FRINK Linked Data Fragments server

Matches in SemOpenAlex for { <https://semopenalex.org/work/W2078201172> ?p ?o ?g. }

• W2078201172 endingPage "1551" @default.
• W2078201172 startingPage "1536" @default.
• W2078201172 abstract "•RNAi and Rab-GAP screen for Aβ regulators identified Rab11 as a positive regulator•Rab11A inhibition decreased both β cleavage of APP and Aβ production•Mechanistically, Rab11 controlled β-secretase recycling to endosomes•Rab11A is linked to late-onset Alzheimer’s disease Alzheimer’s disease (AD) is characterized by cerebral deposition of β-amyloid (Aβ) peptides, which are generated from amyloid precursor protein (APP) by β- and γ-secretases. APP and the secretases are membrane associated, but whether membrane trafficking controls Aβ levels is unclear. Here, we performed an RNAi screen of all human Rab-GTPases, which regulate membrane trafficking, complemented with a Rab-GTPase-activating protein screen, and present a road map of the membrane-trafficking events regulating Aβ production. We identify Rab11 and Rab3 as key players. Although retromers and retromer-associated proteins control APP recycling, we show that Rab11 controlled β-secretase endosomal recycling to the plasma membrane and thus affected Aβ production. Exome sequencing revealed a significant genetic association of Rab11A with late-onset AD, and network analysis identified Rab11A and Rab11B as components of the late-onset AD risk network, suggesting a causal link between Rab11 and AD. Our results reveal trafficking pathways that regulate Aβ levels and show how systems biology approaches can unravel the molecular complexity underlying AD. Alzheimer’s disease (AD) is characterized by cerebral deposition of β-amyloid (Aβ) peptides, which are generated from amyloid precursor protein (APP) by β- and γ-secretases. APP and the secretases are membrane associated, but whether membrane trafficking controls Aβ levels is unclear. Here, we performed an RNAi screen of all human Rab-GTPases, which regulate membrane trafficking, complemented with a Rab-GTPase-activating protein screen, and present a road map of the membrane-trafficking events regulating Aβ production. We identify Rab11 and Rab3 as key players. Although retromers and retromer-associated proteins control APP recycling, we show that Rab11 controlled β-secretase endosomal recycling to the plasma membrane and thus affected Aβ production. Exome sequencing revealed a significant genetic association of Rab11A with late-onset AD, and network analysis identified Rab11A and Rab11B as components of the late-onset AD risk network, suggesting a causal link between Rab11 and AD. Our results reveal trafficking pathways that regulate Aβ levels and show how systems biology approaches can unravel the molecular complexity underlying AD. Alzheimer’s disease (AD) is the most common form of dementia and is characterized by the cerebral deposition of β-amyloid (Aβ) peptides in the form of amyloid plaques (De Strooper, 2010De Strooper B. Proteases and proteolysis in Alzheimer disease: a multifactorial view on the disease process.Physiol. Rev. 2010; 90: 465-494Crossref PubMed Scopus (358) Google Scholar, Frisoni et al., 2011Frisoni G.B. Hampel H. O’Brien J.T. Ritchie K. Winblad B. Revised criteria for Alzheimer’s disease: what are the lessons for clinicians?.Lancet Neurol. 2011; 10: 598-601Abstract Full Text Full Text PDF PubMed Scopus (23) Google Scholar). The amyloid cascade hypothesis postulates that Aβ peptides trigger a series of pathological events leading to neurodegeneration (Huang and Mucke, 2012Huang Y. Mucke L. Alzheimer mechanisms and therapeutic strategies.Cell. 2012; 148: 1204-1222Abstract Full Text Full Text PDF PubMed Scopus (1407) Google Scholar, Selkoe, 2011bSelkoe D.J. Resolving controversies on the path to Alzheimer’s therapeutics.Nat. Med. 2011; 17: 1060-1065Crossref PubMed Scopus (419) Google Scholar). Aβ peptides are liberated from the transmembrane amyloid precursor protein (APP) by the sequential actions of β-secretase and γ-secretase (Thinakaran and Koo, 2008Thinakaran G. Koo E.H. Amyloid precursor protein trafficking, processing, and function.J. Biol. Chem. 2008; 283: 29615-29619Crossref PubMed Scopus (859) Google Scholar, Willem et al., 2009Willem M. Lammich S. Haass C. Function, regulation and therapeutic properties of beta-secretase (BACE1).Semin. Cell Dev. Biol. 2009; 20: 175-182Crossref PubMed Scopus (75) Google Scholar). β-secretase activity is conferred by a transmembrane aspartyl protease, also termed BACE1 (β-site APP-cleaving enzyme 1) (Vassar et al., 1999Vassar R. Bennett B.D. Babu-Khan S. Kahn S. Mendiaz E.A. Denis P. Teplow D.B. Ross S. Amarante P. Loeloff R. et al.Beta-secretase cleavage of Alzheimer’s amyloid precursor protein by the transmembrane aspartic protease BACE.Science. 