A2AR对海马-压后皮层直接环路的调控在TBI后空间工作记忆障碍中的作用及分子调控机制

The dysfunction of spatial reference memory is a severe and long-lasting Sequela after traumatic brain injury (TBI). The underlying mechanisms remain to be elucidated. Preliminary experiments confirmed A2AR knockout or its antagonist can relieve space memory dysfunction after TBI, and found the after the projection fibers from hippocampus to retrosplenial cortex. The level of A2AR in retrosplenial cortex is negatively associated with the spatial reference memory. In addition, there is a potential binding site in A2AR to synaptic related proteins SAP102, and Src kinase can promote the phosphorylation of NR2B, a subunit of NMDA receptor. Thus, we hypothesized that A2AR inhibites NR2B phosphorylation and anchored to postsynaptic membrane via Src kinase and further impair the synaptic plasticity of neurons in retrosplenial cortex and spatial reference memory after TBI. In present project, we plan to confirm the role of A2AR in circuit from hippocampus to retrosplenial in the animals with region-specific A2AR knockout or knockin in TBI model using opotogenetic and neurotropic virus-mediated trans-synaptic neural circuit tracing across synapses. Furthermore, the formation of A2AR/SAP102/Src complex, synaptic plasticity, and spatial reference memory would be examined under the interfering of the antagonist of Src kinase and SAP102-shRNA. Clarifying the mechanisms of spatial memory dysfunction after TBI would provide the explanation for the role of the circuit under physiological function and the compensation function after injury.

空间参考记忆障碍是创伤性脑损伤（TBI）后严重而持久的后遗症，机制复杂尚未阐明。前期证实A2AR敲除或拮抗可缓解TBI后空间记忆障碍，发现海马至压后皮层的投射纤维，且压后皮层A2AR表达水平与空间参考记忆呈负相关。此外，海马A2AR存在突触相关蛋白SAP102的潜在结合位点，另Src激酶可促进NMDA受体亚基NR2B磷酸化，故推测A2AR通过Src依赖方式抑制NR2B磷酸化及膜定位，进而抑制海马-压后皮层的突触可塑性，导致TBI后空间参考记忆障碍。本项目拟以TBI模型，在压后皮层区域特异性A2AR敲除或转入小鼠中，采用光遗传、跨突触网络追踪技术验证A2AR在海马-压后皮层环路中调控作用，检测Src阻断剂和SAP102-shRNA干预下A2AR/SAP102/Src的结合状态，突触结构、兴奋性和空间参考记忆的改变。阐明TBI后空间记忆障碍的机制为生理条件下环路的作用和损伤后功能代偿提供解释。

压后皮层（retrosplenial cortex，RSC）在空间定位、空间信息更新、自我参照等方面的作用也备受关注，创伤性脑损伤（traumatic brain injury，TBI）后RSC损害引起认知功能障碍机制不清。利用光遗传学技术在小鼠 TBI 模型中检测阻断海马-压后皮层投射对电生理和小鼠空间参考记忆能力的改变，证明海马-压后皮层直接环路是调控空间参考记忆的重要神经环路。通过增强磁共振成像等实验证实TBI后压后皮层功能受抑制，而A2AR激动剂则加重空间记忆的损害。并进一步通过海马-压后皮层特异性环路A2AR敲除，采用水迷宫和Y-maze发现小鼠的逃避潜伏期缩短、自发轮替升高，提示工作记忆增强，证实A2AR在压后皮层空间记忆中具有调控作用， 但是这一调控作用与预期的A2AR/SAP102/Src 异聚体的形成关系不显著，但是与A2AR参与的自噬功能有关，A2AR激活可能通过PKA/ERK2/TFEB途径降低溶酶体生物发生，从而削弱自噬通量，进而损害压后皮层参与的空间记忆。该项目的完成为TBI后以A2AR为靶向的治疗提供实验基础和治疗策略。