【作者】 孟冠敏；

【导师】 徐立红；

【作者基本信息】 浙江大学 ， 生物化学与分子生物学， 2011， 博士

【摘要】 水体环境中的藻类大量繁殖已成为世界性的生态问题,除了引发水华造成水体富营养化污染外,还释放大量的藻类毒素对人类和其它生物的健康构成威胁。在蓝绿藻属产生的毒素中,微囊藻毒素(Microcystin, MC)是人类日常生活中接触最多的一类由80多种同类物组成的单环七肽毒素,其中毒性最强的、研究最多的是微囊藻毒素-LR (Microcystin-LR, MC-LR)。MC-LR一般被认为是一种肝毒素,因为MC-LR主要靶向富集于肝脏并引起严重的肝脏毒性效应,包括急性肝出血、肝坏死和并与人类原发性肝癌相关。最近的研究发现,除了肝组织,MC-LR还可以透过血脑屏障和血液-脑脊液屏障而出现于脑组织中,由此推断微囊藻毒素可能还具有神经毒性。越来越多的研究提出微囊藻毒素的潜在神经毒性,然而MC-LR如何发挥神经毒性作用及其内在分子机制尚待揭示。为探索MC-LR的神经毒性作用,我们选用了常被作为神经元细胞模型的高分化型的PC12细胞,首先研究MC-LR对PC12细胞的一系列细胞学效应,包括MC-LR对细胞存活率、细胞凋亡、细胞骨架和氧化应激状态的影响；探讨MC-LR在PC12细胞中如何影响其特异作用靶蛋白PP2A的酶活性、构成PP2A全酶的各亚基的蛋白表达水平；进一步研究了与细胞骨架相关的蛋白tau和HSP27的磷酸化状态,并从信号转导的角度探索该过程中可能的分子机制,从而为进一步阐明MC-LR神经毒性的机制以及提出有效的预防和治疗措施提供新的依据和思路。主要结果：1.在本实验条件下,MC-LR不影响PC12细胞的存活率。2.在本实验条件下,MC-LR不引起PC12细胞发生凋亡。3.在本实验条件下,MC-LR不影响PC12细胞的形态。4. MC-LR对细胞骨架的影响具体表现为微管和微丝结构的紊乱,以及微管的稳定性下降。此外,MC-LR还使PP2A-Ba亚基与微管的结合下降。5. MC-LR可诱导PC12细胞氧化应激水平的升高。6. MC-LR进入PC12细胞后与PP2A-C亚基结合,抑制了PP2A酶活性,并且影响了PP2A各亚基的表达及PP2A-C的翻译后修饰水平。7. MC-LR诱导了微管相关蛋白tau的过度磷酸化并导致tau与细胞骨架的结合下降,还诱导了微丝相关的调控蛋白HSP27的过度磷酸化。8. MC-LR诱导细胞骨架相关蛋白的过度磷酸化的潜在机制可能包括PP2A活性被抑制和p38-MAPK通路的激活。9.抑制p38-MAPK的激活能抑制MC-LR所诱导的细胞骨架相关蛋白的过度磷酸化和细胞骨架的重构。主要结论：1. MC-LR对PC12细胞的PP2A酶活性的影响可能是多重因素综合作用的结果,包括PP2A的不同组成亚基表达的改变以及PP2A-C亚基的翻译后修饰水平的改变。由此本研究在拓展了文献报道的基础上,进一步揭示了MC-LR对其特异靶蛋白PP2A的复杂的作用方式。2.在本研究条件下MC-LR对PC12细胞产生的毒性主要为诱导氧化应激水平的升高和细胞骨架结构的改变。3. MC-LR诱导了调控细胞骨架结构的组装和功能的细胞骨架相关蛋白tau和HSP27的过度磷酸化,这可能导致了细胞骨架的重构。4. MC-LR诱导的细胞骨架相关蛋白tau和HSP27的过度磷酸化的机制可能是因为PP2A活性受到抑制后其介导的脱磷酸化作用减弱,而激活的p38-MAPK介导的磷酸化作用增强。5. p38-MAPK通路的抑制剂能在抑制tau和HSP27的过度磷酸化的同时,对抗MC-LR对PC12细胞骨架产生的毒性效应,由此本研究还为探索有效对抗MC-LR神经毒性的预防和治疗措施提供了新思路。更多还原

