中国科学院昆明动物研究所机构知识库

阿尔茨海默病（Alzheimer’s disease，AD），俗称老年痴呆，是所有痴呆类型中最常见的一种，随着人口的迅速老龄化，其发病率也在不断上升。AD的特征是渐进性记忆丧失和其他认知功能障碍，如运动能力受损、推理和判断能力受损等。AD的主要病理特征包括神经元胞外由β-淀粉样蛋白（β-amyloid，Aβ）聚集形成的老年斑，神经元胞内由于过度磷酸化的微管相关蛋白tau（microtubule-associated protein tau，MAPT）聚集形成的神经纤维缠结，以及突触功能障碍和神经元丢失等。许多AD遗传学研究，如连锁分析、候选基因关联研究、全基因组关联研究（genome-wide association studies，GWAS）和全外显子组测序（whole exome sequencing，WES）等，已经在不同人群（包括我国汉族人群）中鉴定到了许多AD致病基因（如APP、PSEN1、PSEN2和TREM2）以及AD遗传易感基因（如APOE4、ABCA7、BIN1、CR1、CLU和PICALM）。尽管在AD的遗传解析研究方面已经取得了显著的进展，但AD的病理生理学在很大程度上还不清楚。研究显示，基因表达的改变可能在AD发病机制中起着重要作用。为了寻找AD病人与健康对照之间差异表达的基因，许多基因表达分析已经鉴定了在AD病人中表达紊乱的基因。然而，我们注意到只有非常有限数量的差异表达基因能在不同的组织或研究中得到很好的验证。此外，对AD相关差异表达基因的调控机制研究还不够充分。2014年，有两项独立的大样本研究对AD病人大脑中全基因组DNA甲基化图谱进行了扫描，并鉴定到了一些差异性甲基化位点，为研究AD相关差异表达基因的调控机制提供了可靠的依据。在本论文的第一部分，我们利用了整合分析的策略将目前可利用的表观遗传、微阵列、转录组、基因组数据以及AD相关内表型数据结合在一起，以期能鉴定到在不同研究中均能得到验证的受甲基化位点调控的AD差异表达基因（表达上调或者下调），并阐释其调控机制。我们进一步对候选基因在细胞模型中研究其与AD进程的关系，从基因组水平、表观遗传学水平和细胞水平系统地研究了表达改变的调控和作用。经过筛选后，我们发现SERPINF1基因，其编码的蛋白作为参与神经分化的神经营养因子，在AD病人脑组织中显著下调。除了甲基化位点发生改变外，SERPINF1中AD遗传变异位点rs1136287影响到SERPINF1基因的表达水平。这个eSNP（SNP影响基因的表达，也称为表达数量性状基因座[expression Quantitative Trait Loci，eQTL]）rs1136287也与脑脊液（cerebro-spinal fluid，CSF）tau蛋白水平、CSF Aβ42水平、以及白质体积相关。为了进一步研究SERPINF1在基因组水平与AD的相关性，我们对106名中国汉族人群AD患者的SERPINF1基因所有编码区域进行了测序，发现了一个稀有变异p.L305V与AD显著相关。进一步通过基因敲降和过表达方式研究发现，SERPINF1表达水平减少和突变在AD发生发展中发挥作用。外源性SERPINF1蛋白能够逆转Aβ42引起的GABAergic神经元毒性。SERPINF1表达水平减少和突变引起Aβ的生成量增加，以及tau蛋白的磷酸化水平增加，并且该效应由CDK5介导。大鼠脑片电生理实验进一步表明SERPINF1突变p.L305V使得突触传递功能受损。这些结果表明，由表观遗传和遗传改变共同调控的SERPINF1表达水平减少或功能丧失在AD发病进程中起着重要的作用。另一方面，Aβ的过度产生以及清除的不足是导致AD发病的主要病因。自噬是细胞机体清除功能异常蛋白，从而维持自我稳态的主要途径，自噬的激活是对抗Aβ生产过剩引起的神经毒性比较有前景的策略之一。核受体过氧物酶体α（peroxisome proliferator-activated receptor α，PPARα）是一个转录因子，其通过靶定目的基因启动子区域的PPAR反应元件，从而对靶基因进行调控，进而参与脂肪酸代谢以及炎症反应。因此，PPARα在能量代谢、线粒体和过氧化物酶体功能维持方面起着重要作用。PPARα表达量增加，可以通过上调PPARγ的表达，从而对心血管疾病予以改善作用。激活PPARα可以激活肝脏中自噬水平，从而诱导自噬性脂质降解。另外，激活PPARα有助于天然免疫功能发挥。有研究报道，PPARA基因的遗传变异位点与AD显著有关。PPARα也通过影响神经元ADAM10的表达，从而促进APP向α-分泌酶途径进行切割，从而减少神经毒性。他汀类药物，作为PPARα的配体，其通过PPARα-CREB1途径上调BDNF的表达，从而改善小鼠的学习与记忆能力。但PPARα介导的自噬是否与AD的进程有关目前还不清楚。在本论文的第二部分，我们猜想PPARα在神经系统中调控自噬，并且影响AD进程，针对PPARα的药物或化合物，有望改善AD小鼠模型中的症状。经过系统研究，我们发现PPARα的激动剂吉非罗齐（Gemfibrozil，美国食品药品监督管理局已批准用于治疗高血脂症）和匹立尼酸（Wy14643）在人小胶质细胞（HM）和稳定表达APP-p.M671L突变体的人神经胶质瘤U251细胞（U251-APP）中都能激活自噬水平，并且此效应由PPARα介导。对APP/PS1ΔE9小鼠PPARα激动剂吉非罗齐和匹立尼酸给药后，小鼠中的Aβ病理生理学特征得以缓解，小鼠的认知能力得以改善，焦虑症状降低。吉非罗齐和匹立尼酸给药后，APP/PS1ΔE9小鼠海马和皮层组织中可溶性Aβ和以及Aβ斑块沉积度降低，并增强了星形胶质细胞和小胶质细胞向Aβ斑块周围进行聚集，从而对Aβ进行吞噬，此效应与自噬途径的增强紧密相关。本部分研究结果表明，PPARα介导的自噬在对Aβ进行清除从而改善AD小鼠认知功能障碍方面起着重要作用，并且吉非罗齐可能是未来用于治疗AD比较有前景的候选药物。

