Mechanisms of Tat neurotoxicity: the role of mitochondria

 描述(申请人提供)：线粒体生命周期的扰动与神经退行性疾病的因果作用有关，包括帕金森氏症、阿尔茨海默氏症和亨廷顿病。此外，在HIV相关性痴呆(HAD)患者的死后组织中也观察到异常的线粒体，这些患者还表现出树突修剪、神经元凋亡和突触异常，这表明线粒体功能障碍可能在疾病进展中发挥作用。然而，到目前为止，这一假设还没有直接的支持。我的总体目标是证明TAT损害神经元中的线粒体稳态，这可能导致HAD的神经元损伤。这项研究的具体目的是：1)确定TAT如何在体外损害线粒体动力学(即融合、分裂和运输)，2)评估TAT诱导的动态相关蛋白1(Drp1)活性的变化，以及3)确定TAT如何损害有丝分裂。已经证实，TAT导致突触简化和神经元凋亡，在此之前线粒体明显受损，表现为线粒体膜电位下降和活性氧(ROS)释放增加。此外，我的初步数据显示，TAT在体外会损害神经元中线粒体的亚细胞定位。因此，目标1将包括将含有荧光标记线粒体的原代神经元培养物暴露于TAT的研究。通过这些实验，将通过活细胞成像观察线粒体动力学，并通过随机光学重建显微镜(STORM)和扫描电子显微镜(EM)观察线粒体超微结构。在初步研究中，我观察到线粒体损伤以及线粒体的胞体积累。因此，我预计线粒体的逆行运输会增加，线粒体碎裂也会增加。目标2将评估TAT导致线粒体碎裂的机制。原代培养的神经元将暴露在TAT中，然后检测总的和磷酸化的DRp1水平以及钙调神经磷酸酶的活性。由于TAT先前已知可以激活钙调神经磷酸酶，我预计会看到钙调神经磷酸酶活性增加，而磷酸化的Drp1水平降低。目标3将评估TAT对有丝分裂吞噬的影响。为了实现这一目标，我将使用三种互补的技术来评估TAT暴露后原代神经元培养中的有丝分裂：1)亚细胞分离以分离自噬空泡，2)邻近连接试验，以及3)EM以检查自噬的超微结构。综上所述，我预计在TAT暴露后，自噬体内线粒体的定位会增加，但不会因为有丝分裂过程的效率低下而看到整个线粒体水平的下降。这项提议将加强我在线粒体神经生物学和HIV-1神经毒性方面的培训。此外，提出的机制研究可能会提供一种更明确的方法，通过新开发的疗法来针对这种损害。

 DESCRIPTION (provided by applicant): Perturbations to the mitochondrial life cycle have been implicated in a causal role of neurodegenerative diseases, including Parkinson's, Alzheimer's, and Huntington's diseases. Moreover, aberrant mitochondria have also been observed in the post-mortem tissue of HIV associated dementia (HAD) subjects who additionally exhibit dendritic pruning, neuronal apoptosis, and synaptic abnormalities, indicating that mitochondrial dysfunction may play a role in disease progression. However, as yet this hypothesis has no direct support. My overall goal is to demonstrate that Tat impairs mitochondrial homeostasis in neurons, which can contribute to neuronal impairment in HAD. The specific aims of the proposed research are to: 1) determine in vitro how Tat impairs mitochondrial dynamics (i.e. fusion, fission, and trafficking), 2) assess Tat-induced alterations t the activity of dynamin- related protein 1 (Drp1), and 3) determine how Tat impairs mitophagy. It has been established that Tat causes synaptic simplification and neuronal apoptosis, preceded by significant impairment to mitochondria as determined by a decrease in mitochondrial membrane potential and increased reactive oxygen species (ROS) release. Furthermore, my preliminary data show that Tat impairs mitochondrial subcellular localization in neurons in vitro. Thus, Aim 1 will consist of studies in which primary neuronal cultures containing fluorescently labeled mitochondria will be exposed to Tat. From these experiments, mitochondrial dynamics will be observed by live-cell imaging and mitochondria ultrastructure by stochastic optical reconstruction microscopy (STORM) and scanning electron microscopy (EM). In preliminary studies, I have observed mitochondrial impairment as well as somal accumulation of mitochondria. Therefore, I expect to see an increase in retrograde transport of mitochondria and increased mitochondrial fragmentation. Aim 2 will assess a mechanism by which Tat causes mitochondrial fragmentation. Primary neuronal cultures will be exposed to Tat and then probed for levels of total and phosphorylated Drp1 as well as calcineurin activity. Since Tat is known previously to activate calcineurin, I expect to see increased calcineurin activity and decreased levels of phosphorylated Drp1. Aim 3 will assess the impact of Tat on mitophagy. To fulfill this Aim, I will assess mitophagy within primary neuronal cultures following Tat exposure utilizing three complementary techniques: 1) subcellular fractionation to isolate autophagic vacuoles, 2) proximity ligation assay, and 3) EM to examine autophagosomal ultrastructure. Taken together, I expect to observe an increase in mitochondrial localization within autophagosomes following Tat exposure but do not expect to see a decrease in whole mitochondria levels due to inefficiency in the mitophagic process. This proposal will enhance my training in mitochondria neurobiology and HIV-1 neurotoxicity. Moreover, the mechanistic studies proposed will likely present a clearer way to target this damage with newly developed therapies.