Skip to content
← Back to browse
BRAIN Publication-derived

Acetylation changes tau interactome to degrade tau in Alzheimer's disease animal and organoid models

Source Choi et al., 2020 · Department of Biochemistry and Biomedical Sciences, College of Medicine, Seoul National University · 10.1111/acel.13081

👤 Heesun Choi, Haeng Jun Kim, Jinhee Yang, Sehyun Chae, Wonik Lee, Sunwoo Chung, Jisoo Kim, Hyunjung Choi, Hyeseung Song, Chang Kon Lee, Jae Hyun Jun, Yong Jae Lee, Kyunghyeon Lee, Semi Kim, Hye-ri Sim, Young Il Choi, Keun Ho Ryu, Jong-Chan Park, Dongjoon Lee, Sun-Ho Han, Daehee Hwang, Jangbeen Kyung, Inhee Mook-Jung ⏱ 70 days 📋 9 phases 🧫 Patient-Derived iPSC (Alzheimer's disease)

Abstract

This protocol demonstrates the generation and characterization of Alzheimer's disease patient-derived brain organoids and their treatment with CKD-504, an HDAC6 inhibitor, to induce tau degradation through acetylation-dependent chaperone network remodeling. The protocol validates tau reduction and interactome changes in both organoid and ADLP[APT] mouse models, showing therapeutic potential for tau pathology reversal.

Cell source
Patient-Derived iPSC (Alzheimer's disease)
Application
Disease modeling; Drug screening

Protocol overview

50 steps across 9 phases

Generation of human brain cortical organoids from hiPSCs Days 0–70
  1. 1 Obtain patient-derived hiPSCs
  2. 2 Form embryoid bodies (EBs)
  3. 3 Transition floating spheroids to neuronal medium
  4. 4 Replace growth factors to promote neural differentiation
  5. 5 Maintain organoids until differentiation
Treatment of brain organoids with CKD-504 Days 65–70
  1. 1 Prepare CKD-504 treatment solution
  2. 2 Treat organoids with CKD-504
  3. 3 Incubate organoids for tau degradation assay
  4. 4 Harvest organoids for analysis
Animal studies: preventive experimental paradigm in ADLP[APT] mice Starting at 4.5 months of age, duration 4 months
  1. 1 Prepare CKD-504 for intraperitoneal injection
  2. 2 Begin intraperitoneal injection protocol
  3. 3 Perform Y-maze cognitive test
  4. 4 Perform contextual fear conditioning (CFC) test
  5. 5 Harvest brain tissue at study endpoint
Animal studies: therapeutic experimental paradigm in ADLP[APT] mice Starting at 6.5 months of age, duration 2 months
  1. 1 Prepare CKD-504 for therapeutic dosing
  2. 2 Begin intraperitoneal injection in symptomatic mice
  3. 3 Perform Y-maze cognitive test
  4. 4 Perform contextual fear conditioning test
  5. 5 Perform Golgi–Cox staining for dendritic spine analysis
  6. 6 Harvest brain tissue at study endpoint
Biochemical analyses: Western blotting and tau fractionation Post-harvest processing
  1. 1 Prepare tissue lysates or organoid homogenates
  2. 2 Perform tau fractionation using sarkosyl extraction
  3. 3 Perform Western blotting for tau detection
  4. 4 Detect acetylated proteins and tau interactions
  5. 5 Quantify and analyze Western blot data
Immunohistochemical analysis of tau pathology Post-harvest processing
  1. 1 Prepare tissue sections for immunohistochemistry
  2. 2 Block nonspecific binding and permeabilize
  3. 3 Apply primary antibodies against tau and neuronal markers
  4. 4 Apply secondary antibodies and fluorescent detection
  5. 5 Mount and image tissue sections
  6. 6 Quantify tau pathology immunostaining
Tau interactome analysis by mass spectrometry Post-harvest processing
  1. 1 Prepare lysates from treated mouse brains
  2. 2 Perform co-immunoprecipitation with Tau-13 antibody
  3. 3 Wash and elute immunoprecipitated complexes
  4. 4 Prepare samples for liquid chromatography-tandem mass spectrometry (LC-MS/MS)
  5. 5 Perform LC-MS/MS analysis
  6. 6 Analyze MS data and identify differentially interacting proteins
  7. 7 Validate identified interactions by Western blotting
Functional validation: E3 ligase activity and tau degradation assays Post-harvest and in vitro cell culture
  1. 1 Prepare HT22 cells overexpressing tau mutant (P301L) or WT tau
  2. 2 Perform cycloheximide (CHX) chase assay to measure tau turnover
  3. 3 Quantify tau protein remaining at each time point
  4. 4 Measure effect of CKD-504 on tau degradation
  5. 5 Assess effect of E3 ligase overexpression on tau ubiquitination
  6. 6 Analyze tau level changes with E3 ligase expression
Validation of acetylation-dependent tau interactions and degradation In vitro cell culture
  1. 1 Generate tau acetyl-mimic and acetyl-silencing mutants
  2. 2 Transfect HT22 cells with tau mutant plasmids
  3. 3 Assess tau-chaperone interactions in mutants
  4. 4 Measure tau turnover rates in mutants
  5. 5 Test CKD-504 effects on acetyl-mimic and acetyl-silencing mutants
  6. 6 Analyze acetylation levels in tau mutants

Full SOP

🔬

Create a free account to access this protocol

Join OrganMatch to unlock step-by-step procedures, reagent concentrations, QC checklists, and downloadable batch record templates.

Create free account

Already registered? Log in

Attribution

This SOP was authored by Organthis based on the published method in Choi et al., 2020. The originating laboratory holds no rights in this SOP and has not endorsed it unless marked Verified.

This wording is awaiting legal review.

Something wrong with this entry? Report an issue with this protocol

Need a commercial licence?
Use this protocol in your therapeutic or diagnostic pipeline.
Request Commercial Licence