探索女性对阿尔茨海默病更为易感的机制：聚焦于阿尔茨海默病临床前阶段的内嗅皮层变化

Alzheimer’s disease (AD) is an age-related progressive neurodegenerative disorder, the main risk factor is brain aging. The bases of aging are harmful factors such as the limited repair capability, aggregation of extracellular ß-amyloid (Aß) and lack of estrogens, that ultimately defeat protecting factors such as brain reserve and molecules that facilitate synaptic plasticity. Stimulation of neuronal activity may help to shift this balance towards more repair, a phenomenon that is paraphrased as ‘use it or lose it’. The earlier the stimulation of the brain, the better it works. Therefore we focus on the early stages of AD with 2 questions: i) Why is the entorhinal cortex so vulnerable to AD? And ii) Why are women more vulnerable to AD than men? In early AD stages, brain regions affected that store recent memories, including the entorhinal cortex and hippocampus. This early preclinical/pre-symptomatic period is neuropathologically scored as Braak staging for hyperphosphorylated tau protein (p-Tau) as Braak I-II. Animal experimental data show that not just alterations in estrogens, but also in brain-derived luteinizing hormone (LH) and LH receptor (LHR) may play key role in the fact that females are more susceptible to AD than males. This possibility has never been studied in human brain. Moreover, the cholinergic system is crucial for memory processing. We and other groups have found in previous studies that i) the cholinergic system is activated in preclinical AD stages, ii) a transcription factor, early growth response 1 (Egr1) may be, at least partly, responsible for the activation of the brain cholinergic system and other systems in preclinical stages of AD; iii) Egr1 upregulates choline acetyltransferase (ChAT) and acetylcholinesterase (AChE), iv) animal experiments showed that estrogen and LH can directly protect neuronal activity and against aggregation of Aß and p-tau, v) in triple-transgenic AD (3xTg-AD) mice older than 12 month Aß aggregation appeared to be higher in the females than males. We therefore proposed the following hypothesis: During aging, in the entorhinal cortex of individuals with Braak 0-II, local brain estrogen and/or LH decrease more in females than males, causing accumulation of Aß and p-tau to occur earlier in the aging process, and thus an earlier brain degeneration in the course of AD. In this period, the cholinergic innervation in the entorhinal cortex is still intact or activated, so that memory can still be intact for some time. However, in the course of events locally increased Egr1 causes AchE increase, more severe in females than in males due to the local lack of estrogens and/or LH, breaking the balance between cholinergic production and metabolism, thus a decline in cholinergic function related to impairment of memory is earlier in females than males, leading to clinical AD. To validate this hypothesis, we will study 1) in the entorhinal cortex of Braak stage 0-II (≥ 50y) whether there is a sex difference in AD-related proteins (Aß and p-Tau), the Egr1, ChAT and AChE, and local levels of estradiol (E2), LH, estrogen receptor (ER) and LHR; ii) the same parameters in the hippocampal complex of the 3xTg-AD mice during different months of age, subsequently selecting a special age to study the effect of up- or down-regulating Egr1 in the hippocampal complex and manipulating E2 and LH by ovariectomy in the AD mice; and iii) the mechanism of Egr1 regulating AChE expression, with or without Aß, p-Tau, E2 and LH in cell lines. This study will contribute to our understanding the mechanism from aging to neurodegeneration of AD and holds hope for AD postponement and prevention in its early stages.

阿尔茨海默病（AD）是衰老相关神经退行病，女性更为高发，进程可通过对脑内过磷酸化tau蛋白（p-Tau）分布的Braak分期确定。在Braak I-II期，个体无临床症状而内嗅皮层等脑区已出现p-Tau。我们将研究内嗅皮层对AD易感、女性对AD更易感原因。前期研究发现早期生长反应因子1（Egr1）参与AD临床前期脑内胆碱能系统激活。我们将在Braak 0-II期个体（≥50岁）内嗅皮层、不同年龄段三转基因AD小鼠海马复合体研究Aβ、p-Tau、Egr1、胆碱乙酰转移酶、乙酰胆碱酯酶（AChE）、局部雌二醇（E2）、黄体生成素（LH）及其受体水平性别差异；选择合适年龄段AD小鼠分别上、下调控海马复合体内Egr1，摘除卵巢加补充E2、LH研究上述参数改变；于细胞系阐明Egr1调控AChE表达分子机制及其受AD相关蛋白和E2、LH影响。旨在阐明从脑衰老到AD病变发展机制，寻找AD早期诊治靶标。

阿尔茨海默病（AD）是衰老相关性神经退行病，女性更为高发。我们的前期研究发现，AD病程中前额叶中包括早期生长反应因子-1（Egr-1）、乙酰胆碱酯酶（AChE）的表达具有动态先升后降改变。在648例覆盖AD全进程的个体，我们发现80岁以上女性AD病理累积较男性更为严重，而AD临床前期女性内嗅皮层中过磷酸化tau蛋白（p-Tau）表达量显著高于男性。在认知功能完整的老年人内嗅皮层定量研究芳香化酶（ARO）、雌激素受体（ERα、ERβ），雄激素受体、谷氨酸、γ-氨基丁酸、胆碱乙酰转移酶（ChAT）、AChE、Egr1、高尔基体等分子及AD病理蛋白表达，发现性别差异存在于ARO、ERα、GA密度或GABA能和谷氨酸能活性随着年龄增长的变化、80岁前后分子表达，以及分子之间相关性等。在3xTg-AD小鼠模型研究了不同年龄脑中Aβ、p-Tau、Egr-1、AChE分子表达，发现7-8月龄开始β-淀粉样蛋白（Aβ）表达的性别差异；对雌性APPSWE/PS1 AD小鼠模型给与卵巢摘除、雌激素或外周黄体生成素（LH）抑制剂给药，观察对学习记忆行为和海马Aβ表达的影响，发现抑制外周LH较雌激素替代更有效纠正由卵巢摘除导致的学习记忆行为紊乱，该效应与海马Aβ表达无关。在过表达p-Tau、过表达Aβ的SY5Y细胞株分别于前者研究p-TAU 于后者研究Aβ42、Aβ40，并同时研究EGR1、AChE表达以及给药E2的效应，发现EGR1与AChE在过表达p-Tau或Aβ的AD细胞系中变化模式不同，E2对AD病理蛋白均有抑制作用，但是在过表达p-Tau 细胞系，而不是在过表达Aβ的细胞系中发现EGR1及AChE的表达恢复，显示E2减缓pTau相关的EGR1及其调控的胆碱能系统的活性效应较Aβ参与的相关过程更为显著。提示在AD中E2与p-Tau引起的认知功能损伤较与Aβ引起的过程更为相关。研究结果已发表于Brain Pathology，Neuropathol Appl Neurobiol等学术期刊，后续论文在投稿、准备中。本项目的科学发现揭示了了衰老进程中内嗅皮层活性及AD相关改变的显著性别差异以及性激素可能参与该过程的方式，为进一步确定AD早期干预的药物靶点提供了关键线索。