Acetylated α-tubulin介导微管稳定在脑缺血后神经环路保护中的作用与机制研究

Globally, the disability rate of ischemic stroke is as high as 70% or more, and there is no particularly effective treatment in addition to a rehabilitation program. In adulthood, neuronal-circuit reconstruction is so difficult becaude of limited synaptic plasticity after stroke. Only a minority of patients (25%) with acute stroke could recovery after receiving rehabilitation. Previous studies have found that the preservation of more dendritic spines and neural circuits in the acute stage of ischemic stroke is conducive to the recovery of neurological function, but the underlying mechanisms and intervention targets are unclear.Our preliminary data shows that the stability of microtubules decreases after stroke, and the maintenance of microtubule stability during acute stage can specifically preserve mature dendritic spines that have established functional links, further preserve more neural circuits and improve motor dysfunction. Based on this, we hypothesize that maintaining the stability of microtubules is a key target for protecting the mature dendritic spines-neural circuits-motor function after ischemic stroke. In this project, we used the middle cerebral artery occlusion (MCAO) model, combined with living two-photon imaging, behavioral tests, neural circuits tracing, etc.; additionally, conditional knockout/overexpression of MEC17-Acetylated α-tubulin and microtubule stabilizer Epothilone B (EpoB) intervention to stabilize microtubules and explore the role and mechanism of protecting mature dendritic spines and neural circuits after stroke. To provide a new theoretical basis and treatment strategy for promoting functional recovery after ischemic stroke on the neural circuit level.

缺血性脑卒中致残率高达70%以上，除康复锻炼外，仍缺乏有效治疗手段。但成年后突触可塑性低、神经环路重建有限，康复锻炼也仅能使25%患者重返社会。既往研究发现：脑缺血早期保存更多树突棘及神经环路有利于神经功能恢复，但潜在机制与干预靶点不清。我们预实验结果显示：脑缺后微管稳定性下降，早期维持微管稳定性可以特异性保存已经建立功能联系的成熟树突棘，进一步保留更多的神经环路、改善运动功能障碍。据此我们推测：维持微管的稳定性是保护脑缺血后成熟树突棘-神经环路-运动功能的关键靶点。本项目我们采用小鼠脑缺血模型，结合活体的双光子成像、行为学、神经示踪等技术，通过条件性敲除/过表达MEC17-Acetylated α-tubulin及微管稳定剂Epothilone B干预来稳定微管，探索脑缺血后保护成熟树突棘及神经环路的作用和机制。从神经环路层面上为促进脑缺血后神经功能恢复提供新的理论基础和治疗策略。

神经环路破坏是脑卒中病人运动功能障碍的主要原因。运动神经环路包含了突触、神经元胞体和白质纤维束（皮质脊髓束）。本项目利用Thy1-YFP小鼠观察了脑卒中后运动皮层树突棘及基底节皮质脊髓束结构变化，发现了半暗带成熟树突棘丢失和皮质脊髓束退缩小球形成是运动环路破坏的重要病理学改变，退缩小球内含有大量解聚微管和肿胀线粒体。进一步发现，Acetylated α-tubulin (Ac α-tub)表达降低导致微管稳定性下降，是皮层树突棘丢失、白质轴突退变和轴突线粒体转运功能障碍的主要原因，此外，线粒体mPTP通道开放介导的早期线粒体功能障碍也导致脑卒中后轴突退变。过表达MEC17及Tubastatin A干预可以促进Ac α-tub表达，并特异性保护成熟树突棘的结构、减轻轴突损伤、改善轴突线粒体转运，从而改善了脑卒中小鼠运动功能障碍。环孢菌素A（Cyclosporin A, CsA）靶向抑制mPTP开放可以保护线粒体，也能改善脑卒中后轴突损伤、缓解运动功能障碍。.通过该课题的研究，我们发现了脑卒中后树突棘及轴突损伤的2个重要机制（微管稳定型下降和轴突线粒体肿胀），2个重要的干预靶点（Ac α-tub和线粒体mPTP通道）及2种改善脑卒中后运动功能障碍的潜在转化药物（Tubastatin A和环孢菌素A）。