Intracellular mechanisms of gp120 neurotoxicity: role of microtubules

项目摘要/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 他们一生中的神经认知障碍。这些损害统称为与艾滋病毒相关的 神经认知障碍(手)。虽然手部的神经病理已经有了很好的表征，但特异性的 手的发生机制尚不清楚。大量实验证据表明，艾滋病毒 包括包膜蛋白gp120在内的蛋白质引起的神经损害程度与完整病毒相似。 因此，gp120已成为HIV神经毒性的潜在致病因子。然而，gp120的完整机制- 介导的神经毒性仍不清楚。因此，对这些神经毒性机制的研究势在必行。 并阐明潜在治疗干预的靶点。我已经确定gp120内化到神经元中。 通过动力蛋白依赖的内吞作用，内化的gp120可以与III型β-微管蛋白结合，微管蛋白是 神经元微管。此外，gp120导致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，微管蛋白去乙酰化会导致马达的解离。 来自微管的蛋白质kinesin-1和dynein，这会损害轴突运输。初步数据显示， 在gp120存在的情况下，线粒体等重要细胞器的细胞内运输大大减少。 因此，我推测gp120通过以下途径损害细胞器和含货小泡的轴突运输。 微管蛋白的脱乙酰化。为了确认微管蛋白的这种去乙酰化是否构成了gp120的神经毒性作用的基础， 我首先会用Tubacin(AIM 1)药理抑制调节酶HDAC6，防止脱乙酰基 微管蛋白。我将利用HDAC6的siRNA来证实这些结果。使用原代大鼠皮质神经元，我假设 正如其他神经退行性疾病所显示的那样，抑制HDAC6将具有神经保护作用。其次，我 建议确定gp120是否导致kinesin-1和dynein与微管的结合减少(AIM2A)。至 研究这一点，我将使用免疫共沉淀和亚细胞来评估kinesin-1/dynein与微管蛋白的结合。 分离微管相关蛋白。最后，使用生长在原代培养的大鼠皮质神经元 微流室分离轴突，我将使用live评估gp120(AIM 2B)存在下的轴突运输 量子点标记脑源性神经营养因子的成像。我假设gp120会引起一个 运动蛋白-1/动力蛋白与微管的结合减少，因此会损害速度和总距离 由标有BDNF的车辆行驶。这些研究旨在建立gp120介导的神经毒性的新机制。 这损害了通过微管蛋白去乙酰化的轴突运输。此外，在整个拟议的培训过程中，我将获得 擅长各种分子实验方法，重点是马达蛋白和轴突运输。

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.