β-arrestin在记忆巩固和再巩固中作用及机制研究

【作者】 刘星；

【导师】 马兰； 李浩洪；

【作者基本信息】 复旦大学 ， 药理学， 2010， 博士

【摘要】 β-arrestin在哺乳动物体内广泛分布,其亚型包括β-arrestinl和β-arrestin2。其主要作用是作为负调控因子介导七次跨膜受体(7TMR)的内吞和再循环,促进G蛋白偶联受体与G蛋白解偶联,阻断受体依赖的第二信使的激活,从而导致7TMR脱敏。如β-arrestin通过介导μ阿片受体的内吞参与大鼠对吗啡脱敏的形成。近年来许多研究揭示β-arrestin能作为多功能适配器参与受体转运、信号分子的激活和信息传递。当7TMR被激活后,β-arrestin作为支架蛋白促进信号分子在细胞内重新定位。如β-arrestin富集于细胞核内招募转录因子从而促进基因的转录,我们的前期研究结果发现阿片受体激活后β-arrestin1发生核转位后招募p300,上调组蛋白的乙酰化水平,从而促进靶基因的表达。β-arrestin还能富集于细胞膜附近招募并促进激酶磷酸化,有研究表明在肾上腺素受体激动剂的作用下,β-arrestin2和磷酸二脂酶4(PDE4)结合后从胞浆转移至胞膜,促进cAMP的降解。此外β-arrestin通过招募激酶等信号分子,改变其构象并增强激酶活性,将信息从受体传递到效应分子,起到信息传递的作用。如肾上腺素受体被激活后,β-arrestin作为MAPK的支架蛋白,招募MAPK信号通路的成员raf和MEK,导致ERK发生β-arrestin2依赖的持久性激活。目前以上研究大多数停留于细胞水平的机制探讨,其真正的生理功能尚未揭示。鉴于β-arrestin在脑内的广泛分布,本课题根据β-arrestin目前最新的研究进展,试图探讨其在整体水平尤其在神经系统的生理和病理意义。我们采用β-arrestin基因敲除(KO)小鼠进行学习记忆相关行为学测试,并结合分子生物学等检测手段,重点在于揭示β-arrestins在学习记忆中可能发挥的作用。研究结果如下：1)β-arrestin2缺失导致恐惧条件记忆的巩固受损。我们采用恐惧条件记忆模型,单次足底电击和声音刺激联合性训练后β-arrestin2 KO小鼠恐惧条件记忆的短时程记忆和长时程记忆均受抑制；多次强化训练后,β-arrestin2 KO小鼠恐惧条件记忆的获得和学习能力仍显著低于野生型(WT)小鼠,且长时程记忆明显受损。结果提示β-arrestin2参与恐惧条件记忆的巩固。2)恐惧条件记忆的形成过程中杏仁核内PKA的活性受到β-arrestin2调节。单次联合性训练后WT小鼠杏仁核内PKA活性一过性升高,但β-arrestin2 KO小鼠杏仁核内PKA活性无显著变化；PDE4抑制剂通过增加cAMP上调WT小鼠杏仁核内PKA活性,但对β-arrestin2 KO小鼠无明显影响；forskolin通过增加cAMP产物增加WT小鼠杏仁核PKA活性水平。结果提示PKA的活性受到β-arrestin2的调控,并参与恐惧条件记忆的形成。3)杏仁核外侧核β-arrestin2的表达能促进恐惧条件记忆的巩固。