1999; 286: 735-741Crossref PubMed Scopus (3348) Google Scholar), whereas γ-secretase is a multimeric transmembrane protein complex composed of presenilin-1 (PS1)/PS2, nicastrin, Aph-1, and PEN-2 (Annaert and De Strooper, 2002Annaert W. De Strooper B. A cell biological perspective on Alzheimer’s disease.Annu. Rev. Cell Dev. Biol. 2002; 18: 25-51Crossref PubMed Scopus (198) Google Scholar, Selkoe and Wolfe, 2007Selkoe D.J. Wolfe M.S. Presenilin: running with scissors in the membrane.Cell. 2007; 131: 215-221Abstract Full Text Full Text PDF PubMed Scopus (315) Google Scholar). Familial mutations in APP, PS1, or PS2 that increase the production of the amyloidogenic Aβ42 peptide have been associated with early-onset AD (Borchelt et al., 1996Borchelt D.R. Thinakaran G. Eckman C.B. Lee M.K. Davenport F. Ratovitsky T. Prada C.M. Kim G. Seekins S. Yager D. et al.Familial Alzheimer’s disease-linked presenilin 1 variants elevate Abeta1-42/1-40 ratio in vitro and in vivo.Neuron. 1996; 17: 1005-1013Abstract Full Text Full Text PDF PubMed Scopus (1355) Google Scholar, Duff et al., 1996Duff K. Eckman C. Zehr C. Yu X. Prada C.M. Perez-tur J. Hutton M. Buee L. Harigaya Y. Yager D. et al.Increased amyloid-beta42(43) in brains of mice expressing mutant presenilin 1.Nature. 1996; 383: 710-713Crossref PubMed Scopus (1331) Google Scholar). However, there is only limited insight into the cause of late-onset AD (LOAD), which contributes to more than 95% of cases. Genetic modifiers of LOAD may also regulate Aβ production, raising the possibility that genes regulating APP metabolism might impact the risk for AD (Andersen et al., 2005Andersen O.M. Reiche J. Schmidt V. Gotthardt M. Spoelgen R. Behlke J. von Arnim C.A. Breiderhoff T. Jansen P. Wu X. et al.Neuronal sorting protein-related receptor sorLA/LR11 regulates processing of the amyloid precursor protein.Proc. Natl. Acad. Sci. USA. 2005; 102: 13461-13466Crossref PubMed Scopus (497) Google Scholar, Rogaeva et al., 2007Rogaeva E. Meng Y. Lee J.H. Gu Y. Kawarai T. Zou F. Katayama T. Baldwin C.T. Cheng R. Hasegawa H. et al.The neuronal sortilin-related receptor SORL1 is genetically associated with Alzheimer disease.Nat. Genet. 2007; 39: 168-177Crossref PubMed Scopus (928) Google Scholar, Selkoe, 2011aSelkoe D.J. Alzheimer’s disease.Cold Spring Harb. Perspect. Biol. 2011; 3: a004457Crossref Scopus (319) Google Scholar). Several lines of evidence support an important role for membrane trafficking in the amyloidogenic processing of APP and hence in AD pathogenesis (Rajendran and Annaert, 2012Rajendran L. Annaert W. Membrane trafficking pathways in Alzheimer’s disease.Traffic. 2012; 13: 759-770Crossref PubMed Scopus (147) Google Scholar, Thinakaran and Koo, 2008Thinakaran G. Koo E.H. Amyloid precursor protein trafficking, processing, and function.J. Biol. Chem. 2008; 283: 29615-29619Crossref PubMed Scopus (859) Google Scholar). APP and BACE1 are transmembrane proteins that are synthesized in the endoplasmic reticulum (ER), matured in the Golgi complex, and then transported to the plasma membrane and into endosomes via endocytosis (Small and Gandy, 2006Small S.A. Gandy S. Sorting through the cell biology of Alzheimer’s disease: intracellular pathways to pathogenesis.Neuron. 2006; 52: 15-31Abstract Full Text Full Text PDF PubMed Scopus (259) Google Scholar, Thinakaran and Koo, 2008Thinakaran G. Koo E.H. Amyloid precursor protein trafficking, processing, and function.J. Biol. Chem. 2008; 283: 29615-29619Crossref PubMed Scopus (859) Google Scholar). The endolysosomal compartment has been implicated as one of the major sites for Aβ generation (Cataldo et al., 2000Cataldo A.M. Peterhoff C.M. Troncoso J.C. Gomez-Isla T. Hyman B.T. Nixon R.A. Endocytic pathway abnormalities precede amyloid beta deposition in sporadic Alzheimer’s disease and Down syndrome: differential effects of APOE genotype and presenilin mutations.Am. J. Pathol. 