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标题:Stress-Induced Cellular Clearance Is Mediated by the SNARE Protein ykt6 and Disrupted by α-Synuclein
时间:2020-07-28 11:20:40
DOI:10.1016/j.neuron.2019.09.001
PMID:31648898
大小:6449 kb
页数:29 PAGES
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目录:
  • Stress-Induced Cellular Clearance Is Mediated by the SNARE Protein ykt6 and Disrupted by α-Synuclein
    • Introduction
    • Results
      • Lysosomal Dysfunction Occurs Subsequent to α-Syn Aggregation in PD Patient Midbrain Cultures
      • ykt6 Is Disrupted by α-syn and Is Deficient in PD Brain
      • ykt6 Knockdown Preferentially Disrupts Lysosomal Protein Trafficking and Function
      • ykt6 Is Required for the Physiological Response to Lysosomal Stress in Human Midbrain Neurons
      • ykt6 Rescues Lysosomal Function in PD iPSns and Is Enhanced by Blocking Its Farnesylation
      • ykt6 Can Be Therapeutically Targeted by Farnesyltransferase Inhibitors
      • Farnesyltransferase Inhibition Activates ykt6 In Vivo and Reduces Pathological Aggregation in Mice
    • Discussion
    • Supplemental Information
    • Acknowledgments
    • Author Contributions
    • Declaration of Interests
    • References
    • STAR★Methods
      • Key Resources Table
      • Lead Contact and Materials Availability
      • Experimental Model and Subject Details
        • Overview of models employed in this study
        • H4 cell culture
        • Generation of stable-transfected SH-SY5Y cell lines
        • iPS cell culture and neuronal differentiation
        • Control and transgenic α-synuclein mouse lines
      • Method Details
        • CRISPR/Cas9n of iPSCs
          • T7 Endonuclease I Assay for the analysis of off-target effects of α-syn knockout iPSC
        • Biochemistry and cell biology
          • Generation of plasmids
          • Co-immunoprecipitation analysis
          • Separation of lysates into cytosol and membrane fractions
          • Size exclusion chromatography analysis
          • Knockdown of ykt6 shRNA expressing constructs
          • Measurement of farnesyl-ykt6 in SH-SY5Y cells
          • Measurement of Neuroserpin, LAMP1, GCase protein and activity from culture media
          • Measurement of mRNA
          • Analysis of post-ER GCase
          • Sequential biochemical extraction for the analysis of pathological α-syn
          • Western blot analysis
          • Measurement of neuron viability
          • Cell surface biotinylation assay
        • Live-cell assays
          • Live cell proteolysis assay and analysis of constitutive protein secretion
          • Live cell GCase activity assay
          • Live cell ER-Golgi trafficking assay
        • Image analyses
          • Immunofluorescence analysis of cell cultures
          • Immunohistochemistry of DASYN53 mice
          • Quantitative immunohistochemistry of PDGF-α-syn Tg mice
        • Lentivirus treatment of cultures
          • Lentiviral transduction of cell lines and iPSC-neurons
        • FTI treatment of cultures and mice
          • Treatment of cultures with farnesyltransferase inhibitors
          • Treatment of C57BL/6 and DASYN53 mice with farnesyltransferase inhibitors
          • Experimental design of FTI treatment in wild-type α-syn transgenic mice driven by the PDGF-β promoter
        • Pharmacokinetic analysis
        • Behavioral Analysis
          • Behavioral analysis of DASYN53 mice
      • Quantification and Statistical Analysis
      • Data and Code Availability

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