多巴胺受体信号通路对前皮质神经元树突重塑的调控

发布时间：2018-04-19 15:23

本文选题：多巴胺 + 树突重塑　；参考：《南方医科大学》2010年硕士论文

【摘要】： 吸毒是全世界广泛关注的问题,毒品的泛滥直接危害人民的身心健康,并给经济发展和社会进步带来巨大威胁。吸毒最大的危害在于其成瘾性。毒品成瘾(drug addiction)指不择手段、不计后果地强制性获取、使用某种毒品。毒品成瘾一旦形成,即可能成为一种终身性状态,成瘾者不择手段地获取毒品,具有极高的复发性,甚至在戒断多年后仍有可能复发,造成很严重的社会问题。毒品成瘾的形成是一个循序渐进的过程,毒品成瘾过程中,机体在分子、细胞水平发生代偿性变化,这些包括基因表达和神经元形态上的改变,会影响神经元功能和神经通路,进而导致行为学上的异常。可卡因成瘾的过程中,大脑多个部位的结构和功能发生改变,其中重要的是神经元树突发生了重塑(dendrite remodeling),这是可卡因(cocaine)成瘾发生的结构基础。许多研究报道多巴胺信号通路在可卡因介导的神经元树突重塑中发挥重要的调节作用,并且有研究发现发现D1和D3多巴胺受体对下游的长期诱导的靶基因Neogenin和SynaptotagminⅦ起到相反的调节作用；这些基因的长期变化,参与神经元可塑性的形成,并进一步参与毒品给药后的行为学变化。在典型的神经元中,来自胞体的初级树突重复分支建立起一个有特色的树突树(dendritic tree),树突分支会生出微小的突起,称之为树突棘(spine),它是大多数兴奋性突触接受信息的位点,而相应的抑制性突触主要分布在树突树的主干上,树突结构的改变会导致突触信息的变化。Robinson等报道重复施用可卡因会造成纹状体伏核区(nucleus accumbens, NAc)中度棘神经元(medium spiny neuron,MSN)树突增长、分支增加和树突棘的密度增加,而且这种形态变化会在停药后至少维持一个月。结构的改变和学习、长时程增强均有密切联系。结构的改变会带来神经环路的改变、影响神经递质的传递,更重要的是带来突触组织形式的改变。多种信号分子参与了神经元的可塑性变化,多巴胺受体(dopamine receptor)、NMDA (N-methyl-D-aspartate, NMDA)受体、ERK(extracell-ular signal-regulated kinase, ERK)以及Rho蛋白家族等等都可能在其中有重要的调节作用,但是它们如何调节可卡因介导的树突重构的分子机制尚存在很多值得我们研究的地方。我们的策略是通过特异性阻断或者特异性激活上述信号通路,以探究该信号通路是否参与多巴胺诱导的神经元树突重构以及其可能的下游信号分子,进而揭示可卡因成瘾过程中多巴胺信号通路诱导的神经元树突发生重塑可能的分子机制。本课题首先构建了Rho蛋白家族中两个亚型Rac1和RhoA的显性负效突变体(Rac1N17和RhoAN19)和组成型活性突变体(Rac1L61和RhoAL63)的慢病毒,通过感染原代培养的前皮质神经元细胞(Profrontal Cortex Neurons, PFC)验证Rac1和RhoA-GTPasess的生物学活性；进而,在原代培养的前皮质神经元细胞中重复给与多巴胺,在最后一次刺激4d后,固定细胞做免疫荧光,结果显示：多巴胺重复刺激下前皮质区域椎体神经元细胞树突分支总数和树突棘数目增多,神经突触密度增加。进一步,我们采用D1和D3受体的抑制剂与激动剂、Rac1和RhoA的显性负效突变体和组成型活性突变体的慢病毒及Rac1和RhoA的抑制剂,通过特异性的阻断或者激活相应的信号分子,以探究该信号分子是否参与多巴胺诱导的神经元树突重构以及在树突重构中的作用。本研究主要结果如下： 1.构建了Rho家族重组质粒Plenti6/v5-Rac1N17, Plenti6/v5-Rac1L61, Plenti6/v5-RhoAL63, Plenti6/v5-RhoAN19,并且包装制备了相应的慢病毒,感染原代培养的前皮质神经元细胞后,采用G-LISATM系统对慢病毒Plenti6/v5-Rac1N17,Plenti6/v5-Rac1L61, Plenti6/v5-RhoAN19, Plenti6/v5-RhoA-L63进行了功能活性鉴定,经表皮生长因子(Epidermal Growth Factor, EGF)(Rac1-GTPase测定)刺激或者溶血磷脂酸(Lysophosphatidic acid, LPS)(RhoA-GTPase测定)刺激2 min后收集细胞检测相应蛋白活性,结果显示：与Plenti6/v5-EGFP组相比,Plenti6/v5-Rac1N17组Rac1活性降低了29.3%,而Plenti6/v5-Rac1L61组Racl活性则增高了2.3倍；与Plenti6/v5-EGFP组相比,Plenti6/v5-RhoA1N19组RhoA活性降低了42.8%,而Plenti6/v5-RhoA1L63组RhoA活性则增高了1.7倍；结果都具有显著的统计学差异,提示构建的慢病毒具备了特定的生物学活性。 2.建立了多巴胺重复刺激前皮质神经元细胞模型：在原代培养前皮质神经元细胞的第11d,13d,15d应用1μmol/L多巴胺重复刺激。并应用该模型探讨了多巴胺重复刺激对于神经元细胞的树突分支数目、树突棘数目和神经突触密度的影响。