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BRAIN Publication-derived

Alzheimer's Disease Brain Organoids for Drug Screening

Full SOP with Academic or a plan

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

Authors: 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 Duration: 70 days 9 phases Cell source: 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

Phase 1 · Generation of human brain cortical organoids from hiPSCs Days 0-70
  1. a Obtain patient-derived hiPSCs
  2. b Form embryoid bodies (EBs)
  3. c Transition floating spheroids to neuronal medium
  4. d Replace growth factors to promote neural differentiation
  5. e Maintain organoids until differentiation
Phase 2 · Treatment of brain organoids with CKD-504 Days 65-70
  1. a Prepare CKD-504 treatment solution
  2. b Treat organoids with CKD-504
  3. c Incubate organoids for tau degradation assay
  4. d Harvest organoids for analysis
Phase 3 · Animal studies: preventive experimental paradigm in ADLP[APT] mice Starting at 4.5 months of age, duration 4 months
  1. a Prepare CKD-504 for intraperitoneal injection
  2. b Begin intraperitoneal injection protocol
  3. c Perform Y-maze cognitive test
  4. d Perform contextual fear conditioning (CFC) test
  5. e Harvest brain tissue at study endpoint
Phase 4 · Animal studies: therapeutic experimental paradigm in ADLP[APT] mice Starting at 6.5 months of age, duration 2 months
  1. a Prepare CKD-504 for therapeutic dosing
  2. b Begin intraperitoneal injection in symptomatic mice
  3. c Perform Y-maze cognitive test
  4. d Perform contextual fear conditioning test
  5. e Perform Golgi–Cox staining for dendritic spine analysis
  6. f Harvest brain tissue at study endpoint
Phase 5 · Biochemical analyses: Western blotting and tau fractionation Post-harvest processing
  1. a Prepare tissue lysates or organoid homogenates
  2. b Perform tau fractionation using sarkosyl extraction
  3. c Perform Western blotting for tau detection
  4. d Detect acetylated proteins and tau interactions
  5. e Quantify and analyze Western blot data
Phase 6 · Immunohistochemical analysis of tau pathology Post-harvest processing
  1. a Prepare tissue sections for immunohistochemistry
  2. b Block nonspecific binding and permeabilize
  3. c Apply primary antibodies against tau and neuronal markers
  4. d Apply secondary antibodies and fluorescent detection
  5. e Mount and image tissue sections
  6. f Quantify tau pathology immunostaining
Phase 7 · Tau interactome analysis by mass spectrometry Post-harvest processing
  1. a Prepare lysates from treated mouse brains
  2. b Perform co-immunoprecipitation with Tau-13 antibody
  3. c Wash and elute immunoprecipitated complexes
  4. d Prepare samples for liquid chromatography-tandem mass spectrometry (LC-MS/MS)
  5. e Perform LC-MS/MS analysis
  6. f Analyze MS data and identify differentially interacting proteins
  7. g Validate identified interactions by Western blotting
Phase 8 · Functional validation: E3 ligase activity and tau degradation assays Post-harvest and in vitro cell culture
  1. a Prepare HT22 cells overexpressing tau mutant (P301L) or WT tau
  2. b Perform cycloheximide (CHX) chase assay to measure tau turnover
  3. c Quantify tau protein remaining at each time point
  4. d Measure effect of CKD-504 on tau degradation
  5. e Assess effect of E3 ligase overexpression on tau ubiquitination
  6. f Analyze tau level changes with E3 ligase expression
Phase 9 · Validation of acetylation-dependent tau interactions and degradation In vitro cell culture
  1. a Generate tau acetyl-mimic and acetyl-silencing mutants
  2. b Transfect HT22 cells with tau mutant plasmids
  3. c Assess tau-chaperone interactions in mutants
  4. d Measure tau turnover rates in mutants
  5. e Test CKD-504 effects on acetyl-mimic and acetyl-silencing mutants
  6. f Analyze acetylation levels in tau mutants

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Attribution

This SOP was written by Organthis based on the published method in Choi et al., 2020. Once the originating laboratory has confirmed it, the SOP is marked Verified.

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