Alzheimer’s disease (AD) is the most common type of dementia in the elderly, and its prevalence is increasing in the rapidly aging population. AD is characterized by progressive memory loss and other cognitive dysfunctions, such as impaired locomotor ability, reasoning, and judgment. The hallmarks of AD pathology include the presence of extracellular amyloid plaque deposits, composed of β-amyloid (Aβ) peptides, and the intracellular formation of neurofibrillary tangles composed of hyperphosphorylation of tau in the brain, together with synaptic dysfunction and neuronal loss.Numerous genetic studies of AD, such as linkage analyses, candidate gene association studies, genome-wide association studies (GWAS), and whole exome sequencing (WES), have identified many AD causal genes (e.g. APP, PSEN1, PSEN2, and TREM2) and AD-related susceptibility loci (e.g. APOE4, ABCA7, BIN1, CR1, CLU, and PICALM), with independent validations in different populations, including Han Chinese populations. Although remarkable advances has been made in the genetic dissection of AD, the pathophysiology of AD is still largely unknown. Alterations of gene expression may have a pivotal role in the pathogenesis of AD. To seek the potential genes that are differentially expressed between AD patients and healthy controls, many microarrays analyses have been performed and numerous dysregulated genes have been identified. However, we noticed that only very limited number of differentially expressed genes could be replicated among different tissues or studies. Moreover, the regulatory mechanism of AD-related differentially expressed genes has been insufficiently studied. In 2014, two studies surveyed the genome-wide DNA methylation profiling in AD brains with the largest sample size and identified several differentially methylated loci, providing a reliable target for studying the regulatory mechanism of AD-related differentially expressed genes.In the first part of this thesis, we explored the potentially dysregulated genes by using an integrative analysis of the recently available epigenetic, microarray, transcriptomic and genomic datasets, with the aims to map the causal genes that are consistently dysregulated (downregulated or upregulated) in AD within the differentially methylated loci, and to characterize it’s regulatory mechanism.We further confirmed the altered gene expression and its effects in AD cellular models. The regulation and effect of expression alteration were systematically investigated at the genomic, epigenetic, as well as cellular levels.After comprehensive screening, we found that SERPINF1 that encodes pigment-epithelium-derived factor, a 50 kDa secreted glycoprotein of the serpin superfamily, as a neurotrophic factor involved in neuronal differentiation, was consistently downregulated in AD brain tissues. In addition to the differential methylation status as reported, we found that genotype of an AD-risk genetic variant rs1136287 affect the expression level of SERPINF1. This eSNP (SNP affecting gene expression, also called eQTL) rs1136287 was also associated with higher cerebrospinal fluid (CSF) Tau level, lower (CSF) amyloid-b42 (Ab42) level, and lower white matter volume. To dig further at the genomic level, we sequenced all the coding region of SERPINF1 in 106 AD patients, and identified a rare damaging SERPINF1 variant p.L305V in patients, with 5 times occurrence