结果显示：多巴胺重复刺激后神经元细胞的树突分支数目和树突棘数目增多、神经突触密度增加,其结果与PBS对照组相比差异具有统计学意义。 3.应用D1多巴胺受体抑制剂SCH23390(10μmol/L)、D1多巴胺受体激动剂SKF81297(1μmol/L)和D3多巴胺受体抑制剂NGB2904(50μmol/L)以探究D1多巴胺受体和D3多巴胺受体在调节PFC细胞重塑中的作用。在每次加多巴胺刺激前5 min加SCH23390,10 min加NGB2904,制备多巴胺重复刺激模型；同时单独应用D1多巴胺受体激动剂SKF81297刺激15 min,固定细胞做免疫荧光,结果显示：在前皮质区域先应用D1多巴胺受体抑制剂SCH23390后,多巴胺重复刺激引起的神经元形态结构的改变会被显著抑制,即：神经元细胞树突数目和树突棘数目减少,神经突触密度降低；而SKF81297刺激后神经元树突数目、树突棘数目、神经突触密度都显著增加,与多巴胺重复刺激类似,提示D1多巴胺受体在多巴胺重复刺激诱导神经元树突重构中发挥正性调控作用。而先应用D3多巴胺受体抑制剂NGB2904后,神经元细胞树突数目和树突棘数目相比单纯的多巴胺组有所增高,差异具有统计学意义,提示D3多巴胺受体在神经元树突重塑中发挥负性的调控作用,即D1多巴胺受体和D3多巴胺受体在神经元树突重塑中发挥重要的调控作用,并且两者的调控作用是相反的。 4.应用Racl和RhoA的显性负效突变体的慢病毒在前皮质神经元细胞培养的第7d感染细胞,次日换去全液,同样给与多巴胺重复刺激,探讨Rac1和RhoA蛋白是否参与了慢性多巴胺刺激诱导的神经元树突重塑以及其所发挥的作用。固定细胞做免疫荧光,结果显示：在前皮质区域,感染Rac1N17显著抑制了多巴胺重复刺激引起的神经元树突分支数目和树突棘数目的增多以及突触密度的增加,差异具有统计学意义,这提示Rac1在多巴胺刺激诱导的神经元树突重构过程中发挥重要的正性调控作用；相反感染RhoAN19慢病毒后增强了多巴胺刺激引起的神经元树突分支数目、树突棘数目和突触密度的增加,差异具有统计学意义,提示RhoA在多巴胺介导的神经元树突重塑中发挥负性调控作用。 5.同样应用Racl的抑制剂NSC23766以及RhoA的抑制剂Y27632以探究Racl和RhoA是否参与了多巴胺重复刺激诱导的神经元树突重塑。该组前皮质细胞在每次加多巴胺刺激前3 min加NSC23766(100μmol/L)或者Y27632(100μmol/L)。细胞固定后进行免疫荧光染色,结果显示：在前皮质区域,NSC23766显著逆转多巴胺重复刺激引起的树突分支数目、树突棘数目增多和神经突触密度增加,这与病毒感染的结果相同,差异具有统计学意义；而Y27632组与多巴胺组比较则尚无统计学差异。通过上述研究,我们得出以下结论：一、成功构建了Rac1和RhoA蛋白的显性负效突变体和组成型活性突变体的慢病毒,并验证了相应的生物学活性,为下一步研究奠定了基础。二、发现多巴胺重复刺激介导神经元细胞树突分支数目、树突棘数目增多和神经突触数目增加。三、多巴胺诱导的树突重塑过程中,D1、D3多巴胺受体发挥了相反的调控作用,刺激多巴胺受体诱导的神经元树突增长、分支增加和树突棘密度增加主要是通过D1多巴胺受体,而D3多巴胺受体主要起抑制作用。四、多巴胺诱导的树突重塑过程中,Rac1和RhoA蛋白参与介导神经元树突重塑,并且Rac1促进树突分支数目、树突棘数目的增多以及神经突触密度的增加,而RhoA则发挥了相反的调节作用。综上所述,本研究成功构建了Rho蛋白家族中Rac1和RhoA的显性负效突变体和组成型活性突变体,制备了多巴胺重复刺激细胞模型,采用一系列特异性抑制剂、激动剂及突变体,通过阻断多巴胺受体通路、Rho家族信号通路,探究多巴胺诱导的神经元树突重构可能的分子机制。结果表明,D1、D3多巴胺受体和Rac1、RhoA信号通路均参与了多巴胺诱导的神经元树突重塑,并且D1多巴胺受体和Rac1蛋白发挥正性调节作用,而D3多巴胺受体和RhoA蛋白则为负性调节,这提示多巴胺诱导的神经元树突重构是多种信号分子共同作用的结果。上述研究对于我们进一步揭示多巴胺诱导的神经元树突重塑分子机制具有重要的意义,同时也为其临床治疗提供了诸多启示。
[Abstract]:Drug abuse is a widespread concern in the world. The flood of drugs directly endangers the people's physical and mental health and poses a great threat to economic development and social progress. The greatest harm of drug addiction is its addiction. Drug addiction (drug addiction) refers to unscrupulous means, unreckless and strong acquisition, and the use of some kind of drug. Once the drug addiction is formed It can become a life-long state. The addicts