2000; 157: 277-286Abstract Full Text Full Text PDF PubMed Scopus (628) Google Scholar, Haass et al., 1992Haass C. Koo E.H. Mellon A. Hung A.Y. Selkoe D.J. Targeting of cell-surface beta-amyloid precursor protein to lysosomes: alternative processing into amyloid-bearing fragments.Nature. 1992; 357: 500-503Crossref PubMed Scopus (775) Google Scholar, Koo and Squazzo, 1994Koo E.H. Squazzo S.L. Evidence that production and release of amyloid beta-protein involves the endocytic pathway.J. Biol. Chem. 1994; 269: 17386-17389Abstract Full Text PDF PubMed Google Scholar). Recent work has revealed that BACE1 cleavage of APP occurs predominantly in early endosomes, and endocytosis of APP and BACE1 is essential for β cleavage of APP, and Aβ production (Kinoshita et al., 2003Kinoshita A. Fukumoto H. Shah T. Whelan C.M. Irizarry M.C. Hyman B.T. Demonstration by FRET of BACE interaction with the amyloid precursor protein at the cell surface and in early endosomes.J. Cell Sci. 2003; 116: 3339-3346Crossref PubMed Scopus (230) Google Scholar, Rajendran et al., 2006Rajendran L. Honsho M. Zahn T.R. Keller P. Geiger K.D. Verkade P. Simons K. Alzheimer’s disease beta-amyloid peptides are released in association with exosomes.Proc. Natl. Acad. Sci. USA. 2006; 103: 11172-11177Crossref PubMed Scopus (975) Google Scholar, Sannerud et al., 2011Sannerud R. Declerck I. Peric A. Raemaekers T. Menendez G. Zhou L. Veerle B. Coen K. Munck S. De Strooper B. et al.ADP ribosylation factor 6 (ARF6) controls amyloid precursor protein (APP) processing by mediating the endosomal sorting of BACE1.Proc. Natl. Acad. Sci. USA. 2011; 108: E559-E568Crossref PubMed Scopus (182) Google Scholar). The pH of endosomes (pH 4.0–5.0) is optimal for BACE1 activity, which also explains the requirement for endocytosis (Kalvodova et al., 2005Kalvodova L. Kahya N. Schwille P. Ehehalt R. Verkade P. Drechsel D. Simons K. Lipids as modulators of proteolytic activity of BACE: involvement of cholesterol, glycosphingolipids, and anionic phospholipids in vitro.J. Biol. Chem. 2005; 280: 36815-36823Crossref PubMed Scopus (254) Google Scholar, Vassar et al., 2009Vassar R. Kovacs D.M. Yan R. Wong P.C. The beta-secretase enzyme BACE in health and Alzheimer’s disease: regulation, cell biology, function, and therapeutic potential.J. Neurosci. 2009; 29: 12787-12794Crossref PubMed Scopus (451) Google Scholar). In contrast, α-secretase cleavage of APP, which precludes Aβ production, occurs at the plasma membrane (Lichtenthaler, 2011Lichtenthaler S.F. α-secretase in Alzheimer’s disease: molecular identity, regulation and therapeutic potential.J. Neurochem. 2011; 116: 10-21Crossref PubMed Scopus (154) Google Scholar). Components of the γ-secretase complex are also synthesized in the ER, but their assembly and maturation require the coordinated regulation of the ER-Golgi-recycling circuit (Spasic and Annaert, 2008Spasic D. Annaert W. Building gamma-secretase: the bits and pieces.J. Cell Sci. 2008; 121: 413-420Crossref PubMed Scopus (75) Google Scholar). We previously showed that β cleavage of APP occurs in a Rab5-EEA1-positive membrane compartment and that endocytosis is essential for Aβ generation (Rajendran et al., 2006Rajendran L. Honsho M. Zahn T.R. Keller P. Geiger K.D. Verkade P. Simons K. Alzheimer’s disease beta-amyloid peptides are released in association with exosomes.Proc. Natl. Acad. Sci. USA. 2006; 103: 11172-11177Crossref PubMed Scopus (975) Google Scholar). Targeting a transition-state BACE1 inhibitor to endosomes inhibited Aβ production in cultured cells and mice (Rajendran et al., 2008Rajendran L. Schneider A. Schlechtingen G. Weidlich S. Ries J. Braxmeier T. Schwille P. Schulz J.B. Schroeder C. Simons M. et al.Efficient inhibition of the Alzheimer’s disease beta-secretase by membrane targeting.Science. 2008; 320: 520-523Crossref PubMed Scopus (224) Google Scholar). Interestingly, proteins that belong to the retromer family, which transport cargo from early endosomes to the Golgi, have also been implicated in AD (Rogaeva et al., 2007Rogaeva E. Meng Y. Lee J.H. Gu Y. Kawarai T. Zou F. Katayama T. Baldwin C.T. Cheng R. Hasegawa H. et al.The neuronal sortilin-related receptor SORL1 is genetically associated with Alzheimer disease.Nat. Genet. 