Molecular Dissection of the Permeability Transition Pore

线粒体在细胞存活和组织发育中起着关键作用， 能量代谢、细胞内钙稳态调节和细胞凋亡。鉴于这一多因素 它们作为一个完整的系统，调节细胞内Ca ~（2+）代谢和生物能量学功能。在 从正常生理学的角度来看，这种整合反映在钙离子的高储存能力中， +，这可能会保护神经元等细胞 对抗过度活跃期间细胞内Ca 2+的瞬时升高。线粒体ca 2 + 体内平衡必须受到严格的调节，并基于一系列特定的摄取和释放系统。 然而，在体外线粒体内膜（IMM）可以很容易地经历渗透性增加， 分子量约为1，500 Da或更低的溶质。这种渗透性变化，称为 渗透性转换（PT），是由膜孔的开放，线粒体 渗透率转换孔（PTP）。PTP是电压依赖性的，环孢菌素A（CsA）敏感的， 高电导通道的内膜;孔开闭转换是 由多个效应子高度调节，这些效应子可能集中在一组较小的调节位点上。的 PTP长期以来一直被认为是体内线粒体功能障碍的靶点，特别是在 特定人类病理事件的背景。这些怀疑已被检查所证实这些怀疑已被检查所证实 在小鼠中，线粒体CyPD（PTP作用的关键调节因子， CsA）已被淘汰。这些研究证实了PTP在模型中的关键作用， 缺血/再灌注损伤的心脏和大脑，肌肉萎缩症的模型，在 多发性硬化和阿尔茨海默病时发生的轴突损伤。然而，尽管功能 在过去的30年里，传统模型中的候选孔隙组分都没有 经受住了严格且明确的基因测试。在这方面，PBR仍然是唯一的 生物化学鉴定成分 PTP的传统分子模型尚未经过彻底的基因检测。 因此，本申请的总体目标是使用生物化学和遗传工具来批判性地 用多种方法检测外周苯二氮卓受体（PBR）在PTP功能中的作用， 体外和体内试验，我们已经开发，以确认其作用。作为核心组件， PTP的监管机构。我们的研究是基于我们现在已经成功培育的小鼠， 野生型Tspo基因已被替换为 修饰的Tspo基因，含有1 oxP位点。利用这些小鼠，我们的计划是测试PTP功能， 线粒体，细胞和组织缺乏PBR，从而严格评估PBR的作用， PTP功能。重要的是。由于现在已经成功地产生了含有修饰的 含有loxP位点的Tspo基因。我们有理由在两个月内完成这些目标， 多年来提供的资金。

Mitochondria playa pivotal role in cell survival and tissue development by virtue of their role in energy metabolism, regulation of cellular Ca2 + homeostasis and apoptosis. Given this multifactorial role, they regulate cellular Ca2+ metabolism and bioenergetics function as an integrated s~stem. In terms of normal physiology, this integration is reflected in mitochondrion's high capacity to store Ca2 +, which may protect cells like neurons against transient elevation in intracellular Ca2 + during periods of hyperactivity. Mitochondrial Ca2 + homeostasismust be tightly regulated and is based in a series of specific uptake and release systems. Yet, in vitro themitochondrial inner membrane (IMM) can easily undergo a permeability increase to solutes with molecularmasses of about 1,500 Da or lower. This permeability change, called the permeability transition (PT), isregulated by the opening of a membrane pore, the mitochondrial permeability transition pore (PTP). The PTPis voltage-dependent, cyclosporin A (CsA)-sensitive, high-conductance channel of the inner mitochondrialmembrane; pore open-closed transitions are highly regulated by multiple effectors that likely converge on asmaller set of regulatory sites. The PTP has long been implicated as a target for mitochondrial dysfunction invivo, particularly in the context of specific human pathological events. These suspicions have been confirmedby examination of mice in which the expression of mitochondrial CyPD (a key regulator of PTP action and thetarget of CsA) has been eliminated. These studies have confirmed a critical role for the PTP in models of ischemia/reperfusion injury both in the heart and the brain, models of muscular dystrophy, in the axonaldamage occurring during MS, and Alzheimer's disease. However, despite detailed functional characterizationover the last 30 years, none of the candidate pore components in traditional models has withstood critical andunambiguous genetic tests. In this light, the PBR remains the only biochemically identified component in traditional molecular models of the PTP that has not been subjected to thorough genetic testing. Consequently, the overall goal of this application is to use biochemical and genetic tools to critically test the role of the peripheral benzodiazepine receptor (PBR) in PTP function using a variety of in vitro and in vivo tests that we have developed to confirm its role. either as core components or regulators of the PTP. Our studies are based in mice that we have now successfully generated in which the wild-type Tspo gene has been replaced with a modified Tspo gene containing /oxP sites. Using these mice, our plan is to test PTP function in mitochondria, cells and tissues lacking the PBR and thereby rigorously evaluate the role of the PBR in PTP function. Importantly. since mice have now been successfully generated containing a modified Tspo gene containing loxP sites. it is reasonable that we will be able to complete these aims in the two years of funding provided.