微管解聚相关蛋白Stathmin作为恐惧症治疗潜在分子靶点的机制研究

Increasing evidences shew that disruption of fear memory in the restricted time window by drugs or electric stimulus could interfere with fear memory consolidation and reconsolidation, thereby exposing a potential target for the treatment of Phobias.And studies shew that Stathmin,an microtubule depolymerization related protein,is required for acquisition of fear conditioning. Also closely related with long-term potentiation (LTP), in which synaptic plasticity is induced by electrical or chemical stimulation of the lateral nucleus of the amygdala (LA) circuits. However, the mechanisms in the process of fear memory consolidation and reconsolidation have not been clarified. Previously, we successfully established fear-conditioned animal model of rat by a single serious foot-shock stress, and this animal model's contextual fear memory studied according to the process of the classical Pavlovian fear conditioning. We found the mRNA and protein expression level of Stathmin increased obviously in LA of the rat. In this project, we will first microinject into the LA with lentivirus which overexpression or knockout Stathmin,anti-Stathmin antibody, Okadaic acid (Stathmin dephosphorylation reagent),and specific p38MAPK or PKA inhibitors at the process of fear memory consolidation and reconsolidation respectively. And then we will test the mRNA and protein expression level of Stathmin and phosphorylation level of Stathmin by qRT-PCR,Western blot and laser scanning confocal microscope(LSCM). Furthermore,clarify that Stathmin regulates the fear memory consolidation and reconsolidation through the cAMP-PKA and/or ERK/MAPK-signalling cascades. Collectively,this research would throw light on the molecular mechanisms of fear conditioning and exposing a potential target for treatment of phobias.

恐惧记忆巩固及再巩固过程相对不稳定，易受药物等干扰，使其成为恐惧症治疗的重要"时间窗"。研究表明，微管解聚相关蛋白Stathmin在恐惧记忆的获得过程中发挥重要作用，并与恐惧记忆形成的神经细胞学基础长时程增强密切相关；但是，其在恐惧记忆的巩固及再巩固过程中的作用机理尚未阐明。课题组已成功构建恐惧症大鼠模型，并发现Stathmin在模型鼠杏仁核外侧核（LA）mRNA和蛋白水平表达明显增加；本课题拟在模型鼠恐惧记忆巩固及再巩固过程中分别LA区微注射Stathnin基因过表达或干涉的重组慢病毒、抗Stathmin抗体、冈田酸、p38MAPK抑制剂及PKA抑制剂，应用Western Blot、微管体内结合测定、激光扫描共聚焦显微镜等技术，明确Stathmin在恐惧记忆巩固及再巩固过程中的作用机制及是否通过cAMP-PKA和ERK/MAPK信号转导通路实现，为恐惧症生物治疗新分子靶点奠定理论基础。

恐惧记忆巩固及再巩固过程相对不稳定，易受药物等干扰，使其成为恐惧症治疗的重要“时间窗”。研究表明，微管解聚相关蛋白Stathmin在恐惧记忆的获得过程中发挥重要作用，并与恐惧记忆形成的神经细胞学基础长时程增强密切相关；但是，其在恐惧记忆的巩固及再巩固过程中的作用机理尚未阐明。课题组前期成功构建恐惧症大鼠模型，并发现Stathmin在模型鼠杏仁核外侧核（LA）mRNA和蛋白水平表达明显增加。在本课题中成功构建大鼠Stathmin基因过表达和干涉慢病毒载体，并获得高滴度重组慢病毒，可达108 TU/mL；体内外实验均证实，LV-pLenti7.3/V5 DEST-stathmin具有显著的 Stathmin过表达作用,LV- pLenti6 /BLOCK -ITTM -DEST-stathmin-563具有显著的Stathmin基因表达沉默作用；PC12细胞经神经生长因子-2.5s诱导培养48h后，分别加stathmin过表达和干涉重组腺病毒培养48h，干涉stathmin表达，细胞微管形态和β-III-tubulin荧光亮度优于正常对照组和过表达组；动物实验中我们发现，恐惧记忆巩固期完成后6h和恐惧记忆再巩固完成后0.5h、6h，下调Stathmin表达，恐惧症模型鼠冻结时间与对照组和假手术组比较显著缩短，提示在恐惧记忆巩固和在巩固早期，下调Stathmin表达，对恐惧记忆可能具有一定干预作用。课题组进一步研究发现，在恐惧记忆巩固及再巩固早期特异性抑制MAPK信号通路，对恐惧症模型鼠行为学无影响。