人类M1 Muscarinic乙酰胆碱受体调控BACE1水平的新机制

发布时间：2018-05-14 05:07

本文选题：阿尔茨海默症 + β分泌酶BACE1　；参考：《厦门大学》2013年博士论文

【摘要】：阿尔茨海默症(Alzheimer's disease, AD)病人的显著病变之一是大脑中的β分泌酶BACE1切割淀粉样前体蛋白APP产生的Aβ所形成的具有神经毒性的淀粉样斑。此外BACE1也参与精神分裂症(Schizophrenia)的发病。由于BACE1活性及水平受到细胞内多种蛋白的调控,因此寻找调控BACE1的蛋白在AD和精神分裂症的治疗中具有重要意义。我们通过酵母双杂交的实验方法筛选出一种可能与BACE1发生相互作用的蛋白—人类M1Muscarinic乙酰胆碱受体(M1Muscarinic Acetylcholine Receptor, M1mAChR,以下简称M1受体),并在酵母中通过β-gal活性证实了该蛋白可以和BACE1在酵母系统中发生相互作用。M1受体密切调控乙酰胆碱能受体的活性,而乙酰胆碱能神经元细胞在阿尔茨海默症中受到明显损害。许多研究提示M1受体可能成为阿尔茨海默症治疗的新靶点,这就要求我们深入理解M1受体在疾病过程中的参与机制。在此,我们进一步在过表达人类Swedish突变APP的HEK293Swedish以及过表达APP695的N2a695等哺乳动物细胞内,通过免疫生物学技术证实了M1受体可以和非成熟形式的BACE1在内质网中发生相互作用。有研究提示,M1受体的激动剂可通过PKC/MEK信号通路增加非淀粉源途径的APP加工过程,降低Aβ的形成,抑制tau蛋白的过度磷酸化,从而缓解与阿尔茨海默症相伴的认知障碍等症状,因此,特异性的M1受体激动剂在治疗AD方而具有潜在的应用前景。另有研究表明M1受体激动剂AF267可以降低AD模型小鼠大脑中的BACE1水平,但机制不明,并且此激动剂可以非特异地激活M型乙酰胆碱受体其他的同家族成员。另据一项研究发现,在M1受体及其同家族成员M3受体的共同激动剂talsaclidine处理下,BACE1的蛋白水平显著升高,该过程依赖于PKC和MAPK信号通路。为了深入理解M1受体调控BACE1的机制,解释以往报道中颇具争议的观察结果,我们从蛋白-蛋白之间相互作用的角度,开展M1受体对BACE1调控新机制的研究,结果发现：(1)在HEK293Swedish细胞中,瞬时转染M1受体可以剂量相关性地降低BACE1的蛋白水平,从而减少APP经BACE1切割的产物Aβ水平,但并不影响BACE1的mRNA水平；(2)用siRNA下调细胞内的M1受体,可观察到BACE1蛋白的累积；(3)过表达M1受体引起的BACE1蛋白水平下降,可以通过蛋白酶体的抑制剂处理得以补偿性恢复,但却不能被溶酶体抑制剂补偿恢复；(4)M1受体的过表达可以引起BACE1泛素化的增加；(5)当使用抑制剂阻断M1受体下游的MAPK、PKC以及PO3K-Akt等信号通路时,并不影响M1受体过表达造成的BACE1的降解；(6)过表达丧失信号转导功能的C端缺失M1受体突变体同样引起BACE1减少；(7)过表达M1受体的HEK-APP Swedish细胞经M1受体激动剂McN-A-343处理后,其M1受体的分布表现出区室化,其BACE1含量进一步降低；(8)过表达BACE1能够引起M1受体蛋白和(?)mRNA的积累。我们的这些结果表明,M1受体可以通过与BACE1的直接相互作用,介导了BACE1蛋白通过蛋白酶体-泛素途径的降解,从而抑制了Ap的生成和积累。而这一过程不依赖于M1受体的信号途径。我们的研究成果拓展了M1受体调控BACE1的机制,为AD以及精神分裂症的治疗和药物开发提供了重要的理论依据。
[Abstract]:One of the significant pathological changes in Alzheimer ' s disease ( AD ) is the neurotoxic amyloid plaques formed by A.beta . produced by the amyloid precursor protein APP in the brain . In addition , BACE1 is involved in the pathogenesis of schizophrenia . Since BACE1 activity and level are regulated by various proteins in the cell , it is important to find the protein that regulates BACE1 in the treatment of AD and schizophrenia .

It is suggested that M1 receptor may be a new target for the treatment of Alzheimer ' s disease . It is suggested that M1 receptor may be a new target in the treatment of Alzheimer ' s disease .
( 2 ) siRNA down - regulate M1 receptor in cells , and the accumulation of BACE1 protein can be observed ;
( 3 ) The level of BACE1 protein caused by overexpression of M1 receptor can be compensated by the inhibitor treatment of proteasome , but it can not be compensated by lysosomal inhibitor ;
( 4 ) overexpression of M1 receptor can increase the ubiquitination of BACE1 ;
( 5 ) It does not affect the degradation of BACE1 caused by overexpression of M1 receptor when using inhibitors to block signaling pathways downstream of M1 receptor .
( 6 ) The deletion of M1 receptor mutant at the C terminus of overexpression loss signal transduction also resulted in a decrease in BACE1 ;
( 7 ) After treated with the M1 receptor agonist McN - A - 343 , the expression of M1 receptor in the HEK - APP Swedish cell with the M1 receptor was increased , and the BACE1 content of M1 receptor decreased further .
( 8 ) The overexpression of BACE1 can cause the accumulation of M1 receptor protein and ( ? ) mRNA . These results show that M1 receptor can mediate the degradation of BACE1 protein through proteasome - ubiquitin pathway by direct interaction with BACE1 . This process does not depend on the signal pathway of M1 receptor . Our research results extend the mechanism of M1 receptor regulation BACE1 , which provides an important theoretical basis for the treatment and drug development of AD and schizophrenia .

【学位授予单位】：厦门大学
【学位级别】：博士
【学位授予年份】：2013
【分类号】：R749.16

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