Background Alpha-synuclein(αSyn)is a major player in the pathophysiology of synucleinopathies,which include Parkinson’s disease,dementia with Lewy bodies,and multiple system atrophy.To date,there is no disease-modif...Background Alpha-synuclein(αSyn)is a major player in the pathophysiology of synucleinopathies,which include Parkinson’s disease,dementia with Lewy bodies,and multiple system atrophy.To date,there is no disease-modifying therapy available for these synucleinopathies.Furthermore,the intracellular mechanisms by whichαSyn confers toxicity are not yet fully understood.Therefore,it is of utmost importance to investigate the pathophysiology ofαSyninduced toxicity in order to identify novel molecular targets for the development of disease-modifying therapies.Methods We performed the first genome-wide siRNA modifier screening in a human postmitotic neuronal cell model usingαSyn-induced toxicity as a read-out.In a multi-step approach,we identified several genes,whose knockdown protected againstαSyn-induced toxicity.The main hit was further validated by different methods,including immunofluorescence microscopy,qPCR,and Western blot.Furthermore,the main finding was confirmed in mouse primary neurons.Results The highest protection was achieved by knockdown of SNX5,which encodes the sorting nexin 5(SNX5)protein,a component of the retromer complex.The protective efficacy of SNX5 knockdown was confirmed with an independent siRNA system.The protective effect of SNX5 knockdown was further confirmed in primary neurons from transgenic mice,where the knockdown of SNX5 led to amelioration of decrease in synchrony that was observed in untreated and control-siRNA-treated cells.SNX5 protein is a component of the SNX-BAR(Bin/Amphiphysin/Rvs)heterodimer,which is part of the retromer complex.ExtracellularαSyn and overexpression of intracellularαSyn led to fragmentation of the trans-Golgi network,which was prevented by SNX5 knockdown that led to confinement ofαSyn in early endosomes.Conclusion In summary,our data suggest that SNX5 plays an important role in the trafficking and toxicity ofαSyn.Therefore,SNX5 appears to be a target of therapeutic intervention for synucleinopathies.展开更多
The membrane trafficking of cation-independent mannose 6-phosphate receptor（CI-M6PR） between the transGolgi network（TGN） and endosomal compartments is not only critical for maintaining lysosomal function but also ...The membrane trafficking of cation-independent mannose 6-phosphate receptor（CI-M6PR） between the transGolgi network（TGN） and endosomal compartments is not only critical for maintaining lysosomal function but also a well-known event for understanding molecular and cellular mechanisms in retrograde endosome-to-TGN trafficking.Although it has been well established in literature that the C-terminus of bovine CI-M6PR determines its retrograde trafficking,it remains unclear whether the luminal domain of the protein plays a role on these sorting events.In this study,we found that partial deletion of luminal domain of human CI-M6PR mistargeted the mutant protein to nonTGN compartments.Moreover,replacing the luminal domain of both bovine and human CI-M6PR with that from irrelevant membrane proteins such as CD8 or Tac also altered the TGN targeting of the chimeric proteins.On the other hand,only short sequence from HA fused with the transmembrane domain and C-terminus of the receptor,HA-hCIM6PR-tail,resulted in its preferential targeting to TGN as for the full length receptor,strongly suggesting that sorting of the receptor may be influenced by luminal sequence.Furthermore,using this luminal truncated form of HA-hCIM6 PR as a model cargo,we found that the trafficking of the chimeric protein was regulated by the retromer complex through interacting with SNX5.In conclusion,our study strongly suggested that the disrupted luminal domain from hCI-M6PR or other irrelevant membrane proteins interfere with the process of membrane trafficking and TGN targeting of CI-M6PR.展开更多