rate than that in control population. We further attempted to ascertain the roles of SERPINF1 dysregulation and mutation in AD molecular neuropathology by knockdown and overexpression assays. We found that knockdown and mutant of SERPINF1 increased the production of Aβ and phosphorylation of Tau, which might be mediated by CDK5. Furthermore, SERPINF1 treatment reversed Ab42-induced synaptic plasticity deficits in GABAergic interneurons. All these results suggested that SERPINF1 reduction or loss-of-function, regulated by both epigenetic and genetic alterations, paly an essential role in AD pathogenesis.An imbalance between the production and clearance of Aβ has been considered to be actively involved in AD pathogenesis. Autophagy is a major cellular pathway leading to the removal of aggregated proteins and up-regulation of autophagy represents a plausible therapeutic strategy to combat overproduction of neurotoxic Aβ. Nuclear receptor peroxisome proliferator-activated receptor α (PPARα) is a transcription factor that regulates genes involved in fatty acid metabolism and inflammatory processes by binding to PPAR response elements in the promoter region of the genes. Therefore PPARα acts as a key regulator of energy metabolism, and mitochondrial and peroxisomal function. Enhanced PPARα expression protects against cardiovascular disorders though its role of enhancing PPARγ expression. Activation of PPARα reverses the normal suppression of autophagy in the fed state and induces autophagic lipid degradation, and mediates innate host defense. Genetic variants in the PPARA gene have been found to be associated with AD. PPARα also regulates neuronal ADAM10 expression and stimulates APP processing towards the α-secretase pathway. Statins, as ligands of PPARα, increase BDNF expression and improves memory and learning in AD mice via the PPARα-CREB1 pathway. Whether PPARα-mediated autophagy is involved in the pathogenesis of AD remains undetermined.In the second part of this thesis, we hypothesized that PPARα regulates autophagy in the nervous system and the PPARα-mediated autophagy affects AD. We found that pharmacological activation of PPARα by the PPARα agonists Gemfibrozil and Wy14643 induces autophagy in human microglia (HM) cells and U251 human glioma cells stably expressing the human APP mutant (APP-p.M671L) and this effect is PPARα-dependent. Administration of PPARα agonists decreases amyloid pathology and reverses memory deficits and anxiety symptoms in APP/PS1ΔE9 mice. There is a reduced level of soluble Aβ and Aβ plaques in hippocampus and cortex tissues from APP/PS1ΔE9 mice after treatment with either Gemfibrozil or Wy14643, which promoted the recruitment of microglia and astrocytes to the vicinity of Aβ plaques and enhanced autophagosome biogenesis. These results indicate that PPARα is an important factor regulating autophagy in the clearance of Aβ and suggested Gemfibrozil be assessed as a possible treatment for AD.