2007; 39: 168-177Crossref PubMed Scopus (928) Google Scholar, Small et al., 2005Small S.A. Kent K. Pierce A. Leung C. Kang M.S. Okada H. Honig L. Vonsattel J.P. Kim T.W. Model-guided microarray implicates the retromer complex in Alzheimer’s disease.Ann. Neurol. 2005; 58: 909-919Crossref PubMed Scopus (307) Google Scholar). These AD risk genes regulate the residency of APP and BACE1 in the early endosomal compartment, therefore regulating Aβ generation (Siegenthaler and Rajendran, 2012Siegenthaler B.M. Rajendran L. Retromers in Alzheimer’s disease.Neurodegener. Dis. 2012; 10: 116-121Crossref PubMed Scopus (28) Google Scholar). Similarly, proteins of the GGA family have been shown to traffic BACE1 from endosomes to the Golgi, and their depletion led to increased amyloidogenic processing of APP (He et al., 2005He X. Li F. Chang W.P. Tang J. GGA proteins mediate the recycling pathway of memapsin 2 (BACE).J. Biol. Chem. 2005; 280: 11696-11703Crossref PubMed Scopus (200) Google Scholar, Tesco et al., 2007Tesco G. Koh Y.H. Kang E.L. Cameron A.N. Das S. Sena-Esteves M. Hiltunen M. Yang S.H. Zhong Z. Shen Y. et al.Depletion of GGA3 stabilizes BACE and enhances beta-secretase activity.Neuron. 2007; 54: 721-737Abstract Full Text Full Text PDF PubMed Scopus (307) Google Scholar, von Arnim et al., 2006von Arnim C.A. Spoelgen R. Peltan I.D. Deng M. Courchesne S. Koker M. Matsui T. Kowa H. Lichtenthaler S.F. Irizarry M.C. Hyman B.T. GGA1 acts as a spatial switch altering amyloid precursor protein trafficking and processing.J. Neurosci. 2006; 26: 9913-9922Crossref PubMed Scopus (59) Google Scholar). Although APP is known to be routed from endosomes to Golgi via the retromer and retromer-associated proteins including SORL1 and VPS26 (Andersen et al., 2005Andersen O.M. Reiche J. Schmidt V. Gotthardt M. Spoelgen R. Behlke J. von Arnim C.A. Breiderhoff T. Jansen P. Wu X. et al.Neuronal sorting protein-related receptor sorLA/LR11 regulates processing of the amyloid precursor protein.Proc. Natl. Acad. Sci. USA. 2005; 102: 13461-13466Crossref PubMed Scopus (497) Google Scholar, Morel et al., 2013Morel E. Chamoun Z. Lasiecka Z.M. Chan R.B. Williamson R.L. Vetanovetz C. Dall’Armi C. Simoes S. Point Du Jour K.S. McCabe B.D. et al.Phosphatidylinositol-3-phosphate regulates sorting and processing of amyloid precursor protein through the endosomal system.Nat. Commun. 2013; 4: 2250Crossref PubMed Scopus (159) Google Scholar, Small and Gandy, 2006Small S.A. Gandy S. Sorting through the cell biology of Alzheimer’s disease: intracellular pathways to pathogenesis.Neuron. 2006; 52: 15-31Abstract Full Text Full Text PDF PubMed Scopus (259) Google Scholar, Rogaeva et al., 2007Rogaeva E. Meng Y. Lee J.H. Gu Y. Kawarai T. Zou F. Katayama T. Baldwin C.T. Cheng R. Hasegawa H. et al.The neuronal sortilin-related receptor SORL1 is genetically associated with Alzheimer disease.Nat. Genet. 2007; 39: 168-177Crossref PubMed Scopus (928) Google Scholar, Small et al., 2005Small S.A. Kent K. Pierce A. Leung C. Kang M.S. Okada H. Honig L. Vonsattel J.P. Kim T.W. Model-guided microarray implicates the retromer complex in Alzheimer’s disease.Ann. Neurol. 2005; 58: 909-919Crossref PubMed Scopus (307) Google Scholar, Siegenthaler and Rajendran, 2012Siegenthaler B.M. Rajendran L. Retromers in Alzheimer’s disease.Neurodegener. Dis. 2012; 10: 116-121Crossref PubMed Scopus (28) Google Scholar), nothing much is known about BACE1 recycling from endosomes. A better understanding of the specific trafficking mechanisms involved in Aβ production will thus provide further insights into disease pathogenesis and potentially provide novel therapeutic strategies for treating this currently untreatable disease. Rab GTPases regulate many aspects of membrane protein trafficking, acting as membrane organizers on cellular compartments that mediate vesicular trafficking and aid in vesicle fusion (Seabra et al., 2002Seabra M.C. Mules E.H. Hume A.N. Rab GTPases, intracellular traffic and disease.Trends Mol. Med. 2002; 8: 23-30Abstract Full Text Full Text PDF PubMed Scopus (412) Google Scholar). They regulate vesicular trafficking both in the biosynthetic and endocytic routes, enabling cargo sorting within the different membrane compartments (Zerial and McBride, 2001Zerial M. McBride H. Rab proteins as membrane organizers.Nat. Rev. Mol. Cell Biol. 