Hypothesis

Microglial mitochondrial fitness determines the balance between complement‑mediated 'eat me' signaling and CD47‑mediated 'don't eat me' signaling on synapses, such that age‑related decline in microglial oxidative phosphorylation shifts pruning from selective to indiscriminate.

Rationale

• Aging microglia show reduced mitochondrial respiration and increased reliance on glycolysis, which correlates with heightened C1q deposition on synapses 3.
• Healthy mitochondria sustain ATP‑dependent phagocytic checkpoints that favor CD47 surface retention and inhibit complement activation.
• Circadian‑driven glymphatic flux supplies NAD+ precursors that support microglial mitochondrial autophagy; disruption lowers NAD+, impairing mitophagy and tipping the eat‑me/don’t‑me ratio toward elimination.

This links the observed autophagy decline 4 and circadian dysregulation 6,7 to a metabolic checkpoint that governs pruning selectivity, moving beyond the view that autophagy loss merely causes debris accumulation.

Testable Predictions

1. In vivo: Elderly mice with microglia‑specific overexpression of PGC‑1α (a mitochondrial biogenesis regulator) will retain higher synaptic density and show normal CD47 levels on synapses despite age, whereas control aged mice will exhibit synaptic loss and elevated C1q tagging.

   • Measurement: Serial two‑photon imaging of thy1‑YFP synapses; flow cytometry of isolated synaptosomes for C1q and CD47.

2. Ex vivo: Isolated aged microglia treated with mitochondrially targeted NAD+ booster (MitoNR) will display reduced C1q‑mediated phagocytosis of labeled synapses in vitro, an effect blocked by mitochondrial uncoupler FCCP.

   • Readout: pHrodo‑synapse uptake assay.

3. Human: Post‑mortem tissue from cognitively intact older adults will correlate microglial mitochondrial complex I activity (measured by histochemistry) with synaptic CD47 immunoreactivity inversely correlated with cortical C1q load.

Falsification

If enhancing microglial mitochondrial function fails to rescue synaptic CD47 levels or prevent complement‑mediated synapse loss in aged models, the hypothesis that mitochondrial fitness governs the eat‑me/don’t‑me balance is refuted, suggesting alternative drivers (e.g., cytokine signaling) dominate pruning dysregulation.

Implications

Targeting microglial metabolism—via NAD+ supplementation, exercise‑induced mitochondrial signaling, or circadian entrainment—could restore synaptic pruning selectivity without broadly inhibiting phagocytosis, offering a precision strategy to mitigate age‑related cognitive decline.