【Abstract】
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 β-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 (Rab5S34N) construct.
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.
【中文摘要】
背景：在阿爾茲海默病中，GWAS研究鑑定出的Ras and Rab interactor 3(RIN3) 作為小GTP酶的一個鳥苷酸轉換因子（GEF），被發現同時是遲發性AD和散發性早發性AD的風險因子。但是RIN3在AD病理發展過程中的作用尚未發現。
方法：定量PCR和免疫印跡用於檢測RIN3在小鼠各腦區或原代基底前腦膽鹼能神經元（BFCNs）的表達水平。細胞免疫螢光技術用於檢測RIN3在亞細胞結構的定位。重組人源RIN3-flag蛋白純化後用於質譜檢測RIN3的互作蛋白並以免疫細胞螢光、免疫共沉澱及酵母雙雜交技術進行重複驗證。活細胞攝影技術用於觀察澱粉前體蛋白（APP）及β分泌酶1（BACE1）在原代神經元中的軸突運輸改變。
結果：相比野生型，RIN3mRNA表達量在APP/PS1AD小鼠的海馬及皮層有顯著升高。其表達水平升高早在原代BFCNs就已出現，並進一步導致神經元內早期內體增大。另外，RIN3通過其PRD結構域招募另外兩個AD風險基因BIN1和CD2AP到早期內體。在PC12細胞中過表達CD2AP可以導致APP C末端片段（CTFs）升高，而過表達神經源性BIN1則導致tau蛋白過度磷酸化。同時過表達顯性負性Rab5(Rab5S34N)時可抑制RIN3上調帶來的APPCTFs及tau蛋白異常。
結論：RIN3表達量在AD模型小鼠中顯著上調，通過結合BIN１和CD2AP, 上調的RIN3介導了內體運輸障礙、APP剪切異常及tau蛋白過度磷酸化。綜上，我們的工作首次揭示了RIN3在早期AD發展中的病理作用，發現RIN3可作為AD治療的新靶點，為尋找AD治療途徑提供了新思路。