Hypothesis

Age‑dependent decline of the SIRT1‑BMAL1 protein‑protein interaction is the primary molecular trigger that desynchronizes the hippocampal circadian proteome, leading to widespread loss of rhythmic expression in metabolic and oxidative‑stress pathways.

Mechanistic Model

The CLOCK:BMAL1 heterodimer drives transcription of clock‑controlled genes, but its activity is finely tuned by SIRT1‑mediated deacetylation of BMAL1 and associated histones. In young neurons, SIRT1 binds BMAL1 with high affinity, promoting a repressive chromatin state that sharpens transcriptional rhythms. With aging, SIRT1 expression falls and oxidative modifications weaken the SIRT1‑BMAL1 interface. This loss reduces BMAL1 deacetylation, increasing its transcriptional noise and destabilizing the core clock complex. Consequently, downstream targets involved in oxidative phosphorylation (e.g., PINK1, TRAP1, cytochrome c) and ROS scavenging lose rhythmic expression, elevating mitochondrial ROS. Elevated ROS further oxidizes cysteine residues on SIRT1 and BMAL1, creating a positive feedback loop that accelerates network disintegration.

Testable Predictions

1. In middle‑aged mouse hippocampus, the binary interaction between SIRT1 and BMAL1 will show a >70% reduction in AP‑MS spectral counts compared with young mice, whereas other clock protein interactions (e.g., CLOCK‑BMAL1, BMAL1‑CRY1) will change less dramatically.
2. Restoring SIRT1‑BMAL1 binding—either by overexpressing a SIRT1‑resistant BMAL1 mutant (acetyl‑lysine to arginine) or by administering a small‑molecule stabilizer of the SIRT1‑BMAL1 interface—will rescue the rhythmic proteome, increasing the percentage of cycling proteins from ~1.6% back toward young levels (>10%).
3. Mice harboring the acetylation‑resistant BMAL1 knock‑in will exhibit delayed age‑related decline in hippocampal mitochondrial function, lower ROS accumulation, and extended median lifespan relative to wild‑type littermates.

Experimental Approach

• Interactome Mapping: Perform affinity purification coupled to quantitative mass spectrometry (AP‑MS) using anti‑BMAL1 antibody on hippocampal extracts from 3‑month (young) and 12‑month (middle‑aged) C57BL/6J mice. Label samples with TMT10plex to enable accurate ratio calculation of SIRT1‑BMAL1 peptides and other interactors.
• Genetic Rescue: Generate a CRISPR knock‑in line where BMAL1 lysine 537 (a known SIRT1 target) is mutated to arginine (K537R). Validate that this mutation prevents acetylation without affecting DNA binding. Collect hippocampal tissue at 3, 6, 12, and 18 months for RNA‑seq and proteomics to assess circadian rhythmicity (using JTK_CYCLE or RAIN).
• Pharmacological Rescue: Treat middle‑aged mice with a previously identified SIRT1‑BMAL1 stabilizer (e.g., a small molecule identified via virtual screening of the SIRT1 allosteric pocket). Administer daily for 3 months and repeat AP‑MS and behavioral assays (circadian wheel‑running, memory tests).
• Functional Readouts: Measure mitochondrial respiration (Seahorse XF), ROS levels (MitoSOX), and synaptic plasticity (LTP in hippocampal slices). Correlate molecular rescue with physiological outcomes.

Falsifiability: If AP‑MS shows no significant decline in SIRT1‑BMAL1 interaction with age, or if rescuing this interaction fails to restore rhythmic protein expression or improve aging phenotypes, the hypothesis would be refuted. Conversely, a strong correlation between interaction strength, proteomic synchrony, and healthspan would support the model.