Upregulation of RIN3 induces endosomal dysfunction in Alzheimer's disease
Upregulation of RIN3 induces endosomal dysfunction in Alzheimer's disease
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RIN3 的上调诱导阿尔茨海默病的内体功能障碍
DOI：
10.1186/s40035-020-00206-1
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发表时间：
2020-06-18
影响因子：
12.6
通讯作者：
Wu, Chengbiao
中科院分区：
文献类型：
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作者：
Shen, Ruinan;Zhao, Xiaobei;Wu, Chengbiao
背景资料：在阿尔茨海默病（AD）中，GWAS鉴定的风险基因中约有三分之一编码主要在内吞途径中发挥作用的蛋白质。其中，Ras和Rab相互作用因子3（RIN 3）是Rab 5小GTP酶家族的鸟嘌呤核苷酸交换因子（GEF），并且已被暗示是晚发型AD（LOAD）和散发性早发AD（sEOAD）的危险因素。方法：采用定量PCR和免疫印迹法检测RIN 3在小鼠脑组织和培养的基底前脑胆碱能神经元（BFCNs）中的表达水平。免疫染色用于确定RIN 3的亚细胞定位，并观察培养的原代BFCN和PC 12细胞中的内体变化。从HEK 293 T细胞中纯化重组标记的RIN 3蛋白，并用于通过质谱法定义RIN 3相互作用体。通过免疫共沉淀、免疫荧光和酵母双杂交测定来验证RIN 3相互作用伴侣。原代神经元的实时成像用于检查淀粉样前体蛋白（APP）和β-分泌酶1（BACE 1）的轴突运输。免疫印迹法检测APP/PS1小鼠海马和皮层中RIN 3 mRNA的表达、APP加工和Tau磷酸化形式。从E18 APP/PS1小鼠胚胎中培养的基底前脑胆碱能神经元（BFCNs）也显示RIN 3表达增加，伴有早期内体扩大。此外，通过其富含脯氨酸的结构域，RIN 3将另外两种AD风险因子BIN 1（桥接整合子1）和CD 2AP（CD 2相关蛋白）募集到早期内体。有趣的是，RIN 3或CD 2AP的过表达促进APP切割，以增加其在PC 12细胞中的羧基末端片段（CTF）。RIN 3或BIN 1的神经元亚型的上调增加磷酸化Tau水平。因此，RIN 3表达的上调促进APP CTF的积累并增加磷酸化Tau。RIN 3的这些作用通过显性负性Rab 5（Rab 5（S34 N））构建体的表达来挽救。结论：RIN 3在APP/PS1小鼠中表达上调，与内体功能障碍相关。通过与BIN 1和CD 2AP相互作用，RIN 3表达增加改变了AP的轴突运输和加工。结合我们以前的研究，我们目前的工作为RIN 3在调节内体信号和运输中的作用提供了重要的见解。
Background: In Alzheimer's Disease (AD), about one-third of the risk genes identified by GWAS encode proteins that function predominantly in the endocytic pathways. Among them, the Ras and Rab Interactor 3(RIN3) is a guanine nucleotide exchange factor (GEF) for the Rab5 small GTPase family and has been implicated to be a risk factor for both late onset AD (LOAD) and sporadic early onset AD (sEOAD). However, how RIN3 is linked to AD pathogenesis is currently undefined.Methods: Quantitative PCR and immunoblotting were used to measure the RIN3 expression level in mouse brain tissues and cultured basal forebrain cholinergic neuron (BFCNs). Immunostaining was used to define subcellular localization of RIN3 and to visualize endosomal changes in cultured primary BFCNs and PC12 cells. Recombinant flag-tagged RIN3 protein was purified from HEK293T cells and was used to define RIN3-interactomes by mass spectrometry. RIN3-interacting partners were validated by co-immunoprecipitation, immunofluorescence and yeast two hybrid assays. Live imaging of primary neurons was used to examine axonal transport of amyloid precursor protein (APP) and beta-secretase 1 (BACE1). Immunoblotting was used to detect protein expression, processing of APP and phosphorylated forms of Tau.Results: We have shown that RIN3 mRNA level was significantly increased in the hippocampus and cortex of APP/PS1 mouse brain. Basal forebrain cholinergic neurons (BFCNs) cultured from E18 APP/PS1 mouse embryos also showed increased RIN3 expression accompanied by early endosome enlargement. In addition, via its proline rich domain, RIN3 recruited BIN1(bridging integrator 1) and CD2AP (CD2 associated protein), two other AD risk factors, to early endosomes. Interestingly, overexpression of RIN3 or CD2AP promoted APP cleavage to increase its carboxyl terminal fragments (CTFs) in PC12 cells. Upregulation of RIN3 or the neuronal isoform of BIN1 increased phosphorylated Tau level. Therefore, upregulation of RIN3 expression promoted accumulation of APP CTFs and increased phosphorylated Tau. These effects by RIN3 was rescued by the expression of a dominant negative Rab5 (Rab5(S34N)) construct. Our study has thus pointed to that RIN3 acts through Rab5 to impact endosomal trafficking and signaling.Conclusion: RIN3 is significantly upregulated and correlated with endosomal dysfunction in APP/PS1 mouse. Through interacting with BIN1 and CD2AP, increased RIN3 expression alters axonal trafficking and procession of APP. Together with our previous studies, our current work has thus provided important insights into the role of RIN3 in regulating endosomal signaling and trafficking.