HDAC6选择性抑制剂Tubastatin A对脑缺血再灌注损伤的保护作用研究

Histone deacetylase 6 (HDAC6), a member of class IIb HDACs, exclusively exists in the cytoplasm and mainly uses cytoplasmic proteins such as α-tubulin as its substrate. HDAC6 regulates diverse cellular processes, including microtubule transport. HDAC6 is known to be associated with pathological conditions of brain ischemia, which results in depolymerized microtubules and downregulated microtubule-associated motor proteins. In addition, HDAC6 knockdown by shRNA is neuroprotective in cortical neurons subjected to oxygen-glucose deprivation, an in vitro model of brain ischemia. Therefore, inhibition of HDAC6 has emerged as a promising strategy for treatment of ischemic stroke. Our preliminary data have shown that tubastatin A, a novel potent selective HDAC6 inhibitor, significantly improved functional behavioral deficits and reduced brain infarction in ischemic rats. The present study will investigate the effects of HDAC6 knockout in brain ischemic injury and tubastatin A’s protection in HDAC6 knockout mouse model of brain ischemia. We will further study the effects of tubastatin A on α-tubulin acetylation levels, microtubule stability, recruitment of motor proteins to microtubules, and microtubule-dependent mitochondrial axonal transport in brain ischemia models of rats and primary rat cortical neurons, by using Western blot, electron microscope, live cell imaging, etc. This study will demonstrate that HDAC6 is a critical therapeutic target for brain ischemia, and the underlying mechanisms of tubastatin A’s beneficial effects may involve HDAC6 inhibition, preservation of α-tubulin acetylation levels, promotion of motor protein recruitment, maintenance of microtubule stability, and the subsequent microtubule-dependent mitochondrial axonal transport. Our results will warrant clinical trials of tubastatin A for ischemic stroke.

HDAC6通过调控α-tubulin等非组蛋白的乙酰化而影响微管运输等生理过程。脑缺血会上调HDAC6、导致微管解聚、影响微管运输。我们的前期研究证实新型HDAC6选择性抑制剂tubastatin A能改善脑缺血引起的大鼠行为功能障碍、减小脑梗塞体积。本项目将在大鼠、HDAC6基因敲除小鼠和原代神经元上，应用Western blot、电镜、活细胞实时成像等技术，研究基因敲除HDAC6对脑缺血再灌注损伤和tubastatin A保护作用的影响，以及tubastatin A对损伤后α-tubulin乙酰化、微管结构、动力蛋白的微管募集、微管介导的线粒体轴突转运的调节作用。本项目将确证HDAC6是脑缺血损伤的重要靶点，并阐明tubastatin A通过抑制HDAC6、提高α-tubulin的乙酰化、稳定微管结构、改善微管运输而发挥抗脑缺血保护作用，为其临床转化提供药理学研究基础。