endophilin A1在突触结构可塑性中的功能研究

Morphological plasticity of dendritic spines and synapses is essential for the organization and function of neural circuits. Previously we have found that during neuronal development, endophilin A1 modulates spine morphogenesis and synapse formation through binding to p140Cap, however, how endophilin A1-p140Cap activity in dendritic spines is regulated in mature neurons is unclear, and. physiological/pathological functions of endophilin A1 in the central nervous system remains to be explored. In cultured hippocampal neurons, gene knockdown of endophilin A1 or p140Cap caused defects in glycine-induced synaptic plasticity. We also found that the resuce effects of endophilin A1 membrane-binding deficient mutants(KKK-EEE and BAR-ΔH1I) on spine morphology in A1-depleted neurons were obviously hampered, indicating that the regulation of spine morphogenesis by endophilin A1 requires not only binding to p140Cap but also its membrane association. Glycine stimulation to induce plasticity increased the interaction between endophilin A1 and p140Cap and the interaction strength was Ca2+-dependent. A Ca2+ binding-deficient mutant (E264A) of endophilin A1 not only has a lower affinity for p140Cap but also fails to rescue the spine morphology phenotype and the defects in synaptic plasticity caused by endophilin A1 knockdown. Moreover, endophilin A1 knockout mice exhibit lower anxiety behavior. Since endophilin A1 not only binds to the membrane but also interacts with p140Cap, a cytoskeleton regulatory protein, we hypothesize that endophilin A1 in dendritic spines acts as a calcium sensor to function in activity-dependent spine/synaptic structural plasticity and promotes the coordination of plasma membrane and cytoskeleton in spines during synaptic plasticity and therefore is involved in anxiety. We propose to further investigate role(s) of endophilin A1 in synaptic structural plasticity and its potential physiological or pathological functions in cognition and other higher brain functions.

突触结构的动态变化反映了整个神经环路功能的可塑性。先前研究发现endophilin A1通过p140Cap调节树突棘形态发生及突触形成，然而A1参与的生理/病理功能及在突触活动过程中如何被调控尚不清楚。我们发现敲降endophilin A1和p140Cap抑制突触结构可塑性，endophilin A1膜结合缺陷和钙结合缺陷突变体均不能挽救敲降导致的树突棘形态/可塑性缺陷,因此endophilin A1发挥功能需要其膜定位和钙结合能力。此外，A1敲除小鼠焦虑行为明显减弱。因此我们提出假说：突触结构可塑性中，endophilin A1作为钙信号感应器，通过p140Cap时空特异性地调控细胞骨架重塑，介导胞膜与细胞骨架协同改变，促进突触结构可塑性，参与大脑情绪情感、高级认知过程及病理发生发展。我们将运用细胞、生化、电生理及行为手段，研究endophilin A1的生理/病理功能。

突触是中枢神经系统中神经元之间进行信息传递的特化结构，突触结构的动态变化反映了大脑神经环路功能的可塑性。先前研究发现endophilin A1通过p140Cap调节树突棘形态发生及突触形成，然而A1参与的生理/病理功能及在突触活动过程中如何被调控尚不清楚。此研究主要通过细胞分子生物学、形态学、电生理学和行为学等手段，以体外培养的神经元、脑片及整体动物为实验体系，对endophilin A1参与神经系统高级认知功能及其机制进行详细研究。首次发现小鼠海马部位endophilin A1主要表达于CA1和CA3区神经元。比较野生型和基因敲除小鼠的行为学及原代培养海马神经元表型的差异，发现成熟小鼠海马CA1区endophilin A1的功能与A2及A3不同，它通过互作因子p140Cap促进树突棘内F-actin的富集，增强突触可塑性发生，参与空间记忆及条件性恐惧记忆的长时程存储过程。同时我们发现过表达p140Cap虽然可挽救基础状态下树突棘内F-actin的富集，但无法挽救甘氨酸刺激后树突棘内F-actin的富集，因此突触可塑性过程中，树突棘内p140Cap诱导的F-actin时空特异性聚合可能需要endophilin A1相关的分子和/或信号通路的调节，具体的机制尚需进一步深入细致研究。已发表文献显示钙离子可结合endophilin A1的E264位残基，然而我们结果显示钙离子本身和endophilin A1无直接结合，与文献报道不一致，突触可塑性过程中钙信号如何调控endophilin A1的功能，我们仍在继续探索中。同时我们发现敲除endophilin A1对小鼠食物摄取及能量平衡未见明显影响。此外，endophilin A1可能参与抑制性突触的形成，影响神经环路的兴奋/抑制性平衡，由此可能参与神经系统病理性疾病过程，此部分研究尚需进一步探索。