s:** - Test MCU overexpression specifically in layer II neurons in healthy vs

SUMMARY

# s:** - Test MCU overexpression specifically in layer II neurons in healthy vs ## Background and Rationale This falsification experiment investigates the role of mitochondrial calcium uniporter (MCU) overexpression specifically in layer II cortical neurons during neurodegeneration. MCU dysfunction represents a critical convergence point in Alzheimer's disease pathophysiology, where dysregulated mitochondrial calcium homeostasis contributes to synaptic dysfunction and neuronal death. Layer II ne

METHODOLOGY NOTES

**Phase 1: Animal Preparation and Genotyping (Weeks 1-2)** • Obtain 8-week-old wild-type C57BL/6J mice (n=40) and APP/PS1 AD model mice (n=40) • Perform genotyping via PCR to confirm AD transgene status • Acclimate animals to housing conditions for 1 week • Randomly assign to experimental groups: WT+Control AAV (n=20), WT+MCU-AAV (n=20), AD+Control AAV (n=20), AD+MCU-AAV (n=20) **Phase 2: Stereotaxic AAV Injection (Week 3)** • Anesthetize mice with isoflurane (2-3%) • Position in stereotaxic frame and inject AAV9-CaMKII-MCU or AAV9-CaMKII-GFP (control) into entorhinal cortex layer II • Coordinates: AP -4.16mm, ML ±4.2mm, DV -2.8mm from bregma • Inject 1μL viral suspension (1×10^12 vg/mL) at 0.1μL/min using 33-gauge needle • Allow 2-week recovery for viral expression **Phase 3: Calcium Imaging Preparation (Week 5)** • Inject Fura-2 AM (2μM) or Rhod-2 AM (5μM) calcium indicators via tail vein • Prepare acute brain slices (300μm thickness) containing entorhinal cortex • Identify layer I