TOPK抑制HDAC1/2活性调控脑缺血反应性星形胶质细胞表型转化

After cerebral ischemia, astrocytes become reactive and have the ability of differentiation and proliferation. Our preliminary test found that: 1) TOPK, a new member of MAP2K, can combine with histone deacetylase HADC1/2 to increase histone acetylation, which can promote the phenotypic transformation of microglia and neurological function recovery in mice. 2) TOPK overexpression can reduce the level of intermediate filaments GFAP, as well as GFAP-Delta/GFAP-alpha ratio, suggesting that TOPK may inhibit the proliferation and cystoskeletal remodeling of reactive astrocyte. This project intends to verify the hypothesis that TOPK may regulate the phenotype transformation of reactive astrocytes following cerebral ischemia by inhibition of HDAC1/2 activity, 1) upregulates the expression of neurotrophic factor BDNF and GDNF through increasing the histone acetylation at the promoter region of BDNF and GDNF, then promotes the transformation of reactive astrocyte to a neuroprotective phenotype; 2) upregulates the histone acetylation of GFAP promoter, affecting the expression and splicing of different subtypes of GFAP, then promotes cytoskeleton remodeling of reactive astrocyte; 3) promotes the expression of p21 and p57, then inhibit the activity of CDK1/2 and cell cycle activation, then reduces the excessive proliferation of reactive astrocytes. This study will provide a new target for the regulation of the phenotype transformation of reactive astrocytes following cerebral ischemia.

脑缺血后，星形胶质细胞成为反应性星形胶质细胞，具备重新分化和增殖的能力。我们的前期研究发现：①MAP2K家族新成员TOPK可结合组蛋白去乙酰化酶HADC1/2，抑制其活性，增加组蛋白乙酰化，促进小胶质细胞表型转化、改善神经功能。②过表达TOPK可降低GFAP水平、GFAP-δ/GFAP-α比例，提示TOPK可能抑制反应性星形胶质细胞增殖和骨架重塑。本课题拟验证假说：TOPK可能通过抑制脑缺血后反应性星形胶质细胞HDAC1/2活性，①使神经营养因子BDNF和GDNF启动子区组蛋白乙酰化上调，表达增加，促进反应性星形胶质细胞向神经保护型转化；②上调GFAP启动子区组蛋白乙酰化，影响GFAP不同亚型转录和剪接，促进反应性星形胶质细胞骨架重塑；③促进p21和p57表达，进而抑制CDK1/2活性和细胞周期，减轻反应性星形胶质细胞的过度增殖。本研究将为脑缺血反应性星形胶质细胞表型转化的调控提供新靶点。

星型胶质细胞可塑性的不适应性改变可能导致中风急性期的脑水肿和恢复期的胶质瘢痕形成。本课题阐释了TOPK对脑缺血后反应性星形胶质细胞结构重塑、增殖作用及机制。主要结果：①我们检测了脑缺血不同时间点GFAP表达，缺血脑组织GFAPα、GFAPδ、GFAPδ/GFAPα、总GFAP蛋白显著增加，说明在脑修复过程中，存在反应性星形胶质细胞增殖、结构重塑。侧脑室注射TOPK高表达慢病毒，在TOPK敲低慢病毒侧脑室注射的同时腹腔注射HDAC1/2抑制剂FK228、和HDAC抑制剂SAHA，脑缺血14天，TOPK过表达使GFAPα、GFAPδ、GFAP、GFAPδ/ GFAPα水平降低；TOPK敲低使GFAPα、GFAPδ、GFAP、GFAPδ/GFAPα水平升高；即TOPK抑制修复期星形胶质增殖、结构重塑。FK228逆转TOPK敲低作用，作用强于SAHA。采用星形胶质细胞转染TOPK高表达和敲低，OGD处理，星形胶质细胞的骨架结构包括面积、直径、突触数量在缺氧以后均变小，被TOPK高表达逆转。证明TOPK对星胶增殖结构重塑由抑制HDAC1/2活性介导。②为了进一步研究TOPK/HDAC1/2的下游相关执行功能的神经营养因子，我们做了BV2小胶质细胞和原代星型胶质细胞的体外培养，发现在缺血缺氧性损伤早期营养因子IGF-1在小胶质细胞中的分泌减少，在星形胶质细胞中的分泌增加，可能与Pbk/HDAC2/IGF-1这一通路激活相关。③缺氧缺糖损伤显著促进原代神经干细胞向少突胶质和星形胶质细胞新生，抑制向神经元分化。TOPK可能参与到脑缺血后神经干细胞向少突胶质细胞分化的调控过程。④物理治疗-肢体远端缺血后处理治疗可以调节脑缺血再灌注损伤后反应性星形胶质细胞可塑性和抑制STAT3磷酸化，从而促进缺血性卒中后的神经功能恢复。上述研究为进一步研究相关信号通路在缺血后神经再生中的调控机制提供了基础。