Dissection of telomere-independent functions of nuclear and mitochondrial Telomerase Reverse Transcriptase ex vivo and in vivo

端粒酶在衰老过程中起着核心作用。全酶的一个重要组成部分是其催化亚基端粒酶逆转录酶（TERT）。除了在端粒维持中的作用外，它还在细胞核外具有额外的非端粒功能。我们可以表明，在生理条件下，在细胞核和线粒体中发现了TERT，并且细胞核以及线粒体TERT传递针对细胞凋亡的保护。此外，TERT对于呼吸链的活性是必不可少的，这可能是由于其与线粒体DNA结合并保护其免受破坏性外源影响。此外，TERT似乎参与减少细胞活性氧（ROS），这是众所周知的，在老化过程中增加。在身体活动对衰老过程影响的模型系统中，我们可以证明TERT可以以不依赖于端粒的方式抑制衰老。所有先前分析的缺点是几乎所有细胞类型中都表达了TERT，并且使用了完全缺乏TERT的动物和组织，这使得不可能区分细胞核和线粒体中TERT的功能。计划研究的目标是第一次区分离体和体内TERT的核和线粒体功能。为此目的，我们将一方面用编码TERT融合蛋白的转基因装备来自TERT缺陷动物的心血管系统的原代细胞或衍生自这些细胞的细胞系，所述TERT融合蛋白专门转运到细胞核或线粒体。这些细胞将受到刺激，这被称为心血管风险因素，以确定不同细胞区室中的TERT对线粒体功能和ROS产生的影响，以及对细胞凋亡和衰老的保护。对于体内研究，我们产生了普遍表达相同的TERT融合蛋白的转基因小鼠。我们将它们回交到一个TERT缺陷的遗传背景，以获得只在细胞核或线粒体中含有TERT的动物。为了能够研究端粒独立的TERT功能，我们设计了一种育种策略，以避免端粒侵蚀引起的衰老现象。与野生型和TERT缺陷的同窝仔相比，在整个动物和器官水平上的综合表型分析将显示线粒体和核TERT在衰老过程中的功能。此外，我们将研究随着年龄的增长而受损的再生过程以及细胞器特异性TERT对体育锻炼的积极“抗衰老”作用的影响。总的来说，预计的研究将首次揭示细胞核和线粒体中端粒独立的TERT功能的未知方面。

The enzyme telomerase plays a central role in aging processes. An essential component of the holoenzyme is its catalytic subunit telomerase reverse transcriptase (TERT). Besides its role in telomere maintenance it has additional non-telomeric functions also outside the nucleus. We could show that under physiological conditions TERT is found in the nucleus and the mitochondria and that nuclear as well as mitochondrial TERT convey protection against apoptosis. In addition, TERT is essential for the activity of the respiratory chain, which could result from its binding to mitochondrial DNA and protecting it from damaging exogenous influences. Furthermore, TERT seems to be involved in reducing cellular reactive oxygen species (ROS), which are known to increase during aging. In model systems for the influence of physical activity on aging processes we could show that TERT can suppress senescence in a telomere-independent fashion. A drawback of all previous analyses is the expression of TERT in nearly all cell-types and the use of completely TERT-deficient animals and tissues, which makes it impossible to differentiate between the functions of TERT in the nucleus and in the mitochondria. The goal of the projected studies is to discriminate for the first time between nuclear and mitochondrial functions of TERT ex vivo and in vivo. For this purpose we will on one hand equip primary cells from the cardiovascular system of TERT-deficient animals or cell lines derived from these with transgenes encoding TERT fusion proteins, which are exclusively transported to the nucleus or the mitochondria. These cells will be subjected to stimuli, which are known as cardiovascular risk factors, to determine the influence of TERT in the different cellular compartments on mitochondrial function and ROS production as well as on the protection against apoptosis and senescence. For in vivo studies we generated transgenic mice ubiquitously expressing the same TERT fusion proteins. We will backcross them onto a TERT-deficient genetic background to obtain animals containing TERT exclusively in the nucleus or the mitochondria. To be able to study telomere-independent TERT functions, we have devised a breeding strategy to avoid aging phenomena evoked by telomere erosion. A comprehensive phenotyping on the whole animal and organ level in comparison with wildtype and TERT-deficient littermates will show which functions mitochondrial and nuclear TERT have in aging processes. In addition, we will study regeneration processes compromised with age and the influence of organelle-specific TERT on the positive, "anti-aging" effects of physical exercise. Overall, the projected studies will for the first time uncover unknown facets of telomere-independent TERT functions in the nucleus and mitochondria.