The neuronal-specific isoform of BIN1 regulates β-secretase cleavage of APP and Aβ generation in a RIN3-dependent manner.
The neuronal-specific isoform of BIN1 regulates β-secretase cleavage of APP and Aβ generation in a RIN3-dependent manner.
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DOI：
10.1038/s41598-022-07372-4
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发表时间：
2022-03-03
影响因子：
4.6
通讯作者：
Tanzi RE
中科院分区：
文献类型：
--
作者：
Bhattacharyya R;Teves CAF;Long A;Hofert M;Tanzi RE
全基因组关联研究已经确定BIN1（桥接整合子1）和RIN3 （Ras和Rab相互作用子3）是晚发性阿尔茨海默病（LOAD）的遗传危险因素。BIN1的神经元亚型（BIN1V1），而非神经元亚型（BIN1V9），已被证明通过rab5介导的神经元内吞作用调节tau病理和Aβ的产生。BIN1直接与RIN3相互作用，启动rab5介导的内吞作用，这是β-分泌酶（BACE1）介导的β-分泌酶裂解β-淀粉样蛋白前体蛋白（APP）生成淀粉样蛋白β (Aβ)所必需的，而β-淀粉样蛋白是AD老年斑的关键成分。了解BIN1（神经元BIN1V1）和RIN3在β分泌酶介导的APP裂解和Aβ生成中的调节作用，是开发延缓或预防AD进展的新疗法的关键。将BIN1的神经元和非神经元亚型（分别为BIN1V1和BIN1V9）与RIN3一起引入体外细胞系统，检测神经元BIN1V1和非神经元BIN1V9对β分泌酶介导的APP裂解和Aβ生成的依赖作用。用共聚焦显微镜检测BIN1V1和BIN1V9依赖于rin3的亚细胞定位。Western blot分析评估了RIN3和BIN1V1/BIN1V9对β分泌酶介导的APP加工的影响。我们通过FACS富集不含或含RIN3表达BIN1V1的细胞，使用Aβ ELISA法检测Aβ的生成，并通过追踪生物素化或抗体标记的细胞表面APP来评估APP的内化。含有CLAP结构域的神经元BIN1V1和缺乏CLAP结构域的非神经元BIN1V9是大脑中存在的主要亚型。利用共聚焦显微镜，我们发现RIN3对rab5核内体中BIN1V1和BIN1V9的募集有差异调节。我们进一步发现，BIN1V1，而不是BIN1V9，以依赖rin3的方式下调β-分泌酶（BACE1）介导的APP加工。过表达BIN1V1也以依赖于rin3的方式减弱了a β的产生。通过基于细胞的内化实验，我们发现BIN1V1，而不是BIN1V9，延迟APP的内噬作用，而不是BACE1，进入早期内体，从而在空间和时间上将这两种蛋白分离到不同的细胞室，导致BACE1对APP的切割减少，并减少a β的产生-所有这些都以依赖rin3的方式进行。最后，我们发现，在rab5阳性的早期内体中，RIN3可能通过clap结构域将BIN1V1隔离，从而通过延迟APP的内吞噬作用，减弱APP的β分泌酶加工和Aβ的产生。我们的发现提供了两个AD相关分子RIN3和BIN1（神经元BIN1V1）如何相互作用来控制Aβ的产生的新的机制数据，暗示这两个蛋白是预防和治疗AD的潜在治疗靶点。
Genome-wide association studies have identified BIN1 (Bridging integrator 1) and RIN3 (Ras and Rab interactor 3) as genetic risk factors for late-onset Alzheimer’s disease (LOAD). The neuronal isoform of BIN1 (BIN1V1), but not the non-neuronal isoform (BIN1V9), has been shown to regulate tau-pathology and Aβ generation via RAB5-mediated endocytosis in neurons. BIN1 directly interacts with RIN3 to initiate RAB5-mediated endocytosis, which is essential for β-secretase (BACE1)-mediated β-secretase cleavage of β-amyloid precursor protein (APP) to generate Amyloid-β (Aβ), the key component of senile plaques in AD. Understanding the regulatory roles of BIN1 (neuronal BIN1V1) and RIN3 in β-secretase mediated cleavage of APP and Aβ generation is key to developing novel therapeutics to delay or prevent AD progression. Neuronal and non-neuronal isoforms of BIN1 (BIN1V1 and BIN1V9, respectively) were introduced with RIN3 into an in vitro cell-based system to test RIN3-dependent effects of neuronal BIN1V1 and non-neuronal BIN1V9 on β-secretase-mediated cleavage of APP and Aβ generation. Confocal microscopy was performed to examine RIN3-dependent subcellular localization of BIN1V1 and BIN1V9. Western blot analysis was performed to assess the effects of RIN3 and BIN1V1/BIN1V9 on β-secretase mediated processing of APP. We enriched cells expressing BIN1V1 without or with RIN3 via FACS to measure Aβ generation using Aβ ELISA assay, and to evaluate APP internalization by chasing biotinylated or antibody-labeled cell surface APP. Neuronal BIN1V1 containing the CLAP domain and non-neuronal BIN1V9 lacking the CLAP domain are the major isoforms present in the brain. Employing confocal microscopy, we showed that RIN3 differentially regulates the recruitment of both BIN1V1 and BIN1V9 into RAB5-endosomes. We further showed that BIN1V1, but not BIN1V9, downregulates β-secretase (BACE1)-mediated processing of APP in a RIN3-dependent manner. Overexpression of BIN1V1 also attenuated Aβ generation in a RIN3-dependent manner. Using cell-based internalization assays, we show BIN1V1, but not BIN1V9, delays the endocytosis of APP, but not of BACE1, into early endosomes, thereby spatially and temporally separating these two proteins into different cellular compartments, resulting in reduced cleavage of APP by BACE1 and reduced Aβ generation—all in a RIN3-dependent manner. Finally, we show that RIN3 sequesters BIN1V1 in RAB5-positive early endosomes, likely via the CLAP-domain, resulting in attenuated β-secretase processing of APP and Aβ generation by delaying endocytosis of APP. Our findings provide new mechanistic data on how two AD-associated molecules, RIN3 and BIN1 (neuronal BIN1V1), interact to govern Aβ production, implicating these two proteins as potential therapeutic targets for the prevention and treatment of AD.
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影响因子：
82.9
作者：
通讯作者：
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