2001; 2: 107-117Crossref PubMed Scopus (2742) Google Scholar). Rab GTPases switch between an inactive GDP-bound form and an active GTP-bound form, which enables vesicular fusion (Stenmark and Olkkonen, 2001Stenmark H. Olkkonen V.M. The Rab GTPase family.Genome Biol. 2001; 2 (REVIEWS3007)Crossref PubMed Google Scholar). Although GDP-GTP exchange is mediated by Rab-specific GTP exchange factors (GEFs), GTP hydrolysis is achieved by the GTPase-activating proteins (GAPs) (Barr and Lambright, 2010Barr F. Lambright D.G. Rab GEFs and GAPs.Curr. Opin. Cell Biol. 2010; 22: 461-470Crossref PubMed Scopus (340) Google Scholar). In humans, there are 60 Rab proteins and 39 RabGAPs. Overexpression of RabGAPs depletes the active form of Rab proteins and increases the inactive, GDP-bound form, thereby preventing their normal function (Yoshimura et al., 2007Yoshimura S. Egerer J. Fuchs E. Haas A.K. Barr F.A. Functional dissection of Rab GTPases involved in primary cilium formation.J. Cell Biol. 2007; 178: 363-369Crossref PubMed Scopus (282) Google Scholar). Here, we systematically analyzed the role of the cell’s major membrane-trafficking processes in Aβ production by the combined use of RNAi-mediated silencing of all Rab GTPase proteins and an overexpression screen of the RabGAPs. We first developed a multiplexing assay to quantitatively screen for Aβ and soluble ectodomain of APP β (sAPPβ), the two products of the amyloidogenic processing of APP (Figure 1A), from a single sample. Simultaneous measurements of Aβ and sAPPβ from a single sample in the same well would indicate whether a “hit” affected the β cleavage of APP (changing sAPPβ levels) or the γ cleavage/fate of Aβ (changing Aβ levels) (Figure 1B). Thus, intermeasurement variations are avoided, which increases the sensitivity and signal-to-noise ratio allowing high-throughput measurements. We used an electrochemiluminescence (ECL) assay platform because it provides high sensitivity and robust signal detection. In each well of a 384-well plate, one spot of anti-Aβ40 and one of anti-sAPPβ were separately spotted (Figure 1C). Two other spots were coated with BSA for background and nonspecific binding controls. Upon binding to the analytes in the samples, electrochemical signals from the ruthenium-labeled respective secondary antibodies were detected through an image-based capture. Because the captured antibodies are spatially positioned at distinct spots in the well, the values corresponded to the amount of the analyte bound to the specific capture antibody. As a proof of principle, we studied the binding of synthetic Aβ40 and recombinant sAPPβ in the same well. We incubated different concentrations of synthetic Aβ peptides in the assay plate and found a linear dependency of the signal to the amount of peptide used at concentrations lower than 50 ng/ml. At concentrations above 50 ng/ml, we observed saturation (Figure 1D). Similar saturation kinetics was observed with recombinant sAPPβ protein, suggesting that this system perfectly recapitulates binding reactions. When only Aβ was added to the well, background signals were observed for sAPPβ, and vice versa. When both Aβ and sAPPβ were added in the same well, specific signals were detected (Figure 1D), demonstrating a high degree of specificity and no cross-reactivity between the two analytes. In addition, ECL detection allowed us to detect concentrations as low as 10–100 pg of Aβ and sAPPβ. We then proceeded to see if Aβ and sAPPβ could be measured from cultured cells and neurons from transgenic mice. Both Aβ and sAPPβ could be measured from HEK and HeLa cells stably expressing the pathogenic Swedish mutant of APP, which causes familial early-onset AD (Figure 1E). Conditioned medium collected at different times shows that both Aβ and sAPPβ gradually accumulated in a time-dependent manner (Figure 1E). Increase in the volume of the conditioned supernatants produced a linear increase in the measurements demonstrating assay robustness (Figures S1A and S1B). We could also quantify sAPPβ and Aβ in cortical neurons from mice expressing human APP with Arctic/Swedish (Arc/Swe) mutations (Figure 1E). Sample-swapping (Figure 1F) experiments showed that no significant signal was observed for the β-cleaved ectodomain of Swedish APP (sAPPβsw) when measured with the plate coated in