组蛋白乙酰化转移酶GCN5调控Bim表达及蛛网膜下腔出血后早期脑损伤的机制研究

Early brain injury (EBI) after subarachnoid hemorrhage (SAH) is a leading pathological cause of poor outcome, featured with typical apoptosis. We have proved that Bim is a critical factor for neuronal apoptosis, whose high expression was evidenced in EBI brain tissues, but the related mechanism remains elusive. Preliminary data showed that GCN5 activity decreases following SAH, concomitant with Bim upregulation and Caspase 3 activation. In neuron, inhibiting GCN5 transcriptionally upregulates Bim and induces Bim-dependent apoptosis. HDAC inhibitors antagonize these effects. Further, SAH leads to a decrease in CaMK IV activity. Inhibition of CaMKs in neuron inactivates GCN5 and causes its modification shift. Software prediction showed that CaMK IV has a high potential in phosphorylating GCN5 (Ser275). Thus, we proposed our hypothesis for the project: following subarachnoid hemorrhage, CaMK IV activity decreases, which leads to GCN5 dephosphorylation and inactivation and subsequently induces HDAC-dependent Bim upregulation. The upregulated Bim induces apoptosis and EBI. To process the project, several essential methods such as CHIP, kinase reaction, ventricle injection and so on, will be used to get the key evidences that CaMK IV directly phosphorylates GCN5 and GCN5 regulates Bim expression and neuronal apoptosis. The project will elucidate a new mechanism that CaMK-GCN5-Bim pathway regulates EBI following SAH.

蛛网膜下腔出血后的早期脑损伤是预后不良的重要病因，神经元凋亡是其关键特征。我们已证明Bim是促神经元凋亡关键分子，其在早期损伤脑组织中被证实高表达，但机制不清。预实验表明：出血后，随着Bim和Caspase 3激活，乙酰化转移酶GCN5活性下调。神经元中，抑制GCN5诱导Bim转录上调及其依赖的凋亡。而抑制HDAC拮抗这些效应。进一步，出血后使CaMK IV活性下降。抑制CaMK IV使GCN5失活且出现修饰位移。软件预测CaMK IV直接磷酸化GCN5（Ser 275）。据此我们提出假设：出血后，CaMK IV活性下降导致GCN5失活，进而导致HDAC活性依赖的Bim上调，促使凋亡及早期脑损伤发生。本项目拟采用激酶反应、CHIP、脑室注射等方法，获得CaMK IV磷酸化GCN调控Bim表达及神经元凋亡的直接证据；阐明CaMK IV-GCN5-Bim信号通路调控出血后早期脑损伤的新机制。

细胞内乙酰化稳态是由组蛋白乙酰化转移酶（HAT）和组蛋白去乙酰化酶（HDAC）控制的动态平衡过程。当HAT活性下降会导致平衡向去乙酰化倾斜，进而引起胞内乙酰化水平下降及神经元凋亡，但相关机制尚不清楚。本研究中，我们发现用抑制剂CPTH2, MB-3或者siRNAs抑制GCN5活性会引起小脑颗粒神经元凋亡。机制上，抑制GCN5会导致转录因子Egr-1和E2F1激活，继而转录上调BH3-only protein Bim,诱导神经元凋亡。在撤钾和谷氨酸诱导的神经元凋亡模型中，GCN5活性下降是导致Bim表达上调的原因之一，因为用腺病毒载体过表达GCN5会抑制Bim表达及神经元凋亡。而且，在蛛网膜下腔出血后早期脑损伤动物模型中，也发现神经元中GCN5活性下调，Egr-1，E2F1及Bim表达上调，神经元凋亡。用SAHA抑制HDAC活性恢复胞内乙酰化水平，可抑制Bim表达及神经元凋亡，缓解出血引起的早期脑损伤。（结果发表在：Cell Death & Disease, 2017, 8: 0-e2570）. 同时，我们还发现用多种抑制HDAC剂如TSA、SAHA、VPA和M344均能转录下调MKK7，抑制JNK依赖的神经元凋亡。进一步，筛选到HDAC4活性介导MKK7转录表达及凋亡。利用HDAC4特异抑制剂LMK235能有效抑制MKK7/JNK活性和神经元凋亡，缓解出血引起的早期脑损伤。（结果发表在：Frontiers in Cellular Neuroscience, 2019, 13: 0-468）. 总之，我们的研究提示抑制HDAC可能是干预因GCN5活性下降、MKK7/JNK活性依赖的神经元凋亡和出血后早期脑损伤的有效策略。