4 DNMTs enrichment negatively correlates with
relatively low expression of BDNF 33
3-5 5-azacytidine reverses the depressive-like
behaviors through regulation of BDNF 34
3-6 The suppressive effect of microRNAs (miR-206-
3p/5p) on BDNF 3’ UTRs in vitro 35
3-7 The beneficial effects of AM206 on BDNF in vivo 37
3-7-1 Intra-hippocampal administration 37
3-7-2 Intra-nasal administration 37
Chapter Four: Discussion 39
4.1 Overview of this study 40
4.2 BDNF expression and promoter methylation pattern 40
4.3 BDNF and miRNA 40
4.4 Animal model and disease 41
4.5 Conclusion 42
Chapter Five: Figures & Figure legends 43
Chapter Six: References 61
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