capture antibodies, which recognized the β-cleaved ectodomain of wild-type (WT) APP (sAPPβWT). Similarly, no signal was obtained for sAPPβ when we swapped the sAPPβWT supernatants on the sAPPβsw-coated plates. However, robust signals for Aβ were detected in all conditions. Thus, the assay is highly specific for both Aβ and sAPPβ. To study the role of all human Rab proteins in the regulation of amyloidogenic APP processing, we first performed an RNAi screen (Table S1) in cells that robustly produce Aβ and sAPPβ (Rajendran et al., 2006Rajendran L. Honsho M. Zahn T.R. Keller P. Geiger K.D. Verkade P. Simons K. Alzheimer’s disease beta-amyloid peptides are released in association with exosomes.Proc. Natl. Acad. Sci. USA. 2006; 103: 11172-11177Crossref PubMed Scopus (975) Google Scholar, Rajendran et al., 2008Rajendran L. Schneider A. Schlechtingen G. Weidlich S. Ries J. Braxmeier T. Schwille P. Schulz J.B. Schroeder C. Simons M. et al.Efficient inhibition of the Alzheimer’s disease beta-secretase by membrane targeting.Science. 2008; 320: 520-523Crossref PubMed Scopus (224) Google Scholar) and assayed these products using the multiplexing ECL assay system (Figures 2A and 2B ). We included APP, BACE1, and Pen2 (a subunit of the γ-secretase) as positive controls. As expected, silencing of APP and BACE1 decreased both Aβ and sAPPβ levels, whereas silencing of Pen2 decreased Aβ levels, but not sAPPβ levels (Figures 2A and 2B), further validating the assay. Quantification was based on the ECL counts, normalized to the cell viability counts, and relative to that of the scrambled control (medium GC containing siRNA oligo [MedGC]). Apart from Rab36, silencing of the other Rab proteins did not significantly alter cell viability (Figure S1C). The screen identified Rabs that significantly decreased both Aβ and sAPPβ (Figures 2A and 2B), including Rab3A, Rab11A, Rab36, and Rab17 (Figures S2A and S2B). Rab36, a GTPase involved in late endosome positioning, was a top hit in the screen, decreasing both Aβ and sAPPβ (Figure 2C). A secondary validation screen reproduced all the selected hits (Figure S2C). Knockdown efficiency of the relevant genes was confirmed by RT-PCR (Figure S2D). Although our analysis accounts for toxicity, and quantifies Aβ and sAPPβ as a relative count of viable cells, Rab36 depletion was consistently toxic in all the screens (Figure S1C). Therefore, we removed it from further analysis. Rab11A, the major Rab protein involved in the slow recycling of cargo proteins from early endosomes to the cell surface (Sönnichsen et al., 2000Sönnichsen B. De Renzis S. Nielsen E. Rietdorf J. Zerial M. Distinct membrane domains on endosomes in the recycling pathway visualized by multicolor imaging of Rab4, Rab5, and Rab11.J. Cell Biol. 2000; 149: 901-914Crossref PubMed Scopus (817) Google Scholar), was the second top hit in our analysis. In addition, silencing of the isoform, Rab11B, also reduced Aβ levels (Figure 2A). Interestingly, all the isoforms of Rab3, except Rab3C, decreased both Aβ and sAPPβ levels. Rab3 proteins are involved in synaptic function (Schlüter et al., 2004Schlüter O.M. Schmitz F. Jahn R. Rosenmund C. Südhof T.C. A complete genetic analysis of neuronal Rab3 function.J. Neurosci. 2004; 24: 6629-6637Crossref PubMed Scopus (221) Google Scholar) and in the fast axonal transport of APP (Szodorai et al., 2009Szodorai A. Kuan Y.H. Hunzelmann S. Engel U. Sakane A. Sasaki T. Takai Y. Kirsch J. Müller U. Beyreuther K. et al.APP anterograde transport requires Rab3A GTPase activity for assembly of the transport vesicle.J. Neurosci. 2009; 29: 14534-14544Crossref PubMed Scopus (95) Google Scholar). Silencing of Rab3A and Rab3B decreased overall APP levels, suggesting that Rab3 plays a role in the trafficking and maintenance of APP levels (Figure S2E). Silencing of Rab44, Rab6A, and Rab10 decreased Aβ levels without affecting sAPPβ (Figure 2C), implying that these GTPases affect either γ-secretase cleavage or the fate of Aβ. Interestingly, knockdowns of several Rabs also increased both sAPPβ and Aβ levels. To understand how these trafficking pathways contributed to both Aβ and sAPPβ levels, we plotted the normalized values of Aβ and sAPPβ from the screen in a 2D plot and observed a largely correlative curve for both the values, indicating that the Rab proteins