Intracellular mechanisms of gp120 neurotoxicity: role of microtubules

gp120神经毒性的细胞内机制：微管的作用

• 批准号: 9789669
• 负责人: Erin Wenzel
• 金额: $ 1.37万
• 依托单位: GEORGETOWN UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2018
• 资助国家: 美国
• 起止时间: 2018-09-11 至 2019-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9789669
• 关键词: AIDS prevention; Alzheimer' s Disease; Anti-Retroviral Agents; Atrophic; Axon; Axonal Transport; Basal Ganglia; Binding; Biological; Brain; Brain-Derived Neurotrophic Factor; CASP3 gene; Carrier Proteins; Cell Fractionation; Cerebral cortex; Co-Immunoprecipitations; Corpus striatum structure; Data; Deacetylation; Dissociation; Dynamin; Dynein ATPase; Endocytosis; Enzymes; Exhibits; Exposure to; Fractionation; Functional disorder; HDAC6 gene; HIV; HIV 1 Envelope Protein gp120; HIV Envelope Protein gp120; HIV Infections; HIV Seropositivity; HIV-associated neurocognitive disorder; Health; Hippocampus (Brain); Huntington Disease; Image; Impaired cognition; Impairment; Individual; Infection; Inflammation; Inflammatory Response; Kinesin; Label; Lead; Mediating; Microfluidics; Microtubule Alteration; Microtubule Proteins; Microtubule-Associated Proteins; Microtubules; Mitochondria; Molecular; Motor; Nerve Degeneration; Nervous System Trauma; Neuraxis; Neurites; Neurocognitive; Neurocognitive Deficit; Neurodegenerative Disorders; Neuroglia; Neurologic; Neuronal Injury; Neurons; Neurotoxins; Organelles; Pathologic; Patients; Pharmacology; Population; Post-Translational Protein Processing; Protein Isoforms; Proteins; Quantum Dots; Rattus; Reproducibility; Research; Role; Small Interfering RNA; Testing; Therapeutic Intervention; Toxic effect; Training; Travel; Tubulin; Vesicle; Viral Proteins; Virus; Work; antiretroviral therapy; axon injury; base; beta Tubulin; cytotoxic; env Gene Products; functional disability; in vivo; nervous system disorder; neuron apoptosis; neuron loss; neuronal survival; neuropathology; neurotoxic; neurotoxicity; novel; prevent; protein transport; trafficking; tubacin

PROJECT SUMMARY/ABSTRACT Even in the era of combined antiretroviral therapy, up to 50% of HIV-positive patients will demonstrate neurocognitive impairments in their lifetimes. These impairments are collectively known as HIV-associated neurocognitive disorders (HAND). While the neuropathology of HAND has been well-characterized, the specific mechanism by which HAND occurs remains to be clarified. Considerable experimental evidence indicates that HIV proteins, including the envelope protein gp120, cause neurological damage to a similar extent as the full virus. Thus, gp120 has emerged as potential agent underlying HIV neurotoxicity. However, the full mechanism of gp120- mediated neurotoxicity is still unknown. Therefore, it is imperative to investigate these mechanisms of neurotoxicity and elucidate targets for potential therapeutic intervention. I have established that gp120 is internalized into neurons via dynamin-dependent endocytosis and that internalized gp120 can bind to class-III β-tubulin, a component of neuronal microtubules. Moreover, gp120 causes the deacetylation of tubulin, a post-translational modification that impairs the functionality of microtubules. Furthermore, tubulin deacetylation causes a dissociation of the motor proteins kinesin-1 and dynein from microtubules, which impairs axonal transport. Preliminary data indicate that intracellular trafficking of essential organelles, such as mitochondria, is greatly diminished in the presence of gp120. Therefore, I hypothesize that gp120 impairs axonal transport of organelles and cargo-containing vesicles through the deacetylation of tubulin. To confirm whether this deacetylation of tubulin underlies the neurotoxic effect of gp120, I first will inhibit the regulatory enzyme HDAC6 pharmacologically with tubacin (AIM 1) to prevent deacetylation of tubulin. I will confirm these results by utilizing siRNA for HDAC6. Using primary rat cortical neurons, I hypothesize that inhibition of HDAC6 will be neuroprotective, as shown in other neurodegenerative diseases. Secondly, I propose to establish whether gp120 causes decreased binding of kinesin-1 and dynein to microtubules (AIM 2A). To examine this, I will evaluate the binding of kinesin-1/dynein to tubulin using co-immunoprecipitation and sub-cellular fractionation to isolate microtubule associated proteins. Finally, using rat primary cortical neurons grown in a microfluidic chamber to isolate axons, I will evaluate axonal transport in the presence of gp120 (AIM 2B) using live imaging of quantum dot labeled brain-derived neurotrophic factor (BDNF). I hypothesize that gp120 will cause a decrease in kinesin-1/dynein binding to microtubules and therefore will impair both velocity and total distance travelled by the labeled BDNF. These studies aim to establish a new mechanism of gp120-mediated neurotoxicity that impairs axonal transport through tubulin deacetylation. Moreover, throughout the proposed training, I will gain expertise in a variety of molecular experimental approaches with emphasis on motor proteins and axonal transport.

