腺苷A2A受体激活RhoA/ROCK2信号通路破坏神经元骨架促进颅脑创伤后认知障碍的机制研究

The disruption of brain function such as cognition deficit is dominant syndrome after traumatic brain injury (TBI). So far, there is little available therapeutic treatment against the pathogenesis. Neuronal morphological integrity and effective synaptic transmission are basis of cognition. During the period of traumatic brain injury, destruction of neuronal cytoskeleton is the key event for inhibition of the neurite regeneration and the synapse formation and function and thus promotion of cognitive dysfunction. The adenosine A2A receptor (A2AR), one of the most important modulators for both neuropsychiatric and secondary brain injuries, is also involved in cytoskeletal organization. Besides, we previously demonstrated that activation of the A2AR exacerbated cognitive impairment and increased the activity of ROCK2, the key molecule that regulating cytoskeleton formation and cell motility after explosive blast-induced neurotrauma. Therefore, it is possible that activation of A2AR induces neuronal cytoskeleton destruction via ROCK2 signaling and thus exacerbated cognitive impairment following traumatic brain injury. Hence, the first objective of this proposal is to identify that activation of ROCK2 is a vital mechanism involved in A2AR stimulation-promoted neurite damages and synapse abnormalities in cultured cortical and hippocampal neurons. Another objective is to investigate the effects of A2AR activation on cognitive deficits as well as its mechanisms in mice controlled cortical impact model. In this part of study, the A2AR genetic knockout mice and pharmacological treatments are to be used. The expression and activity of RhoA, ROCK2 and downstream substrates, morphology, quantity and function of dentrites, axons and synapses and the cognitive function in both acute and prolonged phases of TBI are to be evaluated. The results will raises the prospect that the A2AR antagonism may be a potential therapeutic strategy for TBI and the consequent cognitive impairment. It will also provide a experimental foundation and a novel opportunity for clinical treatment.

认知障碍是创伤性脑损伤(TBI)的常见后遗症，目前尚缺乏有效对因治疗手段。神经元结构完整和突触有效传递是认知活动的基础，TBI后神经元骨架破坏是抑制神经突再生及突触形成和功能进而引起认知障碍的关键。腺苷A2A受体(A2AR)是神经精神活动及TBI后各种继发性损伤的重要调控因子，我们前期发现颅脑创伤后A2AR能促进ROCK2表达及记忆损害，而RhoA/ROCK2是调节细胞骨架的重要通路，故推测TBI后A2AR可能激活该通路引起神经元骨架破坏而促进认知障碍的发生。本研究拟首先在培养的神经元中证明RhoA/ROCK2活化是A2AR激活破坏神经元突起进而影响突触形态和功能的重要机制；并利用A2AR基因敲除动物和药理学干预，观察调控A2AR对RhoA、ROCK2及下游底物水平和活性以及TBI后树突、轴突和突触形态、数目和功能及认知功能的影响，为临床治疗提供理论依据及新的思路和选择。

腺苷A2A 受体（A2AR）是重要的脑损伤和神经精神调控靶点。我们前期发现抑制A2AR活性可以改善创伤性脑损伤（TBI）后急性继发性损伤，但其在TBI 后慢性认知障碍中的作用及可能机制尚不清楚。本研究拟通过离体实验及A2AR基因敲除小鼠和药理学干预，探讨A2AR活化促进TBI后认知障碍的神经元机制及可能涉及的RhoA/ROCK2信号转导，证明TBI后A2AR活化可通过RhoA/ROCK2通路加重神经元树突、轴突及突触形态和功能损伤而促进认知障碍发生。.研究首先利用小鼠皮质打击和冲击波所致中度TBI，发现激活A2AR促进，而抑制A2AR活性改善了这两种TBI模型所致慢性学习记忆损害。进一步在小鼠原代海马神经元和人神经母细胞瘤细胞系SH-SY5Y体外损伤模型中，利用A2AR调节剂及联合应用ROCK抑制剂法舒地尔进行干预，检测系列等神经突起和突触相关指标、ROCK2表达及其下游底物Tau蛋白磷酸化，发现A2AR活化减少了轴突长度、树突长度和分支及突触素、线粒体表达，增加了ROCK2表达和Tau蛋白S404和S262位点磷酸化，且上述效应可被A2AR拮抗剂和ROCK抑制剂阻断，提示A2AR活化可能通过ROCK2通路促进Tau蛋白磷酸化进而加重神经元突起和突触的损伤。最后通过A2AR KO小鼠和药理学干预，在体观察了TBI后不同时间空间工作记忆和参考记忆等认知功能指标及不同脑区（海马和前额、顶叶、颞叶皮质）骨架相关蛋白表达和形态、突触数量、形态和功能及RhoA、ROCK2、pTau等关键分子表达等指标，在体证明A2AR活化可通过促进RhoA和ROCK2表达及Tau蛋白磷酸化加重TBI后认知障碍。.本项目的完成，完善了A2AR参与继发性脑损伤和认知障碍的理论，为以A2AR及Rho/ROCK为靶点的临床治疗和远期行为学恢复提供了新的思路及实验依据，具有重要的理论意义和潜在的临床价值。发表文章7篇，其中中文CSCD论文3篇；英文论文4篇，包括SCI论文（IF 5.398）1篇，CSCD英文论文2篇，国际会议英文论文1篇。此外，在项目实施过程中，建立完善了动物行为学、光遗传学和在体电生理等神经科学研究平台，为后续研究奠定了良好基础，并获得一项新的国家自然科学基金面上项目资助。培养硕、博士研究生各2名，指导带教实习生和初级技术人员共8人， 1人晋升副研究员，促进了本研究所的学科发展。