responsible for regulating β cleavage are also responsible for controlling Aβ levels and that any perturbation at the level of BACE1 cleavage of APP is the rate-limiting step in Aβ production (Figure 2D). Because RNAi can have off-target effects leading to false-positive results, we complemented our RNAi screen with an overexpression screen of all 39 RabGAPs in the human genome, which suppress Rab function by accelerating the hydrolysis of GTP. In this screen, we overexpressed each RabGAP in the same cellular system used for the RNAi screen and quantitatively measured Aβ, sAPPβ levels, and cell viability (Figures 3 and S1D). Many RabGAPs were found to affect sAPPβ and Aβ levels (Figure 3). RN-TRE, a GAP that has previously been shown to affect amyloidogenic processing of APP (Ehehalt et al., 2003Ehehalt R. Keller P. Haass C. Thiele C. Simons K. Amyloidogenic processing of the Alzheimer beta-amyloid precursor protein depends on lipid rafts.J. Cell Biol. 2003; 160: 113-123Crossref PubMed Scopus (936) Google Scholar), reduced both Aβ and sAPPβ. TBC1D10B, a GAP for Rab35, Rab27A, Rab22A, Rab31, and Rab3A, decreased both Aβ and sAPPβ (Frasa et al., 2012Frasa M.A. Koessmeier K.T. Ahmadian M.R. Braga V.M. Illuminating the functional and structural repertoire of human TBC/RABGAPs.Nat. Rev. Mol. Cell Biol. 2012; 13: 67-73Crossref PubMed Scopus (24) Google Scholar). However, only Rab3A silencing via RNAi decreased Aβ and sAPPβ (Figures 2 and 3). EVI5, a RabGAP for Rab11 (Frasa et al., 2012Frasa M.A. Koessmeier K.T. Ahmadian M.R. Braga V.M. Illuminating the functional and structural repertoire of human TBC/RABGAPs.Nat. Rev. Mol. Cell Biol. 2012; 13: 67-73Crossref PubMed Scopus (24) Google Scholar, Laflamme et al., 2012Laflamme C. Assaker G. Ramel D. Dorn J.F. She D. Maddox P.S. Emery G. Evi5 promotes collective cell migration through its Rab-GAP activity.J. Cell Biol. 2012; 198: 57-67Crossref PubMed Scopus (46) Google Scholar), decreased both Aβ and sAPPβ. In addition, one other RabGAP for Rab11, namely TBC1D15, decreased both Aβ and sAPPβ. Intriguingly, TBC1D14, which alters Rab11 localization and subsequently delays recycling of transferrin to the plasma membrane (Longatti et al., 2012Longatti A. Lamb C.A. Razi M. Yoshimura S. Barr F.A. Tooze S.A. TBC1D14 regulates autophagosome formation via Rab11- and ULK1-positive recycling endosomes.J. Cell Biol. 2012; 197: 659-675Crossref PubMed Scopus (292) Google Scholar), decreased Aβ levels (Figure 3C). These results independently identified Rab11 as an important regulator of sAPPβ and Aβ production. We next analyzed whether the identified Rabs regulated Aβ production through the same trafficking pathway or through distinct mechanisms. To this end, we performed an epistasis mini array profiling (EMAP) screen (Schuldiner et al., 2005Schuldiner M. Collins S.R. Thompson N.J. Denic V. Bhamidipati A. Punna T. Ihmels J. Andrews B. Boone C. Greenblatt J.F. et al.Exploration of the function and organization of the yeast early secretory pathway through an epistatic miniarray profile.Cell. 2005; 123: 507-519Abstract Full Text Full Text PDF PubMed Scopus (697) Google Scholar) wherein we silenced all the hits against all the hits in a 14 × 14 matrix to determine whether a combined knockdown gives rise to an aggravating (noninteracting) or alleviating (interacting) phenotype (i.e., β cleavage of APP). Here, again, single knockdowns of either Rab3A or Rab11A decreased sAPPβ levels (Figure S3). However, when they were silenced together, a further decrease (an aggravating phenotype) was observed, which indicates that Rab3A and Rab11A act independently and contribute to β cleavage via distinct trafficking routes. Most of the genes that were identified as hits did not interact with each other, with the exception that a double knockdown of Rab10/Rab23 or Rab10/Rab25 showed an alleviating phenotype, suggesting that these Rabs regulate the same membrane-trafficking pathways. Because Rab11A was identified as one of the strongest hits in both the RNAi screen and the RabGAP screen, we independently validated this finding in a different cellular model. siRNA-mediated silencing of Rab11A in HEK cells stably exp" @default.
• W2078201172 created "2016-06-24" @default.
• W2078201172 creator A5002376486 @default.
• W2078201172 creator A5006901857 @default.
