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题名	甲醛对阿尔兹海默病的损伤作用
作者	Joshua D. Rizak
学位类别	博士
答辩日期	2015
授予单位	中国科学院研究生院
授予地点	北京
导师	马原野、胡新天
关键词	阿尔兹海默病 甲醛 Aβ聚集 毒性
其他题名	The Implications of Formaldehyde in Alzheimer’s Disease
学位专业	神经生物学
中文摘要	阿尔兹海默病（AD）是一种渐进性的神经退行性疾病，是一种与年龄相关的痴呆。这种疾病与基因因素相关，最终导致脑内老年斑和神经纤维缠结的形成。然而，这两者的直接关系至今未知，而这两者的关系对推进有效的临床治疗以及诊断意义重大。本论文探索了代谢物甲醛（一种与年龄的增长相关并且在AD病人中会显著增长的物质）和AD的病理性变化，如老年斑的形成，tau蛋白的高度磷酸化以及认知损伤之间的关系，最终回答甲醛的毒性是否在AD的病程中起重要作用 短期甲醇暴露因为能引起严重的神经系统后遗症最终导致失明或死亡而被人们所认识。但是其长期毒性还未被系统研究。由于甲醛是甲醇的一种代谢物，所以甲醛与AD病理学之间的这种关联为我们研究甲醇长期暴露的影响提供了新的视角。然而，由于这些短期症状常被联系到甲酸上，即甲醇的终极代谢产物，导致在过去人们关于甲醇的长期影响方面的研究既没有联系到甲醛上，也没有被充分重视。 在本论文的第一个研究中，给小鼠口服甲醇从一个新的角度重新研究甲醇的长期毒性。甲醇作为甲醛的来源，被认为和AD的病程有直接关联。三组小鼠（N=9）给予甲醇水溶液（浓度2%或3.8%）或水为对照供动物自由饮用6周。AD痴呆症状行为学上用Y-迷宫和嗅觉记忆实验分别检测空间记忆和嗅觉记忆。之后分别检测小鼠脑内tau蛋白的磷酸化，淀粉样沉淀的形成以及海马CA1区的神经细胞凋亡情况。口服甲醇的小鼠空间记忆和嗅觉记忆都受到损伤；小鼠在Y-迷宫实验中不能识别新异臂，在嗅觉记忆测试中不能识别柠檬气味。小鼠脑内病理学检测的结果显示口服甲醇组小鼠较对照组小鼠脑内tau蛋白磷酸化程度显著升高并且海马CA1区的神经细胞有更多的凋亡（约10%左右的细胞出现了染色体的压缩现象）。值得注意的是，口服甲醇组小鼠并没有淀粉样沉淀的形成。行为学和免疫组化的结果表明口服甲醇的小鼠有部分类似AD的症状，但是小鼠脑内并没有发现淀粉样沉淀。 第二个研究将上述实验扩展到了非人灵长类恒河猴（猕猴）上，即给年轻雄性猕猴长期自由引用3%的甲醇水溶液。空间延迟任务结果显示口服甲醇的猕猴较实验前出现明显的记忆损伤，并且这种损伤在停药后至少能持续6个月。这一现象与脑脊液中T181和S396位点tau蛋白的高度磷酸化一致，也和脑组织中四个主要脑区（前额叶，顶叶，颞叶，海马）中tau蛋白这两个位点的高度磷酸化以及淀粉样沉淀的形成相一致。脑脊液（CSF）中tau蛋白的磷酸化情况被发现与口服甲醇的状态相关，但磷酸化的变化在停药后的6个月仍然能够维持。这表明口服甲醇可以引起恒河猴长期的持续性的与AD相关的病理学变化。最值得注意的是，与小鼠不同，猕猴中产生了淀粉样沉淀，说明了我们应用的动物物种之间的差异，小鼠对于甲醇的内部防御能力限制了其在AD病理学变化中的应用。然而，在两种动物上的研究都支持甲醇毒性和AD病理学之间的海量证据。 甲醇被代谢成甲醛然后被代谢成甲酸。在急性口服甲醇的猕猴血液中，口服两小时后甲醛含量明显的升高，说明甲醇可以在体内代谢成甲醛。两个额外的体外实验，分别用甲醇以及代谢物甲醛和甲酸培养小鼠胚胎皮层神经细胞及小鼠成神经细胞瘤N2a细胞系，用来研究这些代谢物中哪种物质可以导致AD相关的病理变化（如tau蛋白的高度磷酸化）。这些研究表明是甲醛，而非甲醇或终代谢物甲酸，引起了微管的解体和tau蛋白的高度磷酸化。这些体外的实验表明甲醛很可能是甲醇毒性真正起作用的有害组分，它引起小鼠和猕猴海马中tau蛋白的高度磷酸化和随之而来的记忆损伤。 另外一个体外研究主要关注甲醛在Aβ聚集中的作用。Aβ是AD中淀粉样沉淀的主要组成成分。研究发现高浓度的甲醛可以直接引起稳定的Aβ聚集物产生，这表明甲醛可能在我们口服甲醇的猕猴中观测到的淀粉样沉淀以及AD病人尸检脑组织中淀粉样沉淀的形成中起重要作用。这个研究还进行了更加深入的研究：AD易感基因APOE基因的多态性与甲醛（一种与年龄相关的因素）之间的关系。这两个因素（年龄和APOE基因的多态性）是与痴呆相关的AD发病过程中最引人关注的危险因素，但这两者之间的关系至今未知。甲醛被公认为可以使蛋白质交联的作用，可以和精氨酸，赖氨酸，色氨酸残基以及巯基反应；这表明APOE形式的多态性有可能和甲醛之间的相互作用也各不相同。正因为如此，此研究探索了体外APOE的不同形式对Aβ（β淀粉样肽1-40）聚集的影响。APOE4蛋白（在112和158位点上有精氨酸的亚型）在10 mM甲醛培养下，较APOE3 或APOE2蛋白，可以形成含有更多的Aβ (P < 0.001) 和APOE (P < 0.05) 蛋白含量的聚集物。这个蛋白聚集的形式反映了APOE基因的多态性导致的易感性趋势（APOE4 > APOE3 > APOE2)，并且表明甲醛在人群中不同的APOE基因背景下淀粉样沉淀形成的差异中起作用。这个发现更进一步为甲醛在AD病程中起重要作用增加了证据，并且提供了年龄和APOE这两个AD相关因素之间的关联。 综上所述，小鼠和猕猴行为学上的测试，免疫组化分析，及此研究中的体外实验表明甲醛导致或者参与了AD的病理进程，在猕猴中甲醛可能通过在不同脑区（包括海马）中形成Aβ聚集物和tau蛋白的磷酸化导致微管解体，最终引起记忆损伤的。为了说明这些发现的重要性，论文讨论部分仔细论述了甲醛和AD关系的相关研究，如单碳循环，代谢功能异常，基因因素，年龄相关的甲醛毒性。此论文还探索了未来可能的研究方向.例如, 如何证实甲醛确实在AD的病程中起作用；研究减轻甲醛和/或甲醇毒性的治疗方法。
英文摘要	Alzheimer's disease (AD) is a progressive neurodegenerative disorder causing age-related dementia. It is associated with genetic factors leading to amyloid plaque formation and neurofibrillary tangles within the brain. However, the direct relation between the two factors is unknown, which impedes the development of effective therapeutics and diagnostics. This thesis explores a link recently established between the metabolite formaldehyde (FA), an age-related factor that has been found to be elevated in AD patients, and AD pathologies such as Aβ aggregation, tau hyperphosphorylation and cognitive impairments, ultimately asking whether FA toxicity plays an important role in AD progression. This recently established link between FA, a methanol metabolite, and AD pathology has also provided a new impetus to investigate the chronic effects of methanol exposure. Methanol toxicity is well known for acute neurological sequelae leading to blindness or death. However, this acute sequelae is often associated with formic acid, the