Role of apoE-mediated meningeal lymphatic remodeling in the pathophysiology of Alzheimer’s disease

摘要 年龄和载脂蛋白E ε4（APOE 4）基因等位基因的表达是两个主要的危险因素， 老年痴呆症最近的研究表明，老化导致受损的脑液引流的博纳 在哺乳动物脑膜硬脑膜中发现的真正的淋巴管网络。有趣的是，早期的损失 脑膜淋巴引流与β淀粉样蛋白（Aβ）病理学、血管和小胶质细胞 激活和空间记忆缺陷。然而，在这方面， 关于APOE 4表达对脑膜淋巴功能的影响知之甚少。我们的初步数据 显示APOE 4在人源化基因敲入小鼠中的表达导致脑膜的有害重塑， 淋巴管和减少脑脊液（CSF）引流到颈部淋巴结。有趣的是， 我们还发现脑膜周围淋巴髓样细胞是apoE蛋白的主要局部来源， 在中年APOE 4小鼠中激活。这些观察使我们提出了一个总体假设， APOE 4的表达干扰脑膜先天性髓样细胞和淋巴内皮细胞之间的相互作用， 这将随后导致脑膜炎患者的CSF引流减少，并加剧 神经炎症和脑淀粉样变性的研究进展。在目标1中，我们计划使用本构或 条件性基因敲除小鼠模型，以研究APOE 4的细胞自主和非自主效应 对不同年龄段脑膜淋巴功能障碍的影响，确定潜在的先天免疫转录 标记，并鉴定淋巴生长因子和/或细胞因子的改变。在目标2中，我们将测试 假设脑膜淋巴引流的早期损伤促进了更快的认知衰退， 在APOE 4小鼠中，精细调节脑膜炎细胞因子可改善神经元功能， 以性别依赖的方式进行认知。为了评估这一点，将调节脑膜淋巴引流，使用腺- 促进淋巴管消融或扩张的相关病毒，并评估下游效应 对老年女性和男性APOE的神经元转录组、突触可塑性和行为表现的影响 人源化小鼠。在目标3中，将测试操纵脑膜淋巴功能的治疗结局 在脑淀粉样变性的敲入阿尔茨海默病小鼠模型中，APP-SAA表达APOE 3或 APOE 4.在2个月和10个月大的小鼠中诱导脑膜淋巴管损失或扩张后，我们将 评估不同的实验结果，包括行为表现，神经元活动，脑间质 流体Aβ（通过体内微透析），可溶性和不溶性Aβ聚集体，以及先天性和适应性 大脑及其边缘组织中的免疫细胞激活。总而言之，这项提案将使我们能够发现新的 脑膜淋巴下降的机制，将作为未来项目的基石，并可能导致 新的疗法来治疗APOE 4等位基因携带者，这些携带者患阿尔茨海默病的风险更大。

ABSTRACT Aging and expression of apolipoprotein E ε4 (APOE4) gene alleles represent two major risk factors for Alzheimer’s disease. Recent studies have shown that aging leads to impaired brain fluid drainage by the bona fide lymphatic vascular network that is found in the mammalian meningeal dura. Interestingly, early loss of meningeal lymphatic drainage has been linked to worse amyloid-β (Aβ) pathology, vascular and microglial activation, and spatial memory deficits in Alzheimer’s disease mouse models of brain amyloidosis. However, little is known about the effects of APOE4 expression on meningeal lymphatic function. Our preliminary data shows that expression of APOE4 in humanized knock-in mice leads to a deleterious remodeling of meningeal lymphatic vessels and reduced drainage of cerebrospinal fluid (CSF) into the cervical lymph nodes. Interestingly, we also show that meningeal peri-lymphatic myeloid cells are a main local source of apoE protein and become activated in middle-aged APOE4 mice. These observations led us to raise the overarching hypothesis that expression of APOE4 disturbs the crosstalk between meningeal innate myeloid and lymphatic endothelial cells, which will subsequently lead to reduced CSF drainage by meningeal lymphatics, and exacerbated neuroinflammation and brain amyloidosis in Alzheimer’s disease. In Aim 1, we plan to use constitutive or conditional knockout mouse models to investigate the cell autonomous and non-autonomous effects of APOE4 on meningeal lymphatic dysfunction at different ages, determine the underlying innate immune transcriptional signatures, and identify alterations in lymphatic growth factors and/or cytokines. In Aim 2, we will test the hypothesis that an early impairment in meningeal lymphatic drainage promotes a faster cognitive decay in APOE4 mice, and that fine-tuning meningeal lymphatics in aged APOE4 mice ameliorates neuronal function and cognition in sex-dependent manner. To assess this, will modulate meningeal lymphatic drainage, using adeno- associated viruses that promote lymphatic vessel ablation or expansion, and evaluate the downstream effects on the neuronal transcriptomes, synaptic plasticity, and behavioral performances of aged female and male APOE humanized mice. In Aim 3, the therapeutic outcomes of manipulating meningeal lymphatic function will be tested in a knock-in Alzheimer’s disease mouse model of brain amyloidosis, the APP-SAA, expressing APOE3 or APOE4. After inducing meningeal lymphatic vessel loss or expansion in 2- and 10-month-old mice, we will evaluate different experimental outcomes including behavioral performance, neuronal activity, brain interstitial fluid Aβ (by in vivo microdialysis), soluble and insoluble Aβ aggregates, and the profiles of innate and adaptive immune cell activation in the brain and its border tissues. Altogether, this proposal will allow us to uncover novel mechanisms of meningeal lymphatic decline that will serve as cornerstones for future projects and might lead to novel therapies to treat APOE4 allele carriers that are at a greater risk of developing Alzheimer’s disease.