【Abstract】 Contamination of freshwater and marine environments by cyanobacterial blooms has been a worldwide ecological issue, triggering both water eutrophication and health threats to livestock and humans. Among the various cyanobacteria (blue-green algae), Microcystis aeruginosa is the most common genera producing microcystins (MCs), a family of over80structurally monocyclic heptapeptide toxins, with microcystin-LR (MC-LR) considered to be the most toxic and most commonly encountered variant. MC-LR is thought to act as a hepatotoxin, because it preferentially accumulates in the vertebrate liver, and its presence in contaminated drinking water has been frequently reported to cause acute liver hemorrhage, liver necrosis and primary liver cancer. Recent evidence has demonstrated that MC-LR is also brain-permeable as MC-LR is present in the brain, raising the possibility that MC-LR could exert neurotoxicity. Although the potential neurotoxicity of MC-LR has been proposed, little is known about its molecular basis.The neuroendocrine PC12cell line has been widely utilized as a model system for studying various aspects of neuronal function and neurotoxins. The current study was designed to elucidate the neurological toxicity of the cyanobacterial toxin MC-LR on the PC12cell line and to determine its underlying mechanisms. Studies were undertaken with the objective of evaluating (a) the cellular effects including cell viability, apoptotic rate, cytoskeleton morphology including MT and F-actin architectures, and the level of ROS,(b) protein phosphatase2A activity, the protein profile of PP2A composition subunits including A subunit, C subunit, as well as the regulatory B family subunits, Bpand By, and methylation and phosphorylation modification of PP2A-C,(c) the phosphorylation status of cytoskeletal-associated proteins including tau and HSP27, and the roles of PP2A and p38MAPK signaling in MC-LR-induced cytoskeletal structure remodeling. Our work may further our knowledge of MC-LR-induced neurotoxicity and provide some new theoretical bases and ways for the inhibition of MC-LR-induced neurotoxicity.Results:1.In the present study, MC-LR had no effect on the cell viability of PC12cells.2. In the present study, MC-LR had no effect on neither the apoptotic rate of PC12cells, nor the activation of caspase-33. In the present study, MC-LR had no effect on the cell morphology of PC12cells.4. MC-LR triggered microtubule (MT) and actin cytoskeleton rearrangement, as well as MT destabilization. Additionally, MC-LR induced disruption of PP2A Bcc-MT interaction.5. MC-LR induced the elevation of the ROS level.6. MC-LR directly targeted the PP2A catalytic subunit which was followed by a strongly dose-dependent inhibition of PP2A activity, as well as altered composition subunits levels and post-translational modifications of PP2A.7. MC-LR induced hyperphosphorylation of the neural microtubule-associated protein tau, which correlated with a decrease in cytoskeleton-associated tau. Besides, the phosphorylation of the actin-associated protein HSP27was also increased in MC-LR-treated cells.8. Direct inhibition of protein phosphatase2A (PP2A) activity and indirect activation of the p38mitogen-activated protein kinase (MAPK) could be the mechanisms underlying MC-LR induced hyperphosphorylation of tau and HSP27.9. Pretreatment with the p38MAPK inhibitor SB203580effectively abolished hyperphosphorylation of tau and HSP27, and blocked MC-LR-triggered cytoskeletal alterations.Conclusions:1. PP2A inhibition induced by MC-LR may be the result of the combined effects of alterations of various composition subunits levels that suggesting changes in holoenzyme assembly and PP2A-C post-translational modifications,which provides a new insight into the effect pattern of MC-LR on PP2A.2. Our data showed that the level of ROS as well as the cytoskeleton structure responded relatively sensitively to MC-LR in the neuroendocrine PC12cell line.3. MC-LR induced hyperphosphorylation of the neural microtubule-associated protein tau and the actin-associated protein HSP27, which may be a likely mechanistic link to MC-LR-induced cytoskeletal alterations.4. MC-LR-caused hyperphosphorylation of HSP27and tau may be mediated directly by reduced dephosphorylation from PP2A activity impairment and indirectly by elevated phosphorylation via subsequent p38MAPK activation.5. The protective effect of SB203580on MC-LR-induced cytoskeleton remodeling might be via the conversion of the phosphorylation levels of HSP27and tau, which are involved in cytoskeletal structure maintenance. Thus, a therapeutic strategy based on p38MAPK inhibitors may be effective in counteracting the neurotoxicity of MC-LR.更多还原