通过腺病毒脑立体定位注射的方法,在KO小鼠杏仁核外侧核(LA)表达β-arrestin2后,能形成巩固的恐惧记忆并和β-arrestin2的表达量成正相关；并且LA内PKA活性在联合性训练诱导下明显升高。结果提示KO小鼠LA脑区表达β-arrestin参与并促进恐惧条件记忆巩固的形成。1)β-arrestin2缺失导致物体辨别记忆的再巩固受损。当已经巩固的物体辨别记忆被再次激活后野生型小鼠能对物体形成稳定的长时程记忆,而β-arrestin2KO小鼠记忆再激活后对物体的短时程记忆未见明显影响,但其长时程记忆受到选择性损伤,即β-arrestin2的缺失抑制物体辨别记忆的再巩固。此外,β-arrestin2基因缺失不影响小鼠对物体记忆的巩固。结果提示β-arrestin2是记忆再巩固的形成所必需的。2)β-arrestin1不参与物体辨别记忆再巩固的形成。记忆再激活后β-arrestin1 KO对物体的短时程记忆和长时程记忆均不受影响,即β-arrestin1缺失的小鼠没有表现出明显的物体辨别记忆再巩固的损伤。3)β-肾上腺素受体参与物体辨别记忆再巩固的形成。p-肾上腺素受体(p-AR)的阻断不影响记忆再激活后短时程记忆,但显著抑制记忆再激活后长时程记忆的形成。其中β1-AR的激活在物体辨别记忆再巩固的形成中发挥了主要作用。结果提示β-AR的激活参与物体辨别记忆的再巩固。4)物体辨别记忆再激活导致内嗅皮层EKR的激活是p-肾上腺素受体／β-arrestin2依赖的。首先记忆再激活能诱导野生型小鼠内嗅皮层内侧区ERK活性增高,而侧脑室给予ERK的抑制剂能明显抑制记忆再巩固的形成。其次p-AR抑制剂能显著抑制记忆再激活所诱导的内嗅皮层内侧区ERK的活化。此外,记忆再激活后β-arrestin2 KO小鼠内嗅皮层ERK未见显著激活,而β-arrestin2 KO小鼠在p-AR激动剂异丙肾上腺素(iso)的诱导下内嗅皮层ERK也未见明显激活。结果提示β-AR/β-arrestin2依赖的ERK激活参与物体辨别记忆再巩固的形成。5)内嗅皮层内侧区β-arrestin2的表达能促进物体辨别记忆的再巩固。采用腺病毒立体定位微量注射的方法,在KO小鼠内嗅皮层内侧区表达β-arrestin2。行为学结果显示β-arrestin2 KO小鼠内嗅皮层内侧区表达β-arrestin2促进小鼠对物体辨别记忆再巩固的形成,p-AR抑制剂能显著抑制内嗅皮层表达β-arrestin2的KO小鼠对物体辨别记忆的再巩固。结果提示基因敲除小鼠内嗅皮层内侧区表达β-arrestin2能恢复物体辨别记忆的再巩固。综上所述,本研究在神经系统水平研究了β-arrestin在记忆不同阶段中的作用和涉及的分子机制,发现了β-arrestin在记忆巩固和再巩固中的新功能。本研究首先揭示了β-arrestin2通过调控杏仁核内PKA的活性参与恐惧条件记忆巩固的形成其次发现物体辨别记忆再巩固的形成依赖于内嗅皮层内侧区β-肾上腺素能受体(?)β-arrestin2介导的ERK信号通路的激活。我们的结果首次在整体水平证明了β-arrestin在β-肾上腺素能受体的激活下调控PKA和ERK活性可能发挥的生理功能,为记忆巩固及再巩固过程中可能涉及的脑区环路和信号通路提供了新的作用机制和理论依据,并为临床治疗健忘症和创伤后应激综合症等疾病提供新的线索和治疗依据。更多还原