obtain drugs by means of unscrupulous means of relapse. Even after many years of abstinence, it is possible to relapse and cause serious social problems. The formation of drug addiction is a gradual process. In the course of drug addiction, the body has compensatory changes at the molecular and cell levels in the drug addiction process. These include gene expression and changes in neuronal morphology, which can affect neuronal function and neural pathways, leading to behavioral abnormalities.
In the process of cocaine addiction, the structure and function of multiple parts of the brain are altered, and it is important that the neuron dendrites reshape (dendrite remodeling), which is the structural basis for the occurrence of cocaine (cocaine) addiction. Many studies have reported that dopamine signaling plays an important role in cocaine mediated remolding of neurons. We have found that the D1 and D3 dopamine receptors play the opposite role in the long-term induced target gene Neogenin and Synaptotagmin VII; the long-term changes in these genes participate in the formation of neuronal plasticity and further participate in the behavioral changes after drug delivery. In typical neurons, The primary dendrite repeating branch of the cell body builds a distinctive dendritic tree (dendritic tree). The branch of the dendrite produces a tiny protuberance called the dendritic spines (spine), which is the site for most excitatory synapses to receive information, and the corresponding inhibitory synapses are mainly distributed on the trunk of the tree, and the changes in the dendrite structure will lead to a change in the dendrite structure. Changes in synaptic information.Robinson and other reports that repeated use of cocaine can cause the growth of the dendritic neurons (medium spiny neuron, MSN) in the nucleus accumbens (nucleus accumbens, NAc), the increase of the branch and the density of the dendritic spines, and this morphological change will last for at least one month after the withdrawal of the drug. Structural changes and learning, long time The changes in the structure will bring about the changes in the neural circuits, affect the transmission of neurotransmitters, and more importantly, bring about changes in the form of synapses.
