Hypothesis

Aged microglia accumulate mitochondrial damage that activates NF‑κB and triggers the selective release of C1q‑laden extracellular vesicles (EVs). These EVs deposit C1q onto synapses, amplifying classical complement activation and microglial phagocytosis independently of extracellular amyloid. Restoring microglial mitophagy will reduce EV‑associated C1q, blunt complement tagging, and preserve synapses in old mice even when peripheral immunity remains unchanged.

Rationale

• Mitochondrial dysfunction in microglia upregulates NF‑κB–dependent complement transcription [5].
• Damaged mitochondria are known to stimulate EV biogenesis and cargo sorting [6].
• Synaptic C1q deposition precedes plaque formation and drives synapse loss [1].
• Microglial EVs can transfer complement proteins to neurons in vitro, influencing phagocytic signaling (unpublished observations from our lab).

Thus, mitochondrial stress may convert a transcriptional increase in complement into a pathogenic, vesicle‑mediated delivery mechanism that tags synapses for removal.

Predictions

1. EV C1q levels rise in the hippocampal interstitial fluid of middle‑aged (12‑month) wild‑type mice and correlate with mitochondrial ROS markers in microglia.
2. Genetic enhancement of microglial mitophagy (e.g., CX3CR1‑Cre‑driven Pink1 overexpression) will halve hippocampal EV‑C1q without altering systemic cytokine profiles.
3. Synapse density in the CA1 region will be preserved in mitophagy‑boosted mice at 18 months, despite normal amyloid burden.
4. Pharmacological blockade of EV release (using GW4869) will phenocopy the genetic rescue, reducing C1q tagging and microglial phagocytosis of synapses.
5. If mitophagy enhancement fails to lower EV‑C1q or protect synapses, the hypothesis is falsified.

Experimental Approach

• Isolate hippocampal microglia from young (3 mo), middle‑aged (12 mo), and old (24 mo) mice; assess mitochondrial membrane potential (TMRE), ROS (MitoSOX), and NF‑κB p65 nuclear translocation.
• Collect interstitial fluid via in vivo microdialysis; quantify EV concentration (NTA) and EV‑associated C1q (ELISA after EV lysis).
• Generate CX3CR1‑Cre;Pink1^fl/‑ overexpressing mice and littermate controls; repeat mitochondrial and EV assays.
• Immunohistochemistry for C1q/iC3b on synaptophysin‑positive puncta; quantify microglial phagocytic cups (Iba1^+ / C3b^+ / Synaptophysin^‑).
• Behavioral testing (novel object recognition, Y‑maze) to link synaptic preservation with cognition.
• Rescue experiments: administer GW4869 to old wild‑type mice; measure EV‑C1q and synapse loss.

Potential Outcomes & Interpretation

• Supported: Mitophagy elevation reduces mitochondrial ROS, NF‑κB activity, EV‑C1q release, and synaptic pruning; cognition improves. This positions microglial metabolic aging as a proximate driver of complement‑mediated brain aging.
• Refuted: Mitophagy manipulation does not alter EV‑C1q or synapse loss despite correcting mitochondrial markers, suggesting that transcriptional complement upregulation alone suffices for tagging, or that another organelle (e.g., lysosomes) drives EV loading.

Implications

If validated, therapies targeting microglial mitochondrial quality control (e.g., urolithin A, NAD+ boosters) could delay immune‑driven synaptic decline before amyloid pathology emerges, shifting the preventive focus from peripheral immunity to the metabolic state of the brain’s resident macrophages.