SUMMARY
# s:**
- Test whether HCN1 knockout specifically in EC layer II accelerates or protects against AD pathology
- Measure whether pharmacological HCN1 enha
## Background and Rationale
This experiment investigates the specific role of HCN1 channels in entorhinal cortex layer II neurons and their contribution to Alzheimer's disease pathology progression. HCN1 (Hyperpolarization-activated Cyclic Nucleotide-gated channel 1) channels are critical for neuronal excitability and rhythmic activity, particul
METHODOLOGY NOTES
**Phase 1: Cell Culture Preparation (Days 1-7)**
• Establish iPSC-derived entorhinal cortex layer II neurons from control and AD patient lines (n=6 each)
• Generate HCN1 knockout cell lines using CRISPR/Cas9 with gRNAs targeting exons 2-3 of HCN1
• Validate knockout efficiency by qRT-PCR and Western blot (>95% reduction required)
• Maintain cultures in Neurobasal-A medium with B27 supplement at 37°C, 5% CO2
**Phase 2: Pathology Induction and Treatment (Days 8-35)**
• Treat cells with Aβ42 oligomers (1-5 μM) and tau K18 fibrils (100-500 nM) to induce AD-like pathology
• Apply pharmacological treatments: ZD7288 (HCN1 blocker, 10-100 μM) and ivabradine (HCN1 enhancer, 1-10 μM)
• Establish experimental groups: Control, AD pathology, AD+HCN1 KO, AD+ZD7288, AD+ivabradine
• Monitor cell viability daily using MTT assay and live/dead staining
**Phase 3: Molecular Analysis (Days 21, 28, 35)**
• Collect samples for Western blot analysis of phospho-tau (AT8, PHF-1), total tau, Aβ levels
• Perfor