Many signal molecules are involved in the plasticity of neurons. The dopamine receptor (dopamine receptor), the NMDA (N-methyl-D-aspartate, NMDA) receptor, ERK (extracell-ular signal-regulated kinase, ERK), and the Rho protein family all have important regulatory effects, but how they regulate the weight of cocaine mediated dendrites Our strategy is to explore whether the signaling pathway participates in the dopamine induced neuronal dendrite remodeling and its possible downstream signal molecules by specific blocking or specific activation of the signal pathway, thus revealing the dopamine in the process of cocaine addiction. Signaling pathway induces neuronal remodeling in dendritic cells.
We first constructed the lentivirus of the dominant negative effects mutants (Rac1N17 and RhoAN19) and the constituent active mutants (Rac1L61 and RhoAL63) of two subtypes of the Rho protein family (Rac1N17 and RhoAN19) and the component type active mutants (Rac1L61 and RhoAL63), and verified the biological activity of the Rac1 and it through the primary cultured precultured cortical neurons (Profrontal Cortex Neurons, PFC). Furthermore, dopamine was repeated in the primary cultured cortical neuron cells. After the last stimulation of 4D, the fixed cells were immunofluorescent. The results showed that the number of dendritic branches and the number of dendritic spines in the vertebral neuron cells increased and the density of synapses increased in the anterior cortex of the anterior cortex. Further, we used D1. D3 receptor inhibitors and agonists, Rac1 and RhoA dominant negative effects mutants and constituent active mutants of lentivirus and Rac1 and RhoA inhibitors, through specific blocking or activation of the corresponding signal molecules to explore whether the signal molecules are involved in dopamine induced deity dendrites reconstruction and the reconfiguration of dendrites The main results of this study are as follows:
1. Rho family recombinant plasmids Plenti6/v5-Rac1N17, Plenti6/v5-Rac1L61, Plenti6/v5-RhoAL63, Plenti6/v5-RhoAN19 were constructed, and the corresponding lentivirus was packaged and infected with the primary cultured cortical neurons, and G-LISATM system was used for the lentivirus Plenti6/ v5-Rac1N17, Plenti6/v5-Rac1L61, Plenti6/v5-RhoAN19, Plenti6/. V5-RhoA-L63 performed functional activity identification, stimulated by epidermal growth factor (Epidermal Growth Factor, EGF) (Rac1-GTPase) or lysophosphatidic acid (Lysophosphatidic acid, LPS) (Lysophosphatidic acid, LPS) (RhoA-GTPase assay) stimulated 2 min cells to collect cells to detect the corresponding protein activity. The activity of AC1 was reduced by 29.3% and the activity of Racl in group Plenti6/v5-Rac1L61 increased by 2.3 times. Compared with the Plenti6/v5-EGFP group, the RhoA activity of Plenti6/v5-RhoA1N19 group decreased by 42.8% and the RhoA activity of the Plenti6/v5-RhoA1L63 group increased by 1.7 times; the results all had significant statistical differences, suggesting that the constructed lentivirus had a specific organism. Learning activity.
2. the model of cortical neurons before dopamine repeated stimulation was established: 11d, 13D, and 15d of cortical neurons before primary culture were repeated with 1 micron mol/L dopamine, and the effects of dopamine repeated stimulation on the number of dendritic branches, the number of dendritic spines and the synaptic density were investigated. The results showed that the number of dendritic branches and the number of dendritic spines increased and the synapse density increased after dopamine repeated stimulation, and the difference was statistically significant compared with the PBS control group.
3. the use of D1 dopamine receptor inhibitor SCH23390 (10 micron mol/L), D1 dopamine receptor agonist SKF81297 (1 micron mol/L) and D3 dopamine receptor inhibitor NGB2904 (50 micron) to explore the role of D1 dopamine receptor and D3 dopamine receptor in regulating PFC cell remodeling. 5 doses of dopamine were added before each dopamine stimulation. The D1 dopamine receptor agonist SKF81297 stimulated 15 min and the immobilized cells were immunofluorescent. The results showed that the changes in the morphological structure of neurons caused by dopamine repeated stimulation were significantly inhibited in the precortical region by using the D1 dopamine receptor inhibitor SCH233 90, that is, neurons. The number of dendritic spines and the number of dendritic spines decreased, and the density of synapses decreased, while the number of dendrites, the number of dendritic spines and the synapse density increased significantly after SKF81297 stimulation, similar to the repetitive dopamine stimulation, suggesting that D1 dopamine receptors play a positive role in the reconstruction of neuron dendrites induced by repetitive dopamine stimulation. After using the D3 dopamine receptor inhibitor NGB2904, the number of dendritic cells and the number of dendritic spines increased in comparison with the number of dendritic spines. The difference was statistically significant, suggesting that D3 dopamine receptors play a negative role in the remolding of neuron dendrites, namely, D1 dopamine receptor and D3 dopamine receptor in neuron dendrite weight. Plastic plays an important regulatory role, and the regulation function of the two is the opposite.
