组蛋白乙酰化修饰对脑缺血后海马区神经元BDNF表达的调控作用

An interruption of blood supply to the brain leads to ischemic stroke，which is the leading cause of chronic disability and the second leading cause of death in humans. Despite many studies on ischemic neuronal death, limited advances have been made in developing therapies to counter the ischemia..CA1 pyramidal neurons in the hippocampus are particular vulnerable to ischemic insult whereas CA3 neurons are relatively resistant to ischemia. It has been documented that the expression of brain-derived neurotrophic factor (BDNF) in neurons is an important mechanism of endogenous protection in the brain after ischemic insult. However, it remains unclear the mechanism underlying the low expression of BDNF in the CA1 region compared with CA3. .Epigenetic mechanisms play a role in regulating gene expression and histone acetylation is one kind of these important mechanisms. The dynamic acetylation equilibrium is mostly maintained through the physical and functional interplay between histone acetyltransferases (HAT) and histone deacetylase (HDAC), which were recruited by long non-coding RNAs (lncRNAs). Acetylation can selectively open some gene transcription and lead to changes in the expression of functional proteins..BDNF is expressed throughout the brain, with the highest levels in the hippocampus. Binding of mature BDNF protein to the receptors promotes cell survival, neurite outgrowth, synaptic transmission, plasticity, and cell migration. Rat BDNF transcripts contain 10 exons. Histone modifications at the BDNF promoter can differentially regulate BDNF expression in the brain. Each promoter drives the expression of a distinct mRNA, but each transcript encodes an identical BDNF protein. .Here, we will observe and compare the characteristics of histone methylation of BDNF gene in CA1 and CA3 neurons by using RT-PCR, Western blot, Chromatin immunoprecipitation (ChIP), RNA immunoprecipitation (RIP) and behavioral technology in a transient forebrain ischemic model or oxygen-glucose deprivation (OGD) model. Then, we will simulate the epigenetic mode of CA3 BDNF gene in CA1 area in order to enhance BDNF expression and activate the endogenous protective mechanism in CA1 neurons. This may provide a new therapeutic way for brain injury.

大脑海马CA1区锥体神经元对于短暂缺血性损伤敏感，而CA3区神经元则基本不受影响。神经元受损不同与脑源性神经生长因子（BDNF）表达直接相关，而脑缺血后海马不同区域神经元BDNF表达差异的机制不明。组蛋白乙酰化修饰是一种重要的表观遗传调控方式。组蛋白乙酰化程度可能与长链非编码RNAs的招募以及组蛋白乙酰转移酶和组蛋白去乙酰基酶之间的动态平衡相关。BDNF基因包含10个外显子，不同外显子区组蛋白通过不同程度的乙酰化修饰影响BDNF表达。本申请项目应用免疫印迹、PCR、ChIP和行为学方法，利用在体短暂前脑缺血再灌注模型和培养神经元氧-葡萄糖剥夺模型，观察并比较大鼠海马CA1和CA3区神经元BDNF基因特定区域组蛋白的乙酰化修饰机制，进而在CA1区神经元上模拟CA3区神经元BDNF基因的表观遗传改变模式，增强BDNF蛋白质表达，激活CA1区神经元的内源性保护机制，开辟一条治疗脑损伤的新途径。

脑卒中是目前第二常见的死亡原因和首位致残原因。缺血损伤可导致海马脑区具有内源性保护效应的BDNF蛋白差异化表达，但机制未明。本项目主要利用大鼠短暂脑缺血再灌注模型，应用Western blot方法检测海马神经元BDNF、HDACs（1-11）、p-ERK1/2、NDMA受体、proBDNF等蛋白的表达；qPCR方法检测Bdnf基因各转录本（I-X1）和lncRNA BDNF-AS的表达；应用生物信息学方法发现多种与Bdnf基因调控相关的lncRNAs和miRNA；应用ChIP方法检测Bdnf基因启动子区（p1、p2、p4和p6）组蛋白乙酰化修饰水平（H3K9、H3K14和H4K27）；应用电生理方法检测海马CA1区外向整流氯通道功能；应用组织化学检测海马脑片神经元死亡；应用行为学方法检测慢性口服皮质酮处理大鼠的抑郁样行为。结果发现，缺血损导致的大鼠海马CA1区神经元凋亡和BDNF蛋白表达降低、Bdnf转录本IV表达降低、Bdnf启动子p4区组蛋白H3K27ac水平降低、HDAC（1、2、3、4和7）表达升高、lncRNA BDNF-AS表达降低相关；选择性拮抗HDAC（1、2、3、4、7）功能可以提高脑缺血大鼠CA1区神经元存活率；在DG区，缺血后BDNF表达升高与Bdnf转录本（I、IIc、III、IV、VI和X1）和BDNF-AS表达升高相关；而HDAC（1、2、3、4和7）表达和Bdnf启动子区（p1、p2、p4和p6）组蛋白乙酰化（H3K9ac、H3K14ac和H3K27ac）水平无显著变化；EphA4受体信号通路可负性调控海马脑区BDNF蛋白表达，同时可差异性调控Bdnf基因表达，亦可增强NMDA受体通道和外向整流氯通道功能；糖皮质激素处理可使大鼠海马DG区BDNF表达降低，腹侧DG区proBDNF表达增加，BDNF/proBDNF比值显著降低，Bdnf转录本表达亦发生差异性变化。因此，通过lncRNAs、HDACs和EphA4等机制改变Bdnf基因的表观遗传修饰，可增加BDNF蛋白表达，激活受损神经元的内源性保护机制，降低神经元死亡，最终促进脑卒中患者的后期康复。这将对提高患者的生存质量和降低社会负担具有重要的应用前景。