Phase 1: iPSC-derived Motor Neuron Culture and TDP-43 Manipulation — Week 1-3
Differentiate human iPSCs (control and ALS patient lines with TDP-43 mutations) into motor neurons using established protocols with dual SMAD inhibition (LDN193189 10 μM, SB431542 10 μM) followed by caudalizing factors (retinoic acid 0.1 μM, purmorphamine 1 μM). Generate pure motor neuron cultures (>90% ChAT+/Islet1+) by day 21. Maintain in Neurobasal medium with B27, N2, GDNF (10 ng/mL), BDNF (10 ng/mL), and CNTF (10 ng/mL). For TDP-43 overexpression studies, transfect neurons with TDP-43-GFP plasmids (wild-type or M337V mutant) using Lipofectamine Stem (Thermo Fisher L3015) at 70-80% confluence. Include vector control transfections.
...Phase 1: iPSC-derived Motor Neuron Culture and TDP-43 Manipulation — Week 1-3
Differentiate human iPSCs (control and ALS patient lines with TDP-43 mutations) into motor neurons using established protocols with dual SMAD inhibition (LDN193189 10 μM, SB431542 10 μM) followed by caudalizing factors (retinoic acid 0.1 μM, purmorphamine 1 μM). Generate pure motor neuron cultures (>90% ChAT+/Islet1+) by day 21. Maintain in Neurobasal medium with B27, N2, GDNF (10 ng/mL), BDNF (10 ng/mL), and CNTF (10 ng/mL). For TDP-43 overexpression studies, transfect neurons with TDP-43-GFP plasmids (wild-type or M337V mutant) using Lipofectamine Stem (Thermo Fisher L3015) at 70-80% confluence. Include vector control transfections.
Phase 2: Mitochondrial DNA Release and cGAS Activation Assessment — Week 3-4
Treat motor neurons (n=6 wells per condition) with different concentrations of recombinant TDP-43 protein (0.1, 0.5, 1.0 μM) or induce TDP-43 aggregation using oxidative stress (H2O2 100 μM for 2h). Monitor mitochondrial membrane potential using TMRM (Thermo Fisher T668, 25 nM) and mitochondrial morphology with MitoTracker Green (Thermo Fisher M7514, 200 nM). Extract cytoplasmic DNA using cytoplasmic DNA extraction kit (Norgen 21000) and quantify mtDNA copy number by qPCR targeting cytochrome c oxidase subunit II (COII). Measure cGAMP production using direct ELISA (Cayman Chemical 501700) and monitor cGAS protein levels by Western blot (Cell Signaling 15102, 1:1000).
Phase 3: Pharmacological and Genetic cGAS/STING Inhibition — Week 4-5
Treat neurons with selective small molecule inhibitors: cGAS inhibitor RU.521 (10-50 μM, Selleckchem S8920), STING inhibitor H-151 (0.1-1 μM, Selleckchem S7946), or vehicle control (DMSO <0.1%). For genetic approaches, transfect neurons with validated siRNAs: cGAS siRNA (Dharmacon L-015607-02-0005, 50 nM), STING siRNA (Dharmacon L-024333-00-0005, 50 nM), or non-targeting control. Confirm >70% knockdown efficiency by qRT-PCR and Western blot after 48-72h. Include positive controls with synthetic dsDNA transfection (poly(dA:dT), 1 μg/mL) to activate cGAS/STING pathway.
Phase 4: Inflammatory Pathway Analysis — Week 5-6
Analyze NF-κB activation using multiple approaches: (1) NF-κB p65 nuclear translocation by immunofluorescence (Cell Signaling 8242, 1:800) with quantitative imaging, (2) NF-κB luciferase reporter assays in transfected neurons, (3) phospho-p65 Western blots (Cell Signaling 3033, 1:1000). Monitor Type I interferon responses by measuring: IFN-β mRNA levels (qRT-PCR), IRF3 nuclear translocation (Cell Signaling 4302, 1:800), and phospho-IRF3 (Cell Signaling 4947, 1:1000). Analyze downstream inflammatory gene expression panel including TNF-α, IL-1β, IL-6, CXCL10, and ISG15 using custom TaqMan arrays.
Phase 5: Mitochondrial Dysfunction and Cell Death Assessment — Week 6
Evaluate neuronal viability using multiple assays: MTT reduction (Sigma M5655), LDH release (Promega G1780), and live/dead staining with calcein AM/ethidium homodimer (Thermo Fisher L3224). Measure ATP levels using CellTiter-Glo (Promega G7570) and mitochondrial respiration with Seahorse XF analyzer (oxygen consumption rate, extracellular acidification rate). Assess DNA damage using γH2AX immunofluorescence (Millipore 05-636, 1:1000) and quantify apoptotic cells with cleaved caspase-3 staining (Cell Signaling 9664, 1:400). Monitor cytosolic cytochrome c release as marker of mitochondrial damage.
Phase 6: Rescue Experiments and Mechanism Validation — Week 6-7
Test therapeutic interventions: (1) antioxidants (N-acetylcysteine 1-10 mM, vitamin E 10-100 μM), (2) mitochondrial stabilizers (SS-31 peptide 1-10 μM), (3) anti-inflammatory compounds (Bay 11-7082 NF-κB inhibitor 1-10 μM). Perform rescue experiments with mitochondrial DNA depletion using ethidium bromide (50 ng/mL for 7 days) to create ρ0 cells lacking mtDNA. Validate mechanism by reconstituting cGAS activation with purified mtDNA in cGAS-depleted neurons. Include time-course analysis (1, 4, 8, 24, 48h) to establish temporal relationships between TDP-43 aggregation, mtDNA release, and inflammatory activation.