微管乙酰化状态与tau微管毒性的关系

Microtubules play a critical role in a diverse array of biological processes including cell division, cell growth and motility, intracellular transport and the maintenance of cell shape. The acetylation level of microtubules is closely associated with their in vivo functions and the pathogenesis of neurodegeneration. The microtubule-associated protein tau normally stabilizes microtubules. However, in a group of neurodegenerative diseases called tauopathy including Alzheimer's disease, tau is aggregated in neurofibrillary tangles composed of hyperphosphorylated tau. Microtubule defects are one of the early signs observed in tauopathy brains. To understand the pathogenesis of tauopathy, we overexpressed human tau in Drosophila and observed obvious microtubule defects due to tau overexpression; genetic screen revealed that mutations in histone deacetylase 6 (HDAC6) rescued tau-induced microtubule defects, indicating that increasing the acetylation level of microtubules may protect microtubules from tau's toxicity (Xiong et al., PNAS, 2013). However, others report that overexpression of HDAC6 also ameliorates the PolyQ-associated neurodegeneration by enhancing autophagy-mediated protein degradation of aggregated proteins (Pandey et al., 2007). Thus, how microtubule acetylation affects neurodegenerative processes remains an open question. The goal of this proposal is to better understand the rescue effects of tauopathy by HDAC6 mutations. We will mutate lysine 40 (K40) to glutamine (Q) and to arginine (R) or alanine (A) in alpha-tubulin to mimic acetylated and non-acetylated microtubules, respectively (Dompierre et al., 2007；Akella et al., 2010；Cohen et al., 2011）, via the newly developed transcription activator-like effector nuclease (TALEN) method. We will then analyze the effects these mutations on the microtubule defects and synapse abnormality caused by tau overexpression.The results obtained from these point mutations of alpha-tubulin will be corroborated by pharmacological treatments and manipulating the expression levels of acetylase MEC-17 and deacetylase HDAC6 acting on microtubules. This study will offer novel insights into the neuronal roles for microtubule acetylation and the rescue effects of increased microtubule acetylation on tauopathy.

微管乙酰化水平与其体内功能密切相关，多种神经变性病病理发生都与微管乙酰化稳态失衡有关。研究表明，操作去乙酰化酶表达可以改变不同神经变性病的病理表型。阿尔兹海默症等神经变性病的重要病理特征是微管结合蛋白tau的聚集和微管网络破坏。我们前期研究表明组蛋白去乙酰化酶6（HDAC6）突变可升高微管乙酰化水平，挽救tau过表达导致的微管网络的破坏及神经突触发育的异常（Xiong et al.,2013）。然而，HDAC6除了有多个乙酰化底物外，还有调控自噬等功能。因此，微管乙酰化水平与tau相关神经变性病病理发生的因果关系不明确。我们拟利用最近发展和完善的TALEN技术定点突变alpha-tubulin以模拟微管乙酰化和非乙酰化状态，揭示微管乙酰化水平与神经发育的关系及在tau相关神经变性病病理发生中的作用。本研究将揭示微管在神经系统中的作用机制并为研发防治tau相关神经变性病的药物提供潜在靶点。

Tau是一种微管(microtubules, MT)相关蛋白，在阿尔茨海默病及相关的神经退行性疾病中过度磷酸化并聚集成神经纤维缠结，该类疾病统称为tauopathies（tau蛋白病）。既往研究表明，使用微管稳定药物可改善转基因tauopathy小鼠模型的运输及认知功能缺陷。乙酰化是微管蛋白翻译后修饰的典型特征之一;乙酰化MT可以使微管更稳定。在我们之前的研究中，抑制微管蛋白特异性去乙酰化酶HDAC6可以挽救tau过表达导致的微管和神经肌肉连接(NMJs)的异常。HDAC6也可以作用于其他与微管蛋白无关的生物学过程中的蛋白质。为了进一步验证增加微管蛋白乙酰化可以抑制高表达tau导致的微管的破坏,我们构建了模仿乙酰化的tub84bK40Q突变体,发现增加微管蛋白乙酰化作用可以拯救过表达tau导致的微管的异常及NMJ发育缺陷。我们还发现HDAC6的特异性抑制剂ACY-1215和tubastatin A能够抑制tau蛋白的毒性。由此，增加MT乙酰化和HDAC6抑制剂ACY-1215和tubastatin A可作为干预tau蛋白病的潜在药物。