骨骼锰蓄积与血清Aβ、认知功能的关系及锰致AD的机制研究

Our previous study found manganese(Mn) cumulated in bone, which related with Mn level in brain, and overexposure of Mn was also related with mitochondrion impairment in brain. Recent literature data showed that mitochondrion impairment in astrocyte might be associated with β-amyloid cumulation, which may lead to the irreversible and progressive cognitive impairment and the ultimate Alzheimer’s disease (AD). Thus, there is an urgent need to establish the relationship between AD and Mn exposure in human populations. However, probably because of no available marker of manganese accumulation, such a human-based study is nearly nonexistent. The purpose of this collaborative research is to assess the relationship between Mn exposure and potential biomarkers for AD among Mn-exposed populations. In the process, we will use a newly developed, highly sensitive neutron activation analysis (NAA) technology to noninvasively quantify Mn concentrations in bones of this study cohort. We will also assess the changes in neurobehavioral outcomes and serum levels of Aβ believed to be involved in AD pathogenesis, hoping that Mn levels in bone (BnMn) can be used as an effective novel biomarker for pre-symptomic predication of early neurodegeneration. Our mechanistic approach by in vivo and in vitro experiments will lead to the discovery of whether BnMn contributes to Mn accumulation in brain, particularly in astrocytes, and how this may disturb astrosytes-mediated generation and clearance of Aβ implicated in the pathoetiology of AD. The research, by combining the state-of-the-art technology only available to Zunyi Medical College, human across-sectional epidemiological study, and mechanistic investigation, will likely, for the first time in literature, provide the evidence to support a role of Mn in AD, and therefore bear significant public importance for prevention and treatment of environmental exposure-caused neurodegenerative diseases.

课题组早期研究发现体内锰主要蓄积在骨骼并与脑组织锰水平相关，并证实锰可损伤脑细胞线粒体；而最新研究提示星形胶质细胞线粒体受损可能使脑组织中β-淀粉样肽(Aβ)沉积导致认知损害甚至阿尔兹海默综合征（AD）。另一方面，尽管动物实验表明锰可导致AD，但由于缺乏理想的人体锰过量蓄积的标志物，过量锰促发AD缺乏人群证据。为了评价人群锰蓄积与AD的关系，本研究拟采用中子活化分析（NAA）技术检测锰暴露人群的骨锰含量，观察骨锰水平与认知功能及血清Aβ的关系，评价骨锰是否可用作锰所导致的AD的早期标志物；并进一步通过转基因AD模型小鼠染锰寻找锰致AD的生物学证据，通过动物实验和体外实验查证锰蓄积对Aβ产生与排出，尤其是对星形胶质细胞内化与降解Aβ能力的影响以阐述锰致AD的可能机制。本研究首次使用骨锰作为标志物，结合人群调查及体内外的机制研究，有望首次阐明锰蓄积在AD发病中的角色，为AD的预防提供参考依据。

阿尔茨海默病(Alzheimer’s disease, AD)的发生是否与锰在体内的过量蓄积有关？骨骼锰能否反映脑组织锰的蓄积水平，并与AD样的认知损伤相关？过量锰导致AD样损伤的机制是什么？为了回答以上问题，我们分别采用SD大鼠、APP/PS1双重转基因小鼠染锰实验和N2a细胞染锰实验进行研究。.通过SD大鼠的慢性染锰和脱锰实验，我们观察到锰暴露3月后，全血、脑脊液、海马、皮质、纹状体、股骨锰含量均显著升高（P＜0.05），浓度梯度是股骨>纹状体>海马>皮质>全血>脑脊液；脱锰3月后，染锰组动物除皮质外，以上各组织锰含量仍高于对照组（P＜0.05），锰处理组各组织锰浓度均有不同程度降低，其中股骨锰相对降幅最高（346.55%），依次是全血锰（132.18%）、脑脊液锰（40.17%）、海马锰（23.08%）、皮质锰（22.06%）、纹状体锰（11.34%）。. SD大鼠慢性染锰实验还发现，染锰9月，高锰组逃避潜伏期高于对照组（P<0.05）；染锰12月，高锰组和低锰组逃避潜伏期均高于对照组（P<0.05），高锰组穿越平台次数低于对照组（P<0.05）；染锰APP/PS1双重转基因小鼠出现类似的学习认知改变。 . SD大鼠和APP/PS1双重转基因小鼠染锰后，均观察到血清、脑脊液、海马、皮质中的Aβ1-40、Aβ1-42明显升高，差异均有统计学意义（P<0.05）。APP/PS1小鼠锰暴露组海马LC3Ⅱ/LC3Ⅰ值降低，Beclin1蛋白降低，P62蛋白升高，差异均有统计学意义（均P＜0.05）；细胞实验也表明染锰抑制N2a细胞的自噬功能。此外，染锰组SD大鼠全血LRP1 mRNA、血浆sLRP1均下调（P<0.05）肝脏GOT，GPT升高。. 本项目证实了骨锰与环境锰蓄积之间的关联性，查明骨锰可能是反映锰在体内蓄积和持续性释放的重要器官，扮演“锰池”的作用，并发现过量锰导致“AD样”改变的原因可能是因为锰会导致血清、大脑组织中的Aβ显著升高，加剧了AD病理进程；进一步的研究发现，锰致大脑组织的Aβ蓄积可能与锰导致脑组织的自噬功能障碍有关，并可能与导致LRP1介导的肝脏对Aβ的降解清除障碍有关。本项目为进一步研究锰致AD的分子机制提供了重要的前期数据。