D4R激活恢复衰老受损海马突触可塑性的机制研究

Aging is associated with the impairment of learning and memory, and synaptic plasticity. Currently, the effective means of intervention in aging-related cognitive impairment is lacking. Our study shows that the activation of DA receptor 4 (D4R) restores damaged hippocampal LTP in aged rodents, but the mechanism of such regulation for D4R is unclear. TARPγ8 is a key regulatory protein for LTP. The phosphorylation and expression of TARPγ8 are regulated by CaMKⅡα and Calpain, respectively. Our preliminary data have shown that the expression of TARPγ8 in the aging hippocampus is significantly decreased. Given that CaMKⅡα inactivation and Calpain activation associated with abnormal calcium homeostasis in aging, the applicant has proposed that D4R activation may restore the hippocampal LTP in aging by up-regulation of TRAPγ8 activity and expression. In this project, using in vitro and in vivo electrophysiology, optogenetic and molecular biology techniques, we will determine the aging-related changes in phosphorylation and expression level of TARPγ8 and its related regulatory proteins, and the effects of D4R activation on these changes. We will also determine whether TRAPγ8 is involved in aging-related impairment of LTP and the regulation of D4R activation on aged hippocampal LTP by manipulating the level of phosphorylation and expression of TRAPγ8. This study may provide a new basis for elucidating the mechanism of aging-related LTP damage and a new target for interfering cognitive impairment.

衰老是神经退行性疾病的主要危险因素，存在学习记忆障碍与突触可塑性（LTP）受损。目前对认知损害缺乏有效的干预手段。我们发现多巴胺受体4（D4R）激活恢复了衰老小鼠海马受损的LTP，但其机制尚不清楚。TARPγ8是LTP的关键调节蛋白，其磷酸化与表达分别受CaMKⅡα与Calpain调节。我们前期研究表明衰老海马TARPγ8表达下降，而衰老存在钙稳态异常调节的CaMKⅡα活性下降及Calpain激活。因此，申请人提出D4R可能通过对TRAPγ8活性与表达的上调恢复衰老海马LTP。本项目将采用离体与在体电生理、光遗传及分子生物学技术，探讨TARPγ8及其调节蛋白的活性与表达水平在衰老中的变化及D4R激活对其影响；操控TRAPγ8的磷酸化与表达水平，确定其是否参与衰老海马LTP受损及D4R激活对衰老海马LTP的调节。该研究为阐明衰老相关LTP受损的机制提供新依据，为干预认知功能受损提供新靶标。

钙调蛋白激酶IIα(CaMKIIα)依赖的AMPAR跨膜调节蛋白γ8(TARPγ8)的活化，介导了AMPAR的膜表达，为突触可塑性(LTP)所必须。衰老存在LTP受损，可能与电压依赖性钙通道(VDCC)表达增加，钙蛋白酶(Calpain)活化，CaMKIIα-TARPγ8信号异常有关。多巴胺受体4 (D4R) 激活能改善衰老海马LTP，但相关机制并不清楚。通过记录海马突触LTP，我们发现D4R激活恢复衰老海马LTP与其对VDCC的抑制有关。Western blot结果表明海马TARPγ8以及p-CaMKIIα蛋白表达存在衰老相关的下降，Calpain 1的表达增加；抑制p-CaMKIIα活性减少年轻及老年小鼠TARPγ8的表达；抑制Calpain 1活性对这些小鼠TARPγ8的表达无影响；而阻断VDCC增加了年轻与衰老小鼠海马p-CaMKIIα的表达，仅增加了衰老小鼠海马TARPγ8的表达；D4R激活增加了年轻与衰老海马的p-CaMKIIα表达，仅部分增加了衰老TARPγ8表达。这些结果提示TARPγ8表达依赖于p-CaMKIIα的活性并受到VDCC的控制；而D4R激活相似于VDCC抑制，上调CaMKIIα-TARPγ8信号。.外源性表达TARPγ8于衰老小鼠海马增加了衰老小鼠LTP，进一步证实TARPγ8对LTP的调节作用。为了确定TARPγ8活化是否涉及D4R对衰老LTP的调节，我们将TARPγ8分子的两个CaMKIIα磷酸化位点进行突变，建立了TARPγ8S277A/S281A 基因敲入小鼠(TARPγ8KI)，并证实TARPγ8KI小鼠海马LTP、学习记忆等行为受损；而DR4激活对TARPγ8KI小鼠海马突触LTP无明显作用，提示D4R对LTP的增强可能依赖于CaMKIIα介导的TARPγ8活化。于清醒衰老小鼠我们也证实了其学习与记忆受损与TARPγ8的表达相关，体内注射D4R激动剂改善衰老小鼠学习记忆，恢复了海马LTP。基于上述结果，我们提出D4R激活可能通过抑制VDCC而活化了CaMKIIα-TARPγ8信号，恢复了衰老相关的LTP，改善认知功能。该研究为改善衰老及神经退行性疾病认知功能提供新的新策略。