• W2078201172 creator A5007160724 @default.
• W2078201172 creator A5015618876 @default.
• W2078201172 creator A5020514779 @default.
• W2078201172 creator A5025901197 @default.
• W2078201172 creator A5028735345 @default.
• W2078201172 creator A5029363545 @default.
• W2078201172 creator A5037652352 @default.
• W2078201172 creator A5051824651 @default.
• W2078201172 creator A5052458149 @default.
• W2078201172 creator A5065971643 @default.
• W2078201172 creator A5079565983 @default.
• W2078201172 creator A5089003606 @default.
• W2078201172 creator A5090063659 @default.
• W2078201172 date "2013-12-01" @default.
• W2078201172 modified "2023-10-16" @default.
• W2078201172 title "A Paired RNAi and RabGAP Overexpression Screen Identifies Rab11 as a Regulator of β-Amyloid Production" @default.
• W2078201172 cites W1502820594 @default.
• W2078201172 cites W1537234916 @default.
• W2078201172 cites W1595083713 @default.
• W2078201172 cites W1681258590 @default.
• W2078201172 cites W1968900405 @default.
• W2078201172 cites W1969438 @default.
• W2078201172 cites W1974507908 @default.
• W2078201172 cites W1977570151 @default.
• W2078201172 cites W1978340451 @default.
• W2078201172 cites W1978605742 @default.
• W2078201172 cites W1979751563 @default.
• W2078201172 cites W1992515094 @default.
• W2078201172 cites W1993168711 @default.
• W2078201172 cites W2002181764 @default.
• W2078201172 cites W2005681172 @default.
• W2078201172 cites W2011424449 @default.
• W2078201172 cites W2014064711 @default.
• W2078201172 cites W2019821944 @default.
• W2078201172 cites W2023705774 @default.
• W2078201172 cites W2031337312 @default.
• W2078201172 cites W2040732053 @default.
• W2078201172 cites W2042600422 @default.
• W2078201172 cites W2042688172 @default.
• W2078201172 cites W2047198245 @default.
• W2078201172 cites W2047418563 @default.
• W2078201172 cites W2050946031 @default.
• W2078201172 cites W2053210112 @default.
• W2078201172 cites W2054174655 @default.
• W2078201172 cites W2054615702 @default.
• W2078201172 cites W2058880763 @default.
• W2078201172 cites W2061014101 @default.
• W2078201172 cites W2062793212 @default.
• W2078201172 cites W2067811139 @default.
• W2078201172 cites W2067914995 @default.
• W2078201172 cites W2068208310 @default.
• W2078201172 cites W2070596032 @default.
• W2078201172 cites W2073206959 @default.
• W2078201172 cites W2076185491 @default.
• W2078201172 cites W2082387821 @default.
• W2078201172 cites W2084121214 @default.
• W2078201172 cites W2094625991 @default.
• W2078201172 cites W2095745901 @default.
• W2078201172 cites W2099634214 @default.
• W2078201172 cites W2101468172 @default.
• W2078201172 cites W2104720558 @default.
• W2078201172 cites W2106745747 @default.
• W2078201172 cites W2106903768 @default.
• W2078201172 cites W2110246051 @default.
• W2078201172 cites W2110649177 @default.
• W2078201172 cites W2110747905 @default.
• W2078201172 cites W2112369131 @default.
• W2078201172 cites W2119487442 @default.
• W2078201172 cites W2123941817 @default.
• W2078201172 cites W2126942996 @default.
• W2078201172 cites W2131097779 @default.
• W2078201172 cites W2133019124 @default.
• W2078201172 cites W2139595777 @default.
• W2078201172 cites W2140259754 @default.
• W2078201172 cites W2142672347 @default.
• W2078201172 cites W2147958725 @default.
• W2078201172 cites W2148745491 @default.
• W2078201172 cites W2152148024 @default.
• W2078201172 cites W2154755614 @default.
• W2078201172 cites W2156281143 @default.
• W2078201172 cites W2157860584 @default.
• W2078201172 cites W2159367229 @default.
• W2078201172 cites W2159972803 @default.
• W2078201172 cites W2164646446 @default.
• W2078201172 cites W2166595775 @default.
• W2078201172 cites W2167221791 @default.
• W2078201172 cites W2169555689 @default.
• W2078201172 cites W4231182105 @default.
• W2078201172 cites W4233741900 @default.
• W2078201172 cites W4238609105 @default.
• W2078201172 doi "https://doi.org/10.1016/j.celrep.2013.12.005" @default.
• W2078201172 hasPubMedCentralId "https://www.ncbi.nlm.nih.gov/pmc/articles/4004174" @default.
• W2078201172 hasPubMedId "https://pubmed.ncbi.nlm.nih.gov/24373285" @default.
• W2078201172 hasPublicationYear "2013" @default.

• next