Mechanisms of Tat neurotoxicity: the role of mitochondria

Tat 神经毒性机制：线粒体的作用

• 批准号: 8975549
• 负责人: Summer Johnelle Rozzi Kathol
• 金额: $ 3.09万
• 依托单位: GEORGETOWN UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2014
• 资助国家: 美国
• 起止时间: 2014-12-01 至 2017-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8975549
• 关键词: AIDS Dementia Complex; Alzheimer' s Disease; Autophagosome; Binding; Biological Assay; Calcineurin; Calcineurin inhibitor; Calcium; Calmodulin; Cell Death; Cell Fractionation; Cell physiology; Cells; Data; Disease Progression; Distress; Dynamin; Electron Microscopy; Exhibits; Family; Genetic Transcription; Goals; Guanosine Triphosphate Phosphohydrolases; HIV; HIV-1; Health; Homeostasis; Huntington Disease; Image Analysis; Impairment; In Vitro; Label; Lead; Life; Life Cycle Stages; Ligation; Measures; Membrane; Membrane Potentials; Microscopy; Microtubules; Mitochondria; Mitochondrial Proteins; Movement; N-Methylaspartate; Neurobiology; Neurodegenerative Disorders; Neurons; Optics; Organelles; Parkinson Disease; Pathology; Phosphorylation; Play; Process; Protein Dephosphorylation; Proteins; Quality Control; Rattus; Reactive Oxygen Species; Receptor Activation; Recycling; Research; Role; Scanning Electron Microscopy; Series; Synapses; Techniques; Testing; Tissues; Toxic effect; Training; Trans-Activators; Vacuole; Viral Proteins; insight; live cell imaging; mitochondrial dysfunction; mitochondrial membrane; neuron apoptosis; neuronal cell body; neurotoxic; neurotoxicity; polarized cell; reconstruction; research study; retrograde transport; therapy development; trafficking

 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.

 描述（由申请人提供）：线粒体生命周期的扰动与神经退行性疾病（包括帕金森病、阿尔茨海默病和亨廷顿病）的因果关系有关。此外，在HIV相关性痴呆（HAD）受试者的死后组织中也观察到异常线粒体，其另外表现出树突修剪、神经元凋亡和突触异常，表明线粒体功能障碍可能在疾病进展中起作用。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达特损害神经元线粒体的稳态，这可能有助于HAD的神经元损伤。拟议研究的具体目的是：1）在体外确定达特如何损害线粒体动力学（即融合、分裂和运输），2）评估塔特诱导的动力蛋白相关蛋白1（Drp 1）活性的改变，3）确定达特如何损害线粒体自噬。已经确定，达特导致突触简化和神经元凋亡，之前是线粒体的显著损伤，如通过线粒体膜电位降低和活性氧（ROS）释放增加所确定的。此外，我的初步数据表明，达特损害线粒体在体外神经元亚细胞定位。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达特的研究。从这些实验中，线粒体动力学将观察活细胞成像和线粒体超微结构的随机光学重建显微镜（STORM）和扫描电子显微镜（EM）。在初步研究中，我观察到线粒体损伤以及线粒体的体积累。因此，我希望看到线粒体逆行运输的增加和线粒体碎片化的增加。目的2将评估达特引起线粒体断裂的机制。将原代神经元培养物暴露于达特，然后探测总Drp 1和磷酸化Drp 1的水平以及钙调磷酸酶活性。由于之前已知达特可以激活钙调磷酸酶，我希望看到钙调磷酸酶活性增加和磷酸化Drp 1水平降低。目标3将评估达特对线粒体自噬的影响。为了实现这一目标，我将评估原代神经元培养物内的线粒体自噬达特曝光后，利用三种互补的技术：1）亚细胞分级分离自噬空泡，2）邻位连接试验，和3）EM检查自噬体超微结构。综上所述，我希望观察到达特暴露后自噬体内线粒体定位的增加，但不希望看到由于线粒体吞噬过程中的效率低下而导致的整个线粒体水平的降低。这个建议将加强我在线粒体神经生物学和HIV-1神经毒性方面的训练。此外，提出的机制研究可能会提出一种更清晰的方法，用新开发的疗法来靶向这种损伤。