Age-Related Decrease in Mitofusin 1 Results in Platelet Dysfunction and Thrombosis

项目总结/摘要 衰老是血栓性疾病的最大危险因素之一，如深静脉血栓形成、心肌梗死、 梗塞和中风。血小板通过其活化和聚集在血栓形成中起中心作用。之前 研究表明，血小板活化的年龄依赖性增加，但分子机制仍然未知。 虽然血小板是无核的，但它们含有功能性线粒体和活跃的泛素-蛋白酶体系统 （UPS）。UPS清除旧的和受损的蛋白质，以维持细胞的稳态。我们的初步数据 表明从健康老年（> 75岁）人受试者分离的血小板显示出增加的UPS活性， 与分离的血小板相比，关键线粒体融合蛋白线粒体融合蛋白1（MFN1）的表达降低 来自健康年轻受试者（18 - 35岁）。老年人的血小板也显示出增加 线粒体氧化剂产生和血小板活化。基于这些观察，我们将测试 假设UPS的年龄依赖性变化导致线粒体功能障碍增加 导致线粒体活性氧（mtROS）产生以刺激血小板活化 以及随后的血栓形成。在目标1中，我们将描述来自年轻和老年人的分离血小板中的UPS 健康的人类受试者。将进行伴随的MFN 1水平和血小板活化测量， 确定这些系统如何被UPS中与年龄相关的变化所改变。建立小鼠模型 对于分子机制，我们将在年轻（8 - 10周）和老年（> 1.5岁）中进行相同的测量 野生型（WT）小鼠。这些研究将通过体外细胞培养系统进行补充， CD34+祖细胞可分化为血小板，并用于利用血小板的机制研究。 慢病毒特异性靶向UPS组件。在目标2中，我们产生了血小板特异性MFN 1敲除， (KO)小鼠，以确定血小板中MFN1的减少是否会导致mtROS的增加和对 血栓形成我们将测量血小板形态学、线粒体活性氧产生和线粒体 在年轻和老年血小板特异性MFN1 KO小鼠中的生物能量学，并将其与年轻和老年WT小鼠进行比较。 将使用腺病毒在老年WT和血小板特异性MFN1 KO小鼠中过表达MFN1，以确定其是否 拯救了衰老的表型。此外，将对年轻和老年WT和血小板特异性MFN1（KO）小鼠进行免疫组化。 以确定血小板活化和血栓形成易感性是否增加 当MFN1不存在时。在我们的CD34+细胞培养系统中，慢病毒敲减和MFN 1的过表达将 补充这些机械研究。最后，Aim 3将确定临床使用的药物MitoQ （mtROS清除剂）或硼替佐米（蛋白酶体抑制剂）可以减弱年龄依赖性血小板活化， WT和血小板特异性MFN1 KO鼠模型中的血栓形成。该项目的成功完成将揭开 健康老龄化过程中血小板活化和血栓形成的新机制和新策略 重新利用临床可用的药物来减轻与年龄相关的血栓形成。

PROJECT SUMMARY/ABSTRACT Aging is one of the greatest risk factors for thrombotic diseases such as deep vein thrombosis, myocardial infarction and stroke. Platelets are central to thrombus formation through their activation and aggregation. Prior studies show age-dependent increases in platelet activation, but the molecular mechanisms remain unknown. Though platelets are anucleate they contain functional mitochondria and an active ubiquitin-proteosome system (UPS). The UPS removes old and damaged proteins to maintain cellular homeostasis. Our preliminary data suggests that platelets isolated from healthy aged (>75 years) human subjects show increased UPS activity and decreased expression of the key mitochondrial fusion protein mitofusin 1 (MFN1) compared to platelets isolated from healthy young subjects (18-35 years). Platelets from aged individuals also exhibited increased mitochondrial oxidant production and platelet activation. Based on these observations, we will test the hypothesis that age-dependent changes in the UPS lead to increased mitochondrial dysfunction resulting in mitochondrial reactive oxygen species (mtROS) production to stimulate platelet activation and subsequent thrombosis. In Aim 1, we will characterize the UPS in isolated platelets from young and aged healthy human subjects. Concomitant MFN1 levels and platelet activation measurements will be made to determine how these systems are altered by the age-related changes in the UPS. To establish a murine model for molecular mechanism, we will perform identical measurements in young (8-10 week) and aged (>1.5 year) wildtype (WT) mice. These studies will be complemented by an in vitro cell culture system of human derived CD34+ progenitor cells which can be differentiated into platelets and used for mechanistic studies that utilize lentivirus to specifically target the UPS components. In Aim 2, we generated platelet-specific MFN1 knockout (KO) mice to determine whether decreased MFN1 in platelets results in increased mtROS and susceptibility to thrombosis. We will measure changes in platelet morphology, mtROS production, and mitochondrial bioenergetics in young and aged platelet-specific MFN1 KO mice and compare it to young and aged WT mice. Adenovirus will be used to overexpress MFN1 in old WT and platelet-specific MFN1 KO mice to determine if it rescues the aged phenotype. Additionally, young and aged WT and platelet-specific MFN1 (KO) mice will be subject to laser-induced vascular injury to determine if platelet activation and thrombosis susceptibility increases when MFN1 is absent. Lentivirus knockdown and overexpression of MFN1 in our CD34+ cell culture system will complement these mechanistic studies. Finally, Aim 3 will determine whether the clinically used drugs MitoQ (mtROS scavenger) or Bortezomib (proteasome inhibitor) can attenuate age-dependent platelet activation and thrombosis in WT and platelet-specific MFN1 KO murine models. Successful completion of this project will unveil a novel mechanism by which platelet activation and thrombosis occur during healthy aging and new strategies to repurpose clinically available drugs to mitigate age-related thrombosis.