methanol end product, and the chronic effects of methanol exposure have gone understudied in the past. In the first study presented, mice were fed with methanol to revisit the chronic effects of methanol toxicity, a FA source, as it pertains to AD progression. Three groups of mice (N = 9) were given either water as a control or a methanol solution (concentrations of 2% or 3.8%) ad libitum over a 6-week period. AD-dementia like symptoms were observed in the mice with Y-maze and olfactory memory tests to assess spatial recognition and olfactory memory, respectively. Then tau hyperphosphorylation, amyloid plague formation and neuronal apoptosis of hippocampus CA1 were measured in the mouse brains. The methanol-fed mice were found to have impaired spatial recognition and olfactory memory in the Y-maze and olfactory memory paradigms; the mice failed to recognize the novel arm in the Y-maze apparatus and failed to establish recognition of a lemon odor in the olfactory test. Immunohistochemical analysis of the mouse brains found increased neuronal Tau phosphorylation in the hippocampus and an increased cellular apoptotic marker in hippocampal CA1 neurons (approx. 10% of neurons displayed chromatin condensation) in the methanol-fed groups. Notably, no changes in amyloid-plaque formation were observed in the methanol-fed groups. The findings of the behavioral tests and immunohistochemical analysis suggested that the methanol-fed mice presented with partial AD-like symptoms, as no amyloid plaques were identified in the mice brains. The second study presented expanded this investigation to the non-human primate, rhesus macaque, through the chronic feeding of young male monkeys with 3% methanol ad libitum. Variable Spatial Delay Response Tasks of the monkeys found that the methanol feeding lead to persistent memory decline in the monkeys. This memory decline lasted 6 months beyond the feeding regimen. This change coincided with increases in Tau protein phosphorylation at residues T181 and S396 in cerebral spinal fluid (CSF) during feeding as well as with increases in Tau phosphorylated aggregates and amyloid plaques in four brain regions post mortem: the frontal lobe, parietal lobe, temporal lobe and the hippocampus. Tau phosphorylation in the CSF was found to be dependent on methanol feeding status, but phosphorylation changes in the brain were found to be persistent 6 months after the methanol feeding stopped. This suggested the methanol feeding caused long-lasting and persistent pathological changes that were related to AD development in the monkey. Most notably, the presence of amyloid plaque formations in the monkeys highlighted a marked difference in animal systems used in AD investigations, suggesting that the innate defenses in mice against methanol toxicity may have limited previous investigations into AD pathology. Nonetheless, the findings from both animal studies support a growing body of evidence that links methanol toxicity to AD pathology. Methanol is metabolized to FA and then to formic acid. In monkeys fed a single bolus feeding of methanol, FA levels in the blood increased within 2 hrs indicating that methanol toxicity produces its metabolite FA. Two additional in vitro experiments were performed in mouse embryonic cerebral cortex neurons and mouse neuroblastoma N2a cells to evaluate the effects of methanol metabolites on pathological markers related to AD and to determine which methanol metabolite caused AD associated changes (e.g. tau phosphorylation). These studies found that formaldehyde, but not