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BRAIN Publication-derived

Optogenetics-Assisted Alpha-Synuclein Aggregation Induction System (OASIS) for Parkinson's Disease Drug Discovery and Validation

Source Kim et al., 2023 · Johns Hopkins University School of Medicine · 10.1016/j.stem.2023.05.015

👤 Min Seong Kim, Eun A. Ra, Sin Ho Kweon, Bo Am Seo, Han Seok Ko, Yohan Oh, Gabsang Lee ⏱ 210 days 📋 11 phases 🧫 Patient-Derived iPSC (Parkinson's disease)

Abstract

OASIS is an optogenetics-based system that uses blue light to rapidly induce pathological alpha-synuclein aggregation in midbrain dopaminergic neurons differentiated from PD patient iPSCs and in midbrain organoids. The system enables high-content imaging drug screening to identify neuroprotective compounds that reverse PD-associated pathology.

Cell source
Patient-Derived iPSC (Parkinson's disease)
Application
Disease modeling and drug screening

Protocol overview

82 steps across 11 phases

Generation of opto-α-synuclein knockin iPSC lines Days 1-4
  1. 1 Dissociate SNCA triplication PD hiPSCs into single cells
  2. 2 Prepare nucleofection mixture
  3. 3 Perform nucleofection
  4. 4 Plate nucleofected cells on puromycin-resistant MEFs
  5. 5 Select homologous recombinants with puromycin
Differentiation of hiPSCs to midbrain dopaminergic neurons Days 1-30
  1. 1 Plate dissociated hiPSCs on Geltrex
  2. 2 Initiate neural induction
  3. 3 Add floor plate specification factors (Days 1-7)
  4. 4 Add CHIR99021 (Days 3-11)
  5. 5 Gradual medium replacement (Days 5-11)
  6. 6 Expand neural progenitors (Day 11)
  7. 7 Maturation to dopaminergic neurons (Days 11-30+)
Generation of midbrain organoids Days 1-90
  1. 1 Dissociate PD hiPSCs into single cells
  2. 2 Generate embryoid bodies (Day 1)
  3. 3 Switch to brain organoid generation medium (Day 2)
  4. 4 Add mesencephalic specification factors (Day 4)
  5. 5 Embed organoids in Geltrex (Day 7)
  6. 6 Transfer to petri dishes (Day 7)
  7. 7 Transfer to maturation medium (Day 9)
  8. 8 Mature organoids on orbital shaker (Days 9-90)
  9. 9 Prepare organoids for analysis (Day 90)
Blue light illumination and opto-α-synuclein aggregation induction Days 1-7
  1. 1 Culture opto-α-syn mDA neurons
  2. 2 Set up blue light illumination
  3. 3 Illuminate with blue light (12-well/24-well plates)
  4. 4 Illuminate with blue light (96-well plates for screening)
  5. 5 Monitor aggregate formation
Immunocytochemistry and aggregate detection Days 1-2
  1. 1 Fix cells in paraformaldehyde
  2. 2 Permeabilize and block cells
  3. 3 Incubate with primary antibodies
  4. 4 Incubate with secondary antibodies
  5. 5 Thioflavin S staining (alternative for amyloid aggregates)
  6. 6 Analyze with fluorescence microscopy
High-content imaging screening assay Days 1-2
  1. 1 Seed opto-α-syn SH-SY5Y cells
  2. 2 Incubate cells overnight
  3. 3 Add test compounds
  4. 4 Induce aggregation with blue light
  5. 5 Fix cells
  6. 6 Permeabilize and stain with 5G4 antibody
  7. 7 Automated high-content imaging
  8. 8 Image analysis and AIS calculation
  9. 9 Hit selection
Validation of hit compounds in midbrain dopaminergic neurons Days 1-7
  1. 1 Treat opto-α-syn mDA neurons with hit compounds
  2. 2 Illuminate with blue light for 7 days
  3. 3 Assess 5G4+ aggregate reduction
  4. 4 Assess TH+ neuron survival
  5. 5 Assess cleaved caspase-3 levels
  6. 6 Western blot analysis
  7. 7 Dose-response curve analysis
Validation of hit compounds in midbrain organoids Days 1-7
  1. 1 Treat opto-α-syn midbrain organoids with hit compounds
  2. 2 Illuminate midbrain organoids with blue light
  3. 3 Assess 5G4+ aggregate reduction in organoids
  4. 4 Assess TH+ neuron survival in organoids
  5. 5 Assess non-dopaminergic neuron populations
  6. 6 Cytokine array analysis
Validation in α-synuclein preformed fibril (PFF) models Days 1-14
  1. 1 Prepare mouse primary cortical neurons
  2. 2 Treat primary neurons with α-synuclein PFF
  3. 3 Treat with BAG or CDC compounds
  4. 4 Biochemical extraction of Triton X-100 insoluble fractions
  5. 5 Western blot analysis of pathological α-synuclein
  6. 6 Immunofluorescence analysis
In vivo validation in α-synuclein PFF mouse model Days 1-210
  1. 1 Prepare and anesthetize mice
  2. 2 Stereotaxic intrastriatal α-synuclein PFF injection
  3. 3 Post-operative recovery and monitoring
  4. 4 Prepare BAG 956 dosing solution
  5. 5 Oral administration of BAG or vehicle (Days 31-210)
  6. 6 Grip strength test (Day 180)
  7. 7 Rotarod test (Day 180)
  8. 8 Open field test (Days 175-176)
  9. 9 Elevated plus maze (Days 177-178)
  10. 10 Fear conditioning test (Days 179-182)
  11. 11 Perfusion and brain collection (Day 210)
  12. 12 Stereological counting of TH+ dopaminergic neurons
  13. 13 Immunohistochemistry for TH and pathological α-synuclein
  14. 14 Quantify striatal TH+ fiber density
  15. 15 Western blot analysis of ventral midbrain tissue
Autophagy and PI3K-PDK1 pathway analysis Days 1-10
  1. 1 Treat neurons with BAG or FDA-approved PI3K inhibitors
  2. 2 Assess autophagic flux with LC3-II
  3. 3 Assess p62 degradation
  4. 4 Immunofluorescence for LC3+ autophagosome puncta
  5. 5 Bafilomycin A1 autophagy inhibition
  6. 6 Assess PI3K-PDK1 signaling pathway inhibition
  7. 7 In vivo pathway analysis in α-syn PFF mouse brain

Full SOP

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Attribution

This SOP was authored by Organthis based on the published method in Kim et al., 2023. The originating laboratory holds no rights in this SOP and has not endorsed it unless marked Verified.

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