XBP1s通过GFAT1调控HBP/O-GlcNAcylation对蛛网膜下腔出血后早期继发脑损伤的保护作用及机制研究

Subarachnoid hemorrhage (SAH) is a devastating acute neurological disease with high morbidity and mortality. Despite great effort in clinical and animal research, no pharmacologic intervention has yet been proven to improve neurologic outcome for SAH patients. Such a disappointing outcome from a massive research investment points out a necessity to better understand the underlying pathobiology of SAH. Mounting evidence has demonstrated that early brain injury (EBI) plays a critical role in defining the SAH outcome, which activates many stress response pathways. Among them is the unfolded protein response (UPR), which has been considered as a promising target for neuroprotective therapy, as UPR facilitates recovery of key neuronal functions impaired by metabolic stress, ie, folding and processing of proteins in the endoplasmic reticulum (ER). In UPR, one key mediator is XBP1s, a transcriptional factor that regulates expression of genes that code for ER-resident chaperons and importantly, the enzymes, including the rate-limiting enzyme GFAT1, involved in the hexosamine biosynthetic pathway (HBP). HBP produces UDP-GlcNAc, the substrate for O-GlcNAc modification. Notably, O-GlcNAc modification is a protective pathway under a variety of stress conditions, such as brain ischemia and heart ischemia. Our preliminary studies indicated that levels of XBP1s mRNA was significantly increased in the mouse brain after SAH. Critically, this increase was confirmed in SAH patient samples. Further, GFAT1 mRNA was found to be significantly upregulated in SAH patients. Taken together, we hypothesize that SAH upregulates XBP1s expression and thus the level of GFAT1 in the brain, which leads to increased O-GlcNAcylation that confers neuroprotective effect on EBI. To test this hypothesis, in this proposal, we will use not only genetic gain- and loss-of-function strategies (forebrain-specific XBP1 knockout and transgenic mouse models), but also adeno-associated virus-based gene delivery approache, to clarify the role of the XBP1s/HBP/O-GlcNAcylation axis in the fate and function of neurons after SAH. The results from this project is expected to improve our understanding of EBI after SAH and facilitate the development of novel neuroprotective therapies in SAH.

蛛网膜下腔出血（SAH）后早期继发性脑损伤（EBI）是SAH患者高死亡率和高致残率的重要原因。这与内质网应激有关，但其调控机制不明。我们前期研究发现，人和小鼠SAH早期，内质网UPR通路的XBP1s基因转录增加，且HBP通路关键酶GFAT1 mRNA增加；心肌缺血时XBP1s可直接激活GFAT1从而上调HBP通路。同时我们以往研究发现HBP/O-GlcNAcylation通路对缺血性脑卒中具有神经保护作用。据此，申请者推测XBP1s通过GFAT1激活HBP/O-GlcNAcylation信号通路而对SAH后EBI具有保护作用。本项目拟用XBP1前脑定向基因敲除与过表达小鼠为研究对象，建立SAH模型，并结合腺相关病毒转导及siRNA技术，试图阐明XBP1s/GFAT1/HBP/O-GlcNAcylation信号轴在SAH后EBI的作用机制，为临床开发SAH后EBI的神经保护药物提供实验依据。

蛛网膜下腔出血（SAH）后早期继发性脑损伤（EBI）是SAH患者高死亡率和高致残率的重要原因。这与内质网应激有关，但其调控机制不明。我们前期研究发现，人和小鼠SAH早期，内质网UPR通路的XBP1s基因转录增加，且HBP通路关键酶GFAT1 mRNA增加，而在心肌中XBP1s可直接激活GFAT1从而上调HBP通路，同时我们以往研究发现HBP/O-GlcNAcylation通路对缺血性脑卒中具有神经保护作用。. 本研究实验采用XBP1s与GFAT1在离体和在体的沉默和过表达实验，证实了在SAH后，XBP1s的转录增加能够增强HBP/O-GlcNAcylation的糖化作用并对SAH后EBI具有保护作用，而同时沉默GFAT1基因，这种保护作用消失同时受损区域的蛋白O-GlcNAcylation糖化作用显著降低。. 证明了XBP1s/GFAT1/HBP/O-GlcNAcylation信号轴在SAH后EBI的具有保护作用，为临床开发SAH后EBI的神经保护药物提供实验依据。

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