methanol or the methanol end product - formic acid, induced microtubule disintegration and Tau protein hyperphosphorylation. These in vitro experiments suggested that FA was most likely the detrimental component of methanol toxicity related to hippocampal tau-phosphorylation and the subsequent impaired memory witnessed in both monkeys and mice. An additional in vitro study focused on the role of FA in Aβ aggregation, the main component of amyloid plaques in AD. FA was found to directly cause stable Aβ aggregation at high concentrations, suggesting it may have a role in both the plaque formation witnessed in the monkeys fed methanol, presented here, as well as in the plaque formation found in age-related AD dementia patients studied post-mortem. This study also went further to investigate the relationship between Apolipoprotein E (APOE) genetic variation and FA, as an age related factor. These are the two most noted risk factors (aging and APOE genetic variation) associated with the development of Alzheimer’s disease (AD) related dementia, but the relationship between these two pathological factors is not understood. FA is known to have protein cross-linking properties and to form cross-links with larger arginine, lysine and tryptophan residues but also to have thiol reactivity; suggesting that differences in APOE isoforms may interact differently with FA. As such, this study investigated the formation of protein aggregates between amyloid-beta (1-40) peptide (Aβ) with APOE isoforms in vitro. APOE4 protein, the isoform with arginines at residue 112 and 158, was found to form aggregates with more Aβ (P < 0.001) and APOE (P < 0.05) protein content in 10 mM FA than aggregates formed with either APOE3 or APOE2 protein. This aggregation pattern reflected the trend of vulnerability conferred by the APOE genetic variation (APOE4 > APOE3 > APOE2) and suggested that FA may have a role in the differential pattern of amyloid plaque formation in people with differing APOE genetic backgrounds. This finding further adds to the growing body of evidence that FA has a role in AD progression as well as provides a novel link between aging and APOE risk factors; the cornerstones of one of the world’s largest mental health concerns. All told, the behavioral tests in mice and monkeys, their immunohistochemical analysis, and the in vitro experiments performed in these studies indicated the FA induced and/or is involved in the progression of AD pathology and that it may impair memory in primates through the formation of Aβ aggregates in a number of brain regions, including the hippocampus, as well as through related tau-hyperphosphorylation dependent loss of microtubules in tangle formation. To explore the importance of these findings, an extensive discussion of the current literature as it pertains to AD is presented in this thesis, with respect to the one-carbon cycle, metabolic dysfunctions, genetic factors and age-related FA toxicity. This thesis also explores future directives to both confirm the role of FA in AD progression as well as to investigate therapeutics in alleviating the toxic effects of FA and/or methanol. Nonetheless, the findings of these studies and the literature exploration suggest that FA has an important and integral role in the progression of Alzheimer’s disease.
语种	英语
公开日期	2014-10-17
内容类型	学位论文
源URL	[http://159.226.149.42:8088/handle/152453/8098]
专题	昆明动物研究所_神经系统编码 昆明动物研究所_认知障碍病理学
推荐引用方式 GB/T 7714	Joshua D. Rizak. 甲醛对阿尔兹海默病的损伤作用[D]. 北京. 中国科学院研究生院. 2015.

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