人内源性逆转录病毒W家族env基因高表达导致神经细胞中胞内钙离子浓度升高的机制及作用研究

Recent studies showed that there was a close relationship between the HERV-W(human endogenous retrovirus) env and many diseases, such as schizophrenia, MS(Multiple Sclerosis), cancer, SLE(Systemic Lupus Erythematosus ), and so on. And there was also a relationship between the intracellular calcium concentration ([Ca2 +]i) and schizophrenia. While [Ca2+]i might lead to phosphorylation of cyclic adenosine monophosphate responsive element binding protein (CREB), resulting in abnormal nerve cells, and thus lead to many diseases such as schizophrenia.But the relationship among the overexpression of HERV, [Ca2 +]i and phosphorylation of CREB is still clear. Our data showed that the over-expression of env gene in nerve cells might contribute to the abnormity of several signal pathway. For example, the over-expression of env gene in nerve cells might lead to the expression of many factors （such as BDNF, S100B, CXCL10，KCNN3, etc）. And what's more, it is interesting that we found the over-expression of env gene in nerve cells might lead to the increase of [Ca2+]i and phosphorylation of CREB. While [Ca2+]i might be an important factor in the phosphorylation of CREB. From above on, based on the three National Natural Science Foundations and one stanley foundation of which are taken charge by the applicant, we will carry out "Study of the molecular mechanisms of increase of [Ca2+]i in neuro cell caused by the overexpression of HERV-W env" using molecular biology, bioinformatics and other biotechnologies.Therefore we will carry out this study from five areas: 1.The expression of HERV-W env increases [Ca2+]i in neuro cell; 2. The expression of HERV-W env leads to abnormal exchange between intracellular and extracellular Ca2+; 3. The expression of HERV-W env leads to the release of Ca2+ from endoplasmic reticulum calcium pool (release and re-absorption of Ca2+); 4. The expression of HERV-W env leads to the release of Ca2+ from Mitochondrial calcium pool (release and re-absorption of Ca2+);5. Study of the molecular mechanisms of the phosphorylation of CREB caused by the expression of HERV-W env in nerve cells. For this study, we will show the effect of the HERV-W env in the Ca2+ signal pathway in nerve cells. We will also reveal a new pathway of how HERV-W env causes diseases. And all these would provide new ideas about mechanism of how HERV-W env causes diseases and provide new ideas for treatment of some diseases.

研究表明人内源性逆转录病毒（HERV）W家族env基因高表达与精神分裂症等疾病相关，而胞内钙离子浓度（[Ca2+]i）升高可引起环磷腺苷效应元件结合蛋白（CREB）的磷酸化，导致神经细胞异常，进而引发精神分裂症等疾病。我们的研究发现HERV-W env高表达可导致神经细胞中[Ca2+]i升高，并可导致磷酸化CREB表达水平升高。因此，我们拟研究HERV-W env高表达导致神经细胞中细胞膜钙转运系统调节胞内外Ca2+交换异常及胞内钙库释放Ca2+异常，揭示HERV-W env高表达导致的[Ca2+]i升高的机制；同时研究HERV-W env高表达导致[Ca2+]i的升高，进而引起磷酸化CREB表达水平升高的机制，探讨HERV-W env高表达导致的[Ca2+]i的升高在神经细胞中的作用。本项目将为揭示HERV-W env基因高表达在[Ca2+]i/钙信号通路异常中的作用，提供实验依据。

研究表明人内源性逆转录病毒W 家族（HERV）W家族包膜蛋白（env ）基因高表达与精神分裂症等疾病密切相关，而精神分裂症病人的神经细胞中往往存在胞内钙离子浓度（[Ca2+]i）的升高，但机制未明。在本项目中，我们运用了生物信息学、流式细胞术结合荧光标记、全细胞膜片钳、分子生物学等技术手段，开展了HERV-W env基因高表达导致神经细胞中钙离子通道异常及其相关的机制的研究，结果发现HERV-W env高表达可导致神经细胞中[Ca2+]i升高，并可导致磷酸化CREB表达水平升高；发现HERV-W env基因高表达对L型（CACNA1D）、N型（CACNA1B）、T型（CACNA1G）等电压依赖性钙通道蛋白以及细胞膜钙泵基因均有影响，但差异无统计学意义；同时还发现HERV-W env基因高表达对TRPC3等钙池操纵性钙通道基因的表达有明显的影响，具有显著性差异，进一步研究发现HERV-W env基因可以通过上调TRPC3钙离子通道的表达和活性，介导人神经母细胞瘤细胞胞外钙离子内流入胞；发现HERV-W env基因高表达对三磷酸肌醇受体（IP3R）的3个型以及内质网钙泵的表达水平的影响也没有显著性差异；发现HERV-W env基因高表达对线粒体钙库相关基因线粒体钙离子单向吸收体表达水平有明显的上调；研究还发现HERV-W env过表达可以下调精神分裂症断裂基因1（DISC1）基因的表达，进一步研究发现下调DISC1基因表达可以在不影响TRPC3通道蛋白表达的情况下激活TRPC3通道，从而参与到HERV-W env通过TRPC3通道对胞内钙离子浓度的调控；最后，二代测序技术检测HERV-W env过表达的SH-SY5Y细胞基因转录水平变化，结果发现69个基因表达发生上调，48个基因表达发生下调；差异表达的基因主要富集在病毒进程、I型干扰素相关信号通路、天然免疫反应等，通过对低丰度基因的二次检索后发现，HERV-W env过表达可造成TRPC3通道表达上调。通过以上的研究，我们揭示了HERV-W env基因高表达与钙离子通道异常间的关系，阐明了HERV-W env基因高表达导致钙离子通道异常的分子神经生物学机制，为探讨精神分裂症等疾病的发病机制提供实验依据。同时也为精神分裂症等疾病的检测与治疗提供新的靶标。