Understanding Endoplasmic Reticulum-Mitochondrial Cross-Talk in Corneal Endothelial Cells

项目摘要 Fuchs内皮性角膜营养不良(FECD)是一种常见的、复杂的、与年龄相关的退行性疾病 影响大约4%的美国人口的疾病，女性发病率较高。在FECD中， 角膜内皮(CE)细胞丢失伴随着细胞外基质以滴状物的形式沉积。我的 Mentor(Ula V.Jurkunas)明确报告说，在FECD中，氧化-抗氧化失衡是由于次优 NRF-2调节抗氧化防御，包括其转录靶标NAD(P)H苯二酚的下降 脱氢酶1(NQO1)，导致DNA氧化损伤、线粒体功能障碍和细胞凋亡。我的 初步数据还表明，与WT小鼠模型相比，NQO1-/-小鼠的CE显著丢失 FECD的。与氧化应激和线粒体损伤一样，我的合作导师(阿尔伯特·S·君)和其他人也有 提示内质网应激/未折叠蛋白反应(UPR)在FECD发病机制中的作用。我的初选 数据还表明，NQO-/-细胞系中促凋亡的内质网应激标志物显著且更早地激活 体外建立UVA诱导的FECD模型。然而，目前还没有任何将内质网和线粒体联系起来的研究 氧化-抗氧化失衡应激对FECD中CE的影响。这项建议的目标是界定 氧化-抗氧化剂作用下内质网应激对线粒体应激(改变的生物能量学和动力学)的贡献 FECD失衡。在奖项的指导阶段(K99)，我将确定UVA是否会诱导ER 和线粒体应激(目标1a)，内质网应激改变线粒体的生物能量学(目标1b)和动力学 (形态、碎裂、易位)均处于氧化剂-抗氧化剂失衡状态(目标2a)。我会学会诱导 和我的共同导师实验室(Albert Jun)的合作量化内质网应激，分析线粒体生物能量学 和动力学导师Ula Jurkunas和共同导师Pere Puigserer的实验室，学习体外和体内模型 FECD(尤尔库纳斯的实验室)以及哈佛医学院提供的广泛的职业发展活动。 在R00阶段，在导师和共同导师的支持下，我将与 内质网介导的线粒体内源性凋亡通路激活机制的进一步研究 (目标1c)通过钙离子信号和微管重排(目标2b)。总的来说，这些研究将提供新的 对内质网线粒体与角膜内皮生物学的串扰的见解和观点，这将推动 我们对FECD发病机制的认识。K99大奖将允许我接受额外的急诊室培训和 线粒体生物学与角膜内皮细胞生物学的新培训。广泛的资源和 哈佛医学院、尤尔库纳斯、普伊格瑟尔和Jun的实验室提供的职业发展机会，如 以及在K99阶段计划的研究活动，将使我能够实现以下长期目标 成为致力于研究角膜内质网线粒体串扰的独立调查者 内皮生物学。

Project Summary Fuchs endothelial corneal dystrophy (FECD) is a common, genetically complex and age-related degenerative disease affecting approximately 4 % of the U.S.A. population with a higher incidence in females. In FECD, corneal endothelial (CE) cell loss is accompanied by extracellular matrix deposition in the form of guttae. My mentor (Ula V. Jurkunas) has reported explicitly that in FECD, oxidant-antioxidant imbalance due to suboptimum Nrf-2 regulated antioxidant defense, including a decline in its transcriptional target, NAD(P)H quinone dehydrogenase 1 (NQO1), leads to oxidative DNA damage, mitochondrial dysfunction, and apoptosis. My preliminary data also suggests the significant loss of CE in NQO1-/- mice compared to WT in the mouse model of FECD. Alike oxidative stress and mitochondrial damage, my co-mentor (Albert S. Jun) and others have implicated the role of ER stress/ Unfolded protein response (UPR) in the pathogenesis of FECD. My preliminary data also suggests the significant and earlier activation of pro-apoptotic ER stress markers for NQO-/- cell line after UVA-induced FECD model in vitro. However, there has not been any study linking ER and mitochondrial stress under oxidant-antioxidant imbalance for CE in FECD. The objective of this proposal is to define the contribution of ER stress on mitochondrial stress (altered bioenergetics and dynamics) under oxidant-antioxidant imbalance in FECD. During the mentored phase (K99) of the award, I will determine whether UVA induces ER and mitochondrial stress (Aim 1a), ER stress alters mitochondrial bioenergetic (Aim 1b) and dynamics (morphology, fragmentation, translocation) all under oxidant-antioxidant imbalance (Aim 2a). I will learn to induce and quantify ER stress with my co-mentor lab’s collaboration (Albert Jun), analyze mitochondrial bioenergetics and dynamics with mentor Ula Jurkunas and co-mentor Pere Puigserver’s lab, learn in vitro and in vivo model of FECD (Jurkunas’s lab) along with the extensive career development activities offered at Harvard Medical School. During the R00 phase, with mentor and co-mentor’s support, I will perform uncompleted parts of Aim1-2 with the additional investigation of the mechanism of ER mediated-activation of mitochondrial intrinsic apoptotic pathway (Aim 1c) via Ca+2 signaling and microtubular rearrangement (Aim 2b). Collectively, these studies will provide new insights and perspectives into the ER-Mitochondrial cross talk for corneal endothelial biology, which will advance our understanding of FECD pathogenesis. A K99 award will allow me to receive additional training in ER and Mitochondria biology along with novel training in corneal endothelial cell biology. The extensive resources and career development opportunities available at Harvard Medical School, Jurkunas, Puigserver, and Jun’s lab, as well as the research activities planned in the K99 phase, will enable me to achieve the long-term goal of becoming an independent investigator dedicated to the study of ER-Mitochondrial cross talk in corneal endothelial biology.