ApoE Receptor Pathways to Intraneuronal Abeta

描述（由申请人提供）：神经元内淀粉样蛋白-p肽（AP）积累是阿尔茨海默病（AD）发病机制中的早期毒性事件。了解加速或抑制神经元内A¿积累的细胞机制可能为阿尔茨海默病提供新的治疗策略。A¿可以通过受体介导的摄取在神经元内积累。它也可以通过淀粉样蛋白前体蛋白（APR）在内吞途径中向A¿的从头加工而积累。我们最近的研究表明，载脂蛋白E （apoE）受体，低密度脂蛋白受体（LDLR）家族的成员，调节A¿的摄取以及APR内吞运输和加工到A¿。特别是，我们已经证明LRP在大脑中的过度表达会增加细胞相关的A¿。A¿可以直接或间接地通过A¿伴侣如apoE与apoE受体结合。该提案将重点关注两种apoE受体，ldlr相关蛋白（LRP）和LRP1B。这些同源受体都在神经元中高度表达，并结合多种配体，包括A¿，apoE和APP。然而，我们实验室的证据表明，LRP和LRP1B在配体内吞作用中发挥相反的作用。LRP介导快速内吞作用，而LRP1B内吞作用非常缓慢，因此在细胞表面保留配体。我们的总体假设是LRP促进A -吸收、p产生和神经元内A -积累，而LRP1B阻断这些作用，从而抑制A -毒性和阿尔茨海默病的发病机制。我们设计了体内和体外两种方法来验证我们的假设。在Aim 1中，我们计划在动物模型中确定LRP和LRP1B在神经元内积累中的作用。由于常规的LRP敲除在胚胎早期是致命的，我们的实验室产生了条件LRP前脑特异性敲除小鼠。与LRP1B敲除小鼠一起，我们计划测试LRP和LRP1B在脑内输注A¿或与PDAPP淀粉样蛋白模型小鼠繁殖后神经元内A¿积累中的作用。在Aim 2中，我们将定义LRP和LRP1B在初级神经元中apoe依赖性和apoe非依赖性A¿摄取中的相反作用。在apoE缺失或存在的情况下，将评估神经元中LRP和/或LRP1B表达改变对A¿摄取和神经元内积累的影响。在Aim 3中，我们计划分析LRP和LRP1B在APP内吞运输和A¿产生中的相反作用的机制。我们提出的研究将利用我们在体外和体内方法研究受体介导的内吞作用，APP内吞运输和A¿代谢的细胞生物学方面的经验。由于最近的研究已经确定了在A斑块之前，神经元内A¿在认知缺陷中的因果作用，因此了解导致或防止神经元内AB积聚的途径可能会导致AD治疗的特定靶点。

DESCRIPTION (provided by applicant): Intraneuronal amyloid-p peptide (AP) accumulation is an early and toxic event in the pathogenesis of Alzheimer's disease (AD). Understanding cellular mechanisms that accelerate or inhibit intraneuronal A¿ accumulation may provide novel therapeutic strategies for AD. A¿ can accumulate inside neurons via receptor-mediated uptake. It can also accumulate via de novo processing of amyloid precursor protein (APR) to A¿ in the endocytic pathway. Our recent studies have shown that apolipoprotein E (apoE) receptors, members of the low-density lipoprotein receptor (LDLR) family, modulate A¿ uptake as well as APR endocytic trafficking and processing to A¿. In particular, we have demonstrated that LRP overexpression in the brain increases cell-associated A¿. A¿ can bind to apoE receptors either directly or indirectly via A¿ chaperones such as apoE. This proposal will focus on two apoE receptors, the LDLR-related protein (LRP) and LRP1B. These homologous receptors are both highly expressed in neurons and bind multiple ligands including A¿, apoE, and APP. However, evidence from our lab suggests that LRP and LRP1B play opposing roles in ligand endocytosis. While LRP mediates rapid endocytosis, LRP1B endocytoses very slowly and as a consequence, retains ligands at the cell surface. Our overall hypothesis is that LRP facilitates A¿ uptake, p production, and intraneuronal A¿ accumulation, and that LRP1B blocks these effects, thus inhibiting A¿ toxicity and pathogenesis of Alzheimer's disease. We have designed both in vivo and in vitro approaches to test our hypothesis. In Aim 1, we plan to determine the roles of LRP and LRP1B in intraneuronal accumulation in animal models. Because conventional LRP knockout is early embryonic lethal, our lab has generated conditional LRP forebrain-specific knockout mice. Together with the LRP1B knockout mice, we plan to test the roles of LRP and LRP1B in intraneuronal A¿ accumulation after brain A¿ infusion or after breeding with PDAPP amyloid model mice. In Aim 2, we will define the opposing roles of LRP and LRP1B in apoE-dependent and apoE-independent A¿ uptake in primary neurons. Impacts of altered LRP and/or LRP1B expression in neurons on A¿ uptake and intraneuronal accumulation will be assessed in the absence or presence of apoE. In Aim 3, we plan to dissect the mechanisms underlying the opposing roles of LRP and LRP1B in APP endocytic trafficking and A¿ production. Our proposed studies will take advantage of our experience in studying the cell biology of receptor-mediated endocytosis, APP endocytic trafficking, and A¿ metabolism using both in vitro and in vivo approaches. Because recent studies have established the causal role of intraneuronal A¿ in cognitive deficits prior to A¿ plaques, an understanding of the pathways leading to or protecting against intraneuronal AB accumulation may lead to specific targets for AD therapy.