The endosomal trafficking of signaling membrane proteins, such as receptors, transporters and channels, is mediated by the retromer-mediated sorting machinery, composed of a cargo-selective vacuolar protein sorting tr...The endosomal trafficking of signaling membrane proteins, such as receptors, transporters and channels, is mediated by the retromer-mediated sorting machinery, composed of a cargo-selective vacuolar protein sorting trimer and a membrane-deforming subunit of sorting nexin proteins. Recent studies have shown that the isoforms, sorting nexin 5 (SNX5) and SNX6, have played distinctive regulatory roles in retrograde membrane trafficking. However, the molecular insight determined functional differences within the proteins remains unclear. We reported that SNX5 and SNX6 had distinct binding affinity to the cargo protein vesicular monoamine transporter 2 (VMAT2). SNX5, but not SNX6, specifically interacted with VMAT2 through the Phox domain, which contains an alpha-helix binding motif. Using chimeric mutagenesis, we identified that several key residues within this domain were unique in SNX5, but not SNX6, and played an auxiliary role in its binding to VMAT2. Importantly, we generated a set of mutant SNX6, in which the corresponding key residues were mutated to those in SNX5. In addition to the gain in binding affinity to VMAT2, their overexpression functionally rescued the altered retrograde trafficking of VMAT2 induced by siRNA-mediated depletion of SNX5. These data strongly suggest that SNX5 and SNX6 have different functions in retrograde membrane trafficking, which is determined by the different structural elements within the Phox domain of two proteins. Our work provides a new information on the role of SNX5 and SNX6 in the molecular regulation of retrograde membrane trafficking and vesicular membrane targeting in monoamine neurotransmission and neurological diseases.展开更多
基金funded by the Deutsche Forschungsgemeinschaft(DFG,German Research Foundation)under Germany’s Excellence Strategy within the framework of the Munich Cluster for Systems Neurology(EXC 2145 SyNergy-ID 390857198)within the Hannover Cluster RESIST(EXC 2155--project number 39087428)+6 种基金the German Federal Ministry of Education and Research(BMBF,01 KU1403 A EpiPD)the ParkinsonFonds Germany(Hypothesis-free compound screen,alpha-Synuclein fragments in PD)Deutsche Forschungsgemeinschaft(DFG,HO2402/18-1 MSAomics)VolkswagenStiftung(Niedersachsisches Vorab)Petermax-Muller Foundation(Etiology and Therapy of Synucleinopathies and Tauopathies)the German Federal Ministry of Education and Research(BMBF,JPND Consortium‚SynOD"alpha-Synuclein OMICS to identify Drug-targets"(01ED2405A)Harmonisierung und Re-Analyse vorhandener Datensatze aus einem Zellkulturmodel fur Synucleinopathien(01ED2405B)).
文摘Background Alpha-synuclein(αSyn)is a major player in the pathophysiology of synucleinopathies,which include Parkinson’s disease,dementia with Lewy bodies,and multiple system atrophy.To date,there is no disease-modifying therapy available for these synucleinopathies.Furthermore,the intracellular mechanisms by whichαSyn confers toxicity are not yet fully understood.Therefore,it is of utmost importance to investigate the pathophysiology ofαSyninduced toxicity in order to identify novel molecular targets for the development of disease-modifying therapies.Methods We performed the first genome-wide siRNA modifier screening in a human postmitotic neuronal cell model usingαSyn-induced toxicity as a read-out.In a multi-step approach,we identified several genes,whose knockdown protected againstαSyn-induced toxicity.The main hit was further validated by different methods,including immunofluorescence microscopy,qPCR,and Western blot.Furthermore,the main finding was confirmed in mouse primary neurons.Results The highest protection was achieved by knockdown of SNX5,which encodes the sorting nexin 5(SNX5)protein,a component of the retromer complex.The protective efficacy of SNX5 knockdown was confirmed with an independent siRNA system.The protective effect of SNX5 knockdown was further confirmed in primary neurons from transgenic mice,where the knockdown of SNX5 led to amelioration of decrease in synchrony that was observed in untreated and control-siRNA-treated cells.SNX5 protein is a component of the SNX-BAR(Bin/Amphiphysin/Rvs)heterodimer,which is part of the retromer complex.ExtracellularαSyn and overexpression of intracellularαSyn led to fragmentation of the trans-Golgi network,which was prevented by SNX5 knockdown that led to confinement ofαSyn in early endosomes.Conclusion In summary,our data suggest that SNX5 plays an important role in the trafficking and toxicity ofαSyn.Therefore,SNX5 appears to be a target of therapeutic intervention for synucleinopathies.