【Abstract】 Theβ-arrestins are expressing ubiquitously in mammals, includingβ-arrestin 1 andβ-arrestin 2 respectively, which were originally described as molecules that desensitize the seven transmembrane spanning receptors (7TMR) in conjunction with G protein coupled receptor kinases.β-arrestins bind with the receptor, which blocks their interaction with heterotrimeric G proteins. In this way, the G protein dependent signal pathways activation by the activation of 7TMR is terminated. For example, deletion ofβ-arrestin 2 can enhance morphine-induced analgesia and reward in mice by desensitizingμopioid receptor. Recently some research have establisded the new roles ofβ-arrestin by serving as multifunctional adaptors/scaffolding proteins to promote 7TMR trafficking and signaling. By scaffolding the kinases,β-arrestins bring the activited kinases to the target subcellular localization under the control of the receptor. With scaffolds compartmentalized signalling cascades,β-arrestins regulate the biological effects of the kinases by bringing them into close proximity with their ultimate targets. For example, with the activation of opioid receptor,β-arrestin translocates to nuclear and recruits p300 to p27 and c-fos promoter regions. Under the activation ofβ-adrenergic receptors in the plasma membrane,β-arrestin recruited PDEs to limit activation of membrane associated cAMP-activated protein kinase through receptor desensitization and increasing the rate of its degradation at the membrane. Meanwhile, through recruiting the kinases and changing their activity,β-arrestin transfer the signals from receptors to the target molecues. For example, with the activation ofβ-adrenergic receptor,β-arrestin also function as mitogen activated protein kinase (MAPK) scaffolds, bringing together three components of MAPK signaling modules and inducingβ-arrestin dependently sustained ERK activation. However most studies are still limited to the in vitro results and the physiological function ofβ-arrestin dependent signal pathways are unclear.β-arrestin is abundant in the central nerves system, such as cortex, hippocampus, extended amygdala and hypothalamus, while the central functions have not been extensively studied. Based on the recent investigation aboutβ-arrestin, this study tries to explore the physiological and patholoical role ofβ-arrestins in central nerves system. Combined with molecular biological methods, the behaviors of learning and memory were performed in theβ-arrestin wild type and knockout mice to reveal the potential role ofβ-arrestin in learning and memory. Our results showed that:1)β-arrestin 2 deletion impaires consolidation of conditioned fear memory in mice. With one pair of a tone and a foot shock, theβ-arrestin 2 KO mice was impaired in conditioned fear memory retention one hour or one day after training. What’s more, with 5 pairs intense training, the KO mice showed the decrease learning ability and impaired fear conditioned memory retention one day after training. The above results indicated thatβ-arrestin 2 is involved in consolidation of fear conditioning.2) PKA activity change isβ-arrestin 2 dependent in amygdala during the consolidation of fear conditioning. One pair training increased the PKA activity in membrane of WT mice amygdala, but had no effect on that ofβ-arrestin 2 KO mice. Mean while, the PDE4 selective inhibitor could decrease the PKA activity in WT mice amygdala but not inβ-arrestin 2 KO mice. AC activator forskolin could enhance the PKA activity in amygdala of WT mice but could not increase PKA activity further after paired training. The results suggest thatβ-arrestin 2 dependent regulation of PKA activity takes part in the formation of fear conditioned memory.3)β-arrestin 2 expressing in lateral amygdala (LA) restored fear conditioned memory. With microinjection the adenovirus expressingβ-arrestin 2 was delivered into the bilateral amygdala, which restored the impaired fear conditioned memory and the regulation of PKA activity. The results indicated that theβ-arrestin 2 in LA is necessary of the consolidation of fear conditioning.1)β-arrestin 2 is required for memory reconsolidation of object recognition. With the extensive training of two object recognition, theβ-arrestin 2 KO mice did not show the deficiency in memory consolidation. While one day after the re-exposure of orignal two object, the long-term memory was impaired inβ-arrestin 2 KO mice, while the short-term memory was intact after retrieval in the KO mice. At the same time,β-arrestin 2 KO mice also showed the impaired reconsolidation of spatial memory with Morris water maze test. The results show that deficiency ofβ-arrestin 2 leads to the failure the reconsolidation of object recognition.2)β-arrestin 1 is not necessary for memory reconsolidation of object recogniton. Theβ-arrestin 1 KO mice showed intact memory reconsolidation in object recognition.3)β-adrenergic receptor (β-AR) activation participates in memory reconsolidation of object recognition. To testify the effect ofβ-adrenergic receptor, the upstream signals of P-arrestin 2, on reconsolidation of recognition retention, propranolol, theβ-adrenergic receptor antagonist, was introduced in our study. With administration right after retrieval,β-AR, especiallyβ1-AR inhition disrupted reconsolidation expression of WT mice.4)β-AR activation andβ-arrestin 2 are crucial for ERK activation in entorhinal cortex post retrieval. ERK inhibition with U0126 impaired the reconsolidation of object recognition. Meanwhile, the level of ERK phosphorylation greatly increased after retrieval, which could be inhibited by the performation ofβ-AR antagonist andβ-arrestin 2 deficiency. Additionally, the activation of ERK could not be induced byβ-AR agonist inβ-arrestin 2 KO mice.5) Local Expression ofβ-arrestin 2 in ECM restores the impaired reconsolidation in KO mice. The adenovirus expressing green fluorescent protein (GFP) andβ-arrestin 2 were introduced in ECM via a microcannula. The percentages of ECM neurons expressing GFP in KO mice were 23.15±5.7%. The memory retention test showed that KO mice with the expression ofβ-arrestin 2 restored memory reconsolidation, which could be suppressed byβ-AR antagonist.Taken together, the different roles and mechanisms ofβ-arrestin in learning and memory were systematically studied in this research. In the present study, we explored the novel function ofβ-arrestin in memory consolidation and reconsolidation. Firstly, we find thatβ-arrestin 2 is crucially involved in consolidation of fear conditioning through the regulation of PKA activity; Secondly, we show thatβ-arrestin 2/β-adrenergic receptors dependent activation of ERK signal especially in the medial entorhinal cortex is critical for the reconsolidation of object recognition. Ultimately, the understanding of molecular and anatomical mechanisms of memory consolidation and reconsolidation will contribute to the theoretical discussion in ongoing memory maintenance and its relation to other memory processes.更多还原