早老素在脑肿瘤致癫痫发作过程中的分子机制研究

Epileptic seizures are a potentially life-threatening symptom of structural or metabolic brain dysfunction. They are among the most common presenting features of patients with primary brain tumours. Epilepsy associated with brain tumours has commonly been considered to be more difficult to treat than have other types of epilepsy. Several pathophysiological mechanisms are proposed as capable of triggering epileptic seizures in patients with brain tumors. These include the disruption of neuronal connections and inhibition of local network regulation, the impaired glial cell activity, the increased vascular permeability and the abnormal function of the blood brain barrier. In addition, rapidly growing tumors deregulate adjacent areas and result in peritumoral edema and inflammation, necrosis and hemosiderin deposition. These events might initialize abnormal circuits leading to seizure activity in the brain. Sadly, the pathogenic mechanisms that produce the epileptic condition, especially after precipitating event such as brain tumors, are poorly understood. . Presenilins (PS) are part of the γ-secretase complex, an enzymatic complex that cleaves type I transmembrane proteins implicated in cell adhesion, differentiation, survival, and proliferation. PS2 and PS1 are highly homologous transmembrane proteins, and 2 of their main roles in cellular functions are to increase the production of the amyloidogenic Aβ peptide and to regulate Notch signaling. A resent research shows that PS1 mutations may cause severe seizure. To further define the role of PS in the pathophysiology of brain tumor-related epilepsy, we used immunohistochemistry, western blot and whole cell clamp analysis to localize and quantify PS in the hippocampal formation in the novel and highly relevant rat model of edicationrefractory epilepsy and in nonepileptic control animals. We were also to determine the expression and distribution of PS in tissue samples from the refractory cortex of patients who had been surgically treated for brain tumor-related epilepsy. We compared these tissues with histologically normal samples from controls. In our present study, we found PS was increased expressed in human epileptic peritumoral cortex. In addition , we were able to coimmunoprecipitate PS1 and mGLUR5 in U251 cells. It is possible that mGLUR5 is a substrate for PS1, implying PS1 may directly modulate mGLUR5 in U251 cells. Therefore, we hypothesize that the overexpression of PS in brain is mechanistically involved in the pathophysiology of brain tumor-related epilepsy, and propose that loss of PS may represent a novel therapeutic approach for this disease. The results from this study will shed light on new strategic treatment for brain tumor-related epilepsy.

癫痫发作是一种反复发作的威胁生命的症状。目前脑肿瘤致癫痫发作的细胞和分子机制还远未明确，该类癫痫的临床疗效仍欠佳。我们收集多种脑肿瘤致癫痫发作患者临床标本证实了早老素广泛分布于肿瘤及瘤周组织，同时也证实了早老素在癫痫动物模型中表达增加，并且早老素信号可能调控代谢型谷氨酸受体，因此早老素可能参与脑肿瘤致癫痫发作的过程，但进一步的细胞和分子机制还不清楚。鉴于早老素信号与其他信号之间的广泛相互作用，本项目拟以此为基础，在整体、细胞和分子水平上深入研究早老素信号调控代谢型谷氨酸受体及与Aβ相互关系，揭示早老素信号在脑肿瘤致癫痫发生发展过程中的分子机制，进一步阐明脑肿瘤致癫痫发作过程中以早老素为主调控其产物Aβ及代谢型谷氨酸受体的分子基础及其意义。这些研究从理论上可以构建脑肿瘤致癫痫发作的细胞和分子调控网络，在实际应用上则可为脑肿瘤致癫痫发作提供新的有效靶点，为治疗该类癫痫奠定基础，具有重要意义。

研究背景.癫痫发作在脑肿瘤的病人中是常见的，癫痫发作是由多种因素造成的。脑肿瘤导致的癫痫发作最终会演变成难治性癫痫，目前仍然没有合理的药物控制该种类型的癫痫发作。我们研究过程中发现早老素和脑肿瘤致癫痫发作密切相关，其中早老素可以通过影响TXNIP影响癫痫或脑肿瘤发病过程中的代谢，影响脑肿瘤致癫痫的发病过程。.研究内容、.一、观察早老素及TXNIP在脑肿瘤致癫痫发作过程中的改变：收集动物模型及临床标本，利用多种方法分析PS1、PS2、Aβ、TXNIP、REDD1和代谢相关标记物在皮层和海马的表达变化；.2. 验证早老素影响TXNIP在脑肿瘤致癫痫发作过程中的作用：利用细胞培养、膜片钳等方法研究过表达PS对TXNIP及细胞糖代谢的影响；.3. 验证早老素是否通过调控TXNIP影响细胞的糖代谢诱发癫痫：利用细胞培养、RNAi 和免疫共沉淀等多种方法判定PS 信号调控TXNIP的作用位点和途径；给予Aβ抑制剂观察PS信号对TXNIP及相关分子的影响；利用癫痫模型比较不同发作时期相关分子变化，进一步证明早老素通过影响TXNIP影响代谢通路对动物生物学的调控关系；利用免疫组织化学方法分析TXNIP及相关分子在肿瘤及瘤周中的表达结果，验证其的表达是否具有临床相关性。.结果、.结合我们的结果，我们认为PS 致癫痫影响在脑肿瘤中主要来源于“肿瘤本身”和 “瘤周组织”，这二部分PS 的异常增多可能与脑肿瘤相关的癫痫发作密切相关。我们已完成的研究结果证实PS可以通过影响TXNIP进而影响细胞的糖代谢进而影响组织的兴奋性，并可以促进肿瘤细胞的增殖和侵袭维持肿瘤细胞正常特性。实验western 结果证明了早老素、TXNIP及相关代谢分子和脑肿瘤及癫痫均密切相关，可能促进在胶质瘤致癫痫发作，并且免疫共沉淀结果初步证实TXNIP 和RDDD1 在胶质瘤细胞中相互结合，免疫组织化学证实两个分子在临床标本中均有临床相关性。.意义.这些表明，在脑肿瘤致癫痫发作的过程中PS 可能通过TXNIP参与代谢相关的传导通路，干预该信号的传导可能治疗该类癫痫。