基金supported by the National Nature Science Foundation of China to Y.Liu(Grant No.31371436 and No.8157051134)Y.Huang(Grant No.81500678)the laboratory start-up grant from Nanjing Medical University to Y.Liu
文摘The membrane trafficking of cation-independent mannose 6-phosphate receptor（CI-M6PR） between the transGolgi network（TGN） and endosomal compartments is not only critical for maintaining lysosomal function but also a well-known event for understanding molecular and cellular mechanisms in retrograde endosome-to-TGN trafficking.Although it has been well established in literature that the C-terminus of bovine CI-M6PR determines its retrograde trafficking,it remains unclear whether the luminal domain of the protein plays a role on these sorting events.In this study,we found that partial deletion of luminal domain of human CI-M6PR mistargeted the mutant protein to nonTGN compartments.Moreover,replacing the luminal domain of both bovine and human CI-M6PR with that from irrelevant membrane proteins such as CD8 or Tac also altered the TGN targeting of the chimeric proteins.On the other hand,only short sequence from HA fused with the transmembrane domain and C-terminus of the receptor,HA-hCIM6PR-tail,resulted in its preferential targeting to TGN as for the full length receptor,strongly suggesting that sorting of the receptor may be influenced by luminal sequence.Furthermore,using this luminal truncated form of HA-hCIM6 PR as a model cargo,we found that the trafficking of the chimeric protein was regulated by the retromer complex through interacting with SNX5.In conclusion,our study strongly suggested that the disrupted luminal domain from hCI-M6PR or other irrelevant membrane proteins interfere with the process of membrane trafficking and TGN targeting of CI-M6PR.
基金This work was supported by the National Natural Science Foundation of China(Grant Nos.31371436 and 8157051134 to Y.L.)by the laboratory start-up grant from Nanjing Medical University(to Y.L.).
文摘The endosomal trafficking of signaling membrane proteins, such as receptors, transporters and channels, is mediated by the retromer-mediated sorting machinery, composed of a cargo-selective vacuolar protein sorting trimer and a membrane-deforming subunit of sorting nexin proteins. Recent studies have shown that the isoforms, sorting nexin 5 (SNX5) and SNX6, have played distinctive regulatory roles in retrograde membrane trafficking. However, the molecular insight determined functional differences within the proteins remains unclear. We reported that SNX5 and SNX6 had distinct binding affinity to the cargo protein vesicular monoamine transporter 2 (VMAT2). SNX5, but not SNX6, specifically interacted with VMAT2 through the Phox domain, which contains an alpha-helix binding motif. Using chimeric mutagenesis, we identified that several key residues within this domain were unique in SNX5, but not SNX6, and played an auxiliary role in its binding to VMAT2. Importantly, we generated a set of mutant SNX6, in which the corresponding key residues were mutated to those in SNX5. In addition to the gain in binding affinity to VMAT2, their overexpression functionally rescued the altered retrograde trafficking of VMAT2 induced by siRNA-mediated depletion of SNX5. These data strongly suggest that SNX5 and SNX6 have different functions in retrograde membrane trafficking, which is determined by the different structural elements within the Phox domain of two proteins. Our work provides a new information on the role of SNX5 and SNX6 in the molecular regulation of retrograde membrane trafficking and vesicular membrane targeting in monoamine neurotransmission and neurological diseases.
