BIN1 deficiency enhances ULK3-dependent autophagic flux and reduces dendritic size in mouse hippocampal neurons

Genome-wide association studies identified variants around the <i>BIN1</i> (bridging integrator 1) gene locus as prominent risk factors for late-onset Alzheimer disease. In the present study, we decreased the expression of BIN1 in mouse hippocampal neurons to investigate its neuronal function. <i>Bin1</i> knockdown via RNAi reduced the dendritic arbor size in primary cultured hippocampal neurons as well as in mature Cornu Ammonis 1 excitatory neurons. The AAV-mediated <i>Bin1</i> RNAi knockdown also generated a significant regional volume loss around the injection sites at the organ level, as revealed by 7-Tesla structural magnetic resonance imaging, and an impaired spatial reference memory performance in the Barnes maze test. Unexpectedly, <i>Bin1</i> knockdown led to concurrent activation of both macroautophagy/autophagy and MTOR (mechanistic target of rapamycin kinase) complex 1 (MTORC1). Autophagy inhibition with the lysosome inhibitor chloroquine effectively mitigated the <i>Bin1</i> knockdown-induced dendritic regression. The subsequent molecular studydemonstrated that increased expression of ULK3 (unc-51 like kinase 3), which is MTOR-insensitive, supported autophagosome formation in BIN1 deficiency. Reducing ULK3 activity with SU6668, a receptor tyrosine kinase inhibitor, or decreasing neuronal ULK3 expression through AAV-mediated RNAi, significantly attenuated <i>Bin1</i> knockdown-induced hippocampal volume loss and spatial memory decline. In Alzheimer disease patients, the major neuronal isoform of BIN1 is specifically reduced. Our work suggests this reduction is probably an important molecular event that increases the autophagy level, which might subsequently promote brain atrophy and cognitive impairment through reducing dendritic structures, and ULK3 is a potential interventional target for relieving these detrimental effects.<b>Abbreviations</b>: AV: adeno-associated virus; Aβ: amyloid-β; ACTB: actin, beta; AD: Alzheimer disease; Aduk: Another Drosophila Unc-51-like kinase; AKT1: thymoma viral proto-oncogene 1; AMPK: AMP-activated protein kinase; AP: autophagosome; BafA1: bafilomycin A₁; BDNF: brain derived neurotrophic factor; BIN1: bridging integrator 1; BIN1-iso1: BIN1, isoform 1; CA1: cornu Ammonis 1; CA3: cornu Ammonis 3; CLAP: clathrin and adapter binding; CQ: chloroquine; DMEM: Dulbecco’s modified Eagle medium; EGFP: enhanced green fluorescent protein; GWAS: genome-wide association study; MAP1LC3B/LC3B: microtubule-associated protein 1 light chain 3 beta; MRI: magnetic resonance imaging; MTOR; mechanistic target of rapamycin kinase; MTORC1: MTOR complex 1; PET: positron emission tomography; qRT-PCR: real-time quantitative reverse transcription PCR; ROS: reactive oxygen species; RPS6KB1: ribosomal protein S6 kinase B1; TFEB: transcription factor EB; ULK1: unc-51 like kinase 1; ULK3: unc-51 like kinase 3.

全基因组关联研究（Genome-wide association studies, GWAS）已将<i>BIN1</i>（桥接整合因子1，bridging integrator 1）基因位点附近的变异确定为晚发性阿尔茨海默病的重要风险因素。
本研究通过下调小鼠海马神经元中BIN1的表达，以探究其神经元功能。经RNA干扰（RNAi）介导的<i>Bin1</i>敲低，可降低原代培养海马神经元以及成熟海马CA1（Cornu Ammonis 1）兴奋性神经元的树突分支面积。借助7特斯拉结构磁共振成像（magnetic resonance imaging, MRI）检测发现，腺相关病毒（adeno-associated virus, AAV）介导的<i>Bin1</i> RNAi敲低可在器官层面导致注射位点周围出现显著的区域体积丢失；同时，巴恩斯迷宫实验显示小鼠空间参考记忆能力受损。
出乎意料的是，<i>Bin1</i>敲低同时激活了巨自噬/自噬（macroautophagy/autophagy）以及MTOR（雷帕霉素机制靶标，mechanistic target of rapamycin kinase）复合物1（MTOR complex 1, MTORC1）。使用溶酶体抑制剂氯喹（chloroquine, CQ）抑制自噬，可有效缓解<i>Bin1</i>敲低诱导的树突回缩。后续分子生物学实验证实，不依赖MTOR的ULK3（unc-51样激酶3，unc-51 like kinase 3）表达上调，可在BIN1缺失的情况下促进自噬体形成。使用受体酪氨酸激酶抑制剂SU6668降低ULK3活性，或通过AAV介导的RNAi敲低神经元ULK3表达，可显著减轻<i>Bin1</i>敲低诱导的海马体积丢失与空间记忆衰退。
在阿尔茨海默病患者中，BIN1的主要神经元亚型表达特异性降低。本研究结果提示，这种表达降低可能是提升自噬水平的重要分子事件，后续或通过破坏树突结构促进脑萎缩与认知损伤，而ULK3或是缓解这些有害效应的潜在干预靶点。
<b>缩写说明</b>：
AAV：腺相关病毒（adeno-associated virus）；Aβ：淀粉样β蛋白；ACTB：肌动蛋白β（actin, beta）；AD：阿尔茨海默病（Alzheimer disease）；Aduk：果蝇另一种Unc-51样激酶（Another Drosophila Unc-51-like kinase）；AKT1：胸腺瘤病毒原癌基因1（thymoma viral proto-oncogene 1）；AMPK：AMP活化蛋白激酶（AMP-activated protein kinase）；AP：自噬体（autophagosome）；BafA1：巴弗洛霉素A₁（bafilomycin A₁）；BDNF：脑源性神经营养因子（brain derived neurotrophic factor）；BIN1：桥接整合因子1（bridging integrator 1）；BIN1-iso1：BIN1亚型1（BIN1, isoform 1）；CA1：海马CA1区（cornu Ammonis 1）；CA3：海马CA3区（cornu Ammonis 3）；CLAP：网格蛋白与接头蛋白结合域（clathrin and adapter binding）；CQ：氯喹（chloroquine）；DMEM：杜尔贝科改良伊格尔培养基（Dulbecco’s modified Eagle medium）；EGFP：增强型绿色荧光蛋白（enhanced green fluorescent protein）；GWAS：全基因组关联研究（genome-wide association study）；MAP1LC3B/LC3B：微管相关蛋白1轻链3β（microtubule-associated protein 1 light chain 3 beta）；MRI：磁共振成像（magnetic resonance imaging）；MTOR：雷帕霉素机制靶标（mechanistic target of rapamycin kinase）；MTORC1：MTOR复合物1（MTOR complex 1）；PET：正电子发射断层扫描（positron emission tomography）；qRT-PCR：实时定量逆转录聚合酶链反应（real-time quantitative reverse transcription PCR）；ROS：活性氧（reactive oxygen species）；RPS6KB1：核糖体蛋白S6激酶B1（ribosomal protein S6 kinase B1）；TFEB：转录因子EB（transcription factor EB）；ULK1：unc-51样激酶1（unc-51 like kinase 1）；ULK3：unc-51样激酶3（unc-51 like kinase 3）。