4. using the dominant negative effect mutant of Racl and RhoA in the 7d infected cells cultured in the precortical neuron cells, the following day was changed to the full liquid, and the same was given to the dopamine repeated stimulation. It was discussed whether the Rac1 and RhoA proteins were involved in the dendrite remolding induced by chronic dopamine stimulation and the role it played. The results showed that in the anterior cortex, infection of Rac1N17 significantly inhibited the number of dendritic branches and the number of dendritic spines caused by dopamine repeated stimulation and the increase of the density of synapses. The difference was statistically significant, suggesting that Rac1 plays an important role in the process of dendritic reconstruction induced by dopamine. Positive regulation, and the number of dendritic branches caused by dopamine stimulation, the number of dendritic spines and the increase of synapse density, and the difference between the number of dendritic spines and the density of synapse were increased after RhoAN19 infection, suggesting that RhoA plays a negative role in the remolding of dopamine mediated dendrites.
5. the same Racl inhibitor NSC23766 and the inhibitor Y27632 of RhoA were used to explore whether Racl and RhoA were involved in the remolding of neuron dendrites induced by dopamine repetitive stimulation. In this group, the anterior cortical cells were 3 min plus NSC23766 (100 u mol/L) or Y27632 (100 mu mol/L) before each dopamine stimulation. Immunofluorescence staining was performed after the cells were fixed. The results showed that in the anterior cortex, NSC23766 significantly reversed the number of dendritic branches caused by dopamine repetitive stimulation, the increase of dendritic spines and the increase of synapse density, which was the same as the results of virus infection, and the difference was statistically significant, but there was no statistical difference between the Y27632 group and the dopamine group.
Through the above studies, we draw the following conclusions: first, the dominant negative and constituent active mutants of Rac1 and RhoA proteins were successfully constructed, and the corresponding biological activity was verified. Two, the number of dendritic branches, dendritic spines, and the number of dendritic spines were found. Increase in number and number of synapses increased. Three, during the remolding process of dopamine induced dendrites, D1, D3 dopamine receptors play an opposite role in stimulating the growth of neuron dendrites induced by dopamine receptors, and the increase in the density of branches and dendrites is mainly through the D1 dopamine receptor, while D3 dopamine receptors mainly inhibit the dendrites. Four, during the remolding process of dopamine induced dendrites, Rac1 and RhoA proteins are involved in mediating the remolding of neuron dendrites, and Rac1 promotes the number of dendritic branches, the number of dendritic spines and the increase of neural synapse density, while RhoA plays the opposite role in regulating the dendrite.
To sum up, this study successfully constructed the dominant negative and active mutants of Rac1 and RhoA in the Rho protein family, prepared a dopamine repetitive stimulation cell model, and used a series of specific inhibitors, agonists and mutants to explore dopamine induced by blocking the dopamine receptor pathway, the Rho family signal pathway. The possible molecular mechanism of neuron dendrite reconstruction shows that D1, D3 dopamine receptors and Rac1, RhoA signaling pathways are involved in dopamine induced dendritic remodeling, and D1 dopamine receptors and Rac1 proteins play a positive regulatory role, while D3 dopamine receptors and RhoA egg white are negatively regulated, which suggests dopamine induced nerves. The reconfiguration of the dendrites is the result of the interaction of various signal molecules. The above study is of great significance for us to further reveal the molecular mechanism of dopamine induced remolding of neuron dendrites, and also provides a great deal of inspiration for its clinical treatment.

【学位授予单位】：南方医科大学
【学位级别】：硕士
【学位授予年份】：2010
【分类号】：R346

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