Experiment Proposal (Crux): What is the role of GPX4-dependent ferroptosis, lipid peroxidation, and iron handling in ALS and mot [ask-aa724961]

AIMS

HYPOTHESES

PROTOCOL SUMMARY

PHASE 1 - Computational Causal Analysis (Weeks 1-4): (1) Extract motor neuron and upper motor neuron populations from SEA-AD snRNA-seq using established markers (CHAT, MNX1, ISL1, SLC5A7). (2) Score cells for mitochondrial dysfunction signature (MT-ND genes, SDH subunits, OPA1, TFAM) and ferroptosis signature (GPX4, SLC7A11, ACSL4, FSP1, LPCAT3). (3) Compute Spearman correlation and pseudotime ordering to establish co-expression patterns. (4) Perform GSEA on mitochondrial-early vs ferroptosis-early motor neuron clusters to determine upstream pathway activation. PHASE 2 - Primary Motor Neuron Validation (Weeks 5-10): (1) Differentiate iPSC-derived motor neurons from SOD1-G93A or C9orf72 lines. (2) Treat parallel cultures at day 7 with: (a) oligomycin (complex V inhibitor) to induce mitochondrial failure alone, (b) RSL3 (GPX4 inhibitor) to induce ferroptosis alone, (c) erastin2 (system Xc- inhibitor), (d) MitoQ + ferrostatin-1 combination, (e) vehicle controls. (3) At 24h, 48h, 72h post-treatment: harvest cells for lipidomics (LC-MS/MS quantifying PE-ox, PC-ox, free PUFA/DHA ratios), measure OCR and ECAR (Seahorse), and collect conditioned media for 4-HNE, MDA, and labile iron pool quantification. PHASE 3 - Biomarker Panel Development (Weeks 8-12): (1) Using Phase 2 data, define biomarker thresholds that distinguish primary ferroptosis (high 4-HNE, low labile iron before OCR decline) from secondary ferroptosis (normal 4-HNE initially, iron elevation only after OCR collapse). (2) Validate biomarker ratios against existing SEA-AD proteomic data for GPX4 and mitochondrial complex subunits. (3) Test iron chelation (deferoxamine, 10-100μM) only in primary ferroptosis model conditions to establish therapeutic index window.

PREDICTED OBSERVATIONS

If H1 is true: motor neurons will show mitochondrial dysfunction genes upregulated 1-2 days before ferroptosis markers appear, with GSEA showing TFAM/NRF2 pathway activation preceding GPX4 suppression. If H2 is true: rescuing mitochondria will prevent ACSL4 induction and lipid peroxidation accumulation, maintaining cell viability without requiring direct ferroptosis inhibitors. If H3 is true: the 4-HNE-to-OCR ratio at 24h will be the strongest discriminator, with elevated 4-HNE preceding 50% OCR decline identifying primary ferroptosis cases.

FALSIFICATION CRITERIA

H1 falsified if: ferroptosis markers appear at same timepoint or earlier than mitochondrial genes in pseudotime analysis; if GPX4 protein is reduced before any measurable OCR decline. H2 falsified if: rescuing mitochondrial function with MitoQ/SS-31 delays but does not prevent ferroptosis marker accumulation and eventual cell death, indicating ferroptosis is independently triggered. H3 falsified if: biomarker ratios show no correlation with treatment response; if all patient-derived motor neuron lines show identical biomarker patterns regardless of genotype, indicating no separable subpopulation for enrichment.

DATASET DEPENDENCIES