项目总结/摘要 即使在联合抗逆转录病毒治疗的时代，高达50%的艾滋病毒阳性患者将表现出 神经认知障碍这些损害统称为艾滋病毒相关的 神经认知障碍（HAND）。虽然HAND的神经病理学已经得到了很好的表征，但特定的 HAND发生的机制仍有待澄清。大量的实验证据表明，艾滋病毒 包括包膜蛋白gp 120在内的蛋白质引起的神经损伤程度与完整病毒相似。 因此，gp 120已成为潜在的代理HIV神经毒性。然而，gp 120的全部机制- 介导的神经毒性仍然未知。因此，研究神经毒性的机制是非常必要的 并阐明潜在治疗干预的目标。我已经确定gp 120被内化到神经元中， 并且内化的gp 120可以结合III类β-微管蛋白， 神经元微管此外，gp 120引起微管蛋白的脱乙酰化，这是一种翻译后修饰， 损害微管的功能。此外，微管蛋白去乙酰化导致马达的解离， 来自微管的蛋白质驱动蛋白-1和动力蛋白，其损害轴突运输。初步数据表明 在GP 120的存在下，基本细胞器如线粒体的细胞内运输大大减少。 因此，我推测gp 120通过以下途径损害细胞器和含货物囊泡的轴突运输： 微管蛋白的脱乙酰化。为了证实微管蛋白的这种去乙酰化是否是gp 120神经毒性作用的基础， 首先，我将用tubacin（AIM 1）抑制调节酶HDAC 6 β，以防止 微管蛋白我将通过利用针对HDAC 6的siRNA来证实这些结果。使用大鼠皮层神经元，我假设 HDAC 6的抑制将是神经保护性的，如在其他神经退行性疾病中所示。其次我 建议确定gp 120是否导致驱动蛋白-1和动力蛋白与微管（AIM 2A）的结合减少。到 为了验证这一点，我将使用免疫共沉淀和亚细胞免疫荧光技术来评估驱动蛋白-1/动力蛋白与微管蛋白的结合。 分级分离以分离微管相关蛋白。最后，使用大鼠原代皮层神经元生长在一个 微流控室分离轴突，我将评估轴突运输在gp 120（AIM 2B）的存在下，使用活 量子点标记的脑源性神经营养因子（BDNF）的成像。我假设gp 120会导致 驱动蛋白-1/动力蛋白与微管结合的减少，因此将损害速度和总距离 由标记的BDNF传播。这些研究旨在建立一种新的gp 120介导的神经毒性机制 通过微管蛋白去乙酰化损害轴突运输。此外，在整个拟议的培训，我将获得 在各种分子实验方法方面的专业知识，重点是马达蛋白和轴突运输。