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HCN1 (Hyperpolarization-activated Cyclic Nucleotide-gated channel 1) channels are critical for neuronal excitability and rhythmic activity, particul","entities":{"genes":["HCN1"],"diseases":["Alzheimer's Disease"]},"model_system":"cell_line","_schema_version":1,"experiment_type":"falsification","primary_outcome":"Comparative analysis of tau pathology development and neuronal survival in HCN1 knockout versus control entorhinal cortex layer II neurons following amyloid-beta and tau exposure.","methodology_notes":"**Phase 1: Cell Culture Preparation (Days 1-7)**\n• Establish iPSC-derived entorhinal cortex layer II neurons from control and AD patient lines (n=6 each)\n• Generate HCN1 knockout cell lines using CRISPR/Cas9 with gRNAs targeting exons 2-3 of HCN1\n• Validate knockout efficiency by qRT-PCR and Western blot (>95% reduction required)\n• Maintain cultures in Neurobasal-A medium with B27 supplement at 37°C, 5% CO2\n\n**Phase 2: Pathology Induction and Treatment (Days 8-35)**\n• Treat cells with Aβ42 oligomers (1-5 μM) and tau K18 fibrils (100-500 nM) to induce AD-like pathology\n• Apply pharmacological treatments: ZD7288 (HCN1 blocker, 10-100 μM) and ivabradine (HCN1 enhancer, 1-10 μM)\n• Establish experimental groups: Control, AD pathology, AD+HCN1 KO, AD+ZD7288, AD+ivabradine\n• Monitor cell viability daily using MTT assay and live/dead staining\n\n**Phase 3: Molecular Analysis (Days 21, 28, 35)**\n• Collect samples for Western blot analysis of phospho-tau (AT8, PHF-1), total tau, Aβ levels\n• Perfor","replication_status":"single_study","extraction_metadata":{"backfill_at":"2026-04-16T01:00:16.909754","needs_review":true,"extraction_notes":"Backfilled from debate_extraction source (no PMID 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