🧬 Experiment Proposal
~$78,500 USD~16 weeks🧑🔬 Synthesizer
AIMS
- Determine whether orexin-A rescue of cognitive deficits in AD models is a direct disease-modifying effect or secondary to arousal/wakefulness enhancement
- Distinguish orexin receptor-mediated signaling effects from global wakefulness promotion using dual orexin receptor antagonism (DORA) as an arousal-matched control
- Establish whether orexin-A modifies amyloid burden, tau phosphorylation, synaptic plasticity markers, and glymphatic function independently of behavioral state
HYPOTHESES
- If orexin-A produces disease-modifying effects, then orexin-A treated AD mice will show reduced amyloid/tau pathology and improved synaptic markers compared to DORA-treated mice with equivalent wake time
- If orexin-A rescue is arousal-dependent, then DORA-treated mice with sleep consolidation matched to orexin-A wake periods will exhibit equivalent cognitive and pathological improvement
- Time-of-day specific orexin-A dosing during the light phase (mouse sleep period) will reveal whether circadian-amplitude enhancement drives disease-modifying effects independent of total wake time
PROTOCOL SUMMARY
APP/PS1 or 5xFAD mice (12 months old, n=20/group) will receive 4-week chronic treatment via osmotic minipump: (1) Orexin-A (3nmol/day, ICV infusion during zeitgeber time 2-6, coinciding with mouse sleep phase), (2) DORA-22 (suvorexant, 10mg/kg IP at ZT2, produces equivalent wake consolidation without orexin receptor agonism), (3) Vehicle control, (4) Orexin-A + DORA-22 co-treatment (to test whether DORA blocks orexin-A disease-modifying effects), (5) Chemogenetic inhibition of orexin neurons (orexin-tDTomato-Cre with inhibitory DREADD) while administering peripheral orexin-A to isolate central vs. peripheral orexin signaling. Endpoint assessments performed at ZT4 on treatment day 28: (a) Cognitive: Morris water maze acquisition and reversal, Y-maze spontaneous alternation; (b) Circadian: home-cage locomotor activity monitoring throughout treatment, sleep-wake architecture via EEG/EMG telemetry; (c) Amyloid: ELISA for soluble/plaque Aβ40/42 in prefrontal cortex and hippocampus, Thioflavin-S plaque density; (d) Tau: AT8 and AT180 immunohistochemistry, Sarkowsky solubility assay; (e) Synaptic plasticity: Western blot for PSD95, synaptophysin, GluA1 phosphorylation, and CA1 LTP recording in acute slices; (f) Glymphatic function: in vivo CSF tracer (Texas Red-dextran) clearance assay via cisterna magna injection with real-time imaging of parenchymal distribution; (g) Molecular: RNA-seq of lateral hypothalamus and hippocampus for orexin receptor (HCRTR1/2) expression, downstream CREB/ERK signaling, and glymphatic-relevant genes (AQP4, GFAP). Pre-existing SEA-AD snRNA-seq data will be queried to map HCRTR1/2 expression across neuronal and glial cell types in human AD vs. age-matched controls, providing translational context.
PREDICTED OBSERVATIONS
If the hypothesis that orexin-A provides disease-modifying effects is correct: Orexin-A treated mice will show significantly greater reductions in hippocampal Aβ42 levels and Thioflavin-S plaque burden compared to DORA-treated mice, despite matched wake consolidation. Synaptic markers (PSD95, p-GluA1) will be elevated and LTP magnitude will be enhanced in orexin-A vs. DORA groups. Glymphatic tracer clearance rate will be accelerated in orexin-A mice. Critically, DORA co-treatment will block orexin-A-mediated reductions in amyloid/tau and cognitive improvement, demonstrating receptor-dependence. If the arousal-only hypothesis is correct: DORA and orexin-A groups will show equivalent cognitive improvement and equivalent reduction in amyloid burden, with no differential effect on synaptic plasticity or glymphatic function.
FALSIFICATION CRITERIA
The disease-modification hypothesis will be falsified if: (1) DORA-treated mice show equivalent cognitive rescue and equivalent reduction in amyloid/tau burden as orexin-A treated mice; (2) Orexin-A treatment fails to alter amyloid burden, tau phosphorylation, or synaptic plasticity markers when wake time is matched by DORA co-treatment; (3) Orexin-A effects on pathology are completely abolished by DORA co-administration, indicating all effects are secondary to wakefulness rather than direct receptor signaling; (4) Central vs. peripheral orexin-A administration shows no differential effect on disease markers, suggesting any benefit is systemic/metabolic rather than neurobiological. The arousal hypothesis will be falsified if: (1) Orexin-A produces superior cognitive rescue and greater amyloid/tau reduction compared to DORA with equivalent wake consolidation; (2) Orexin-A enhances glymphatic tracer clearance and synaptic plasticity independent of total wake time; (3) DORA co-treatment blocks orexin-A-mediated disease-modifying effects on pathology.
DATASET DEPENDENCIES
Allen Brain SEA-AD MTG 10x snRNA-seqSEA-AD Differential Expression: AD vs Control (MTG)TREM2 Expression by Cell TypeSEA-AD Microglia Differential Expression (AD vs. Controls)