Hypothesis

Aging neurons are actively removed when their metabolic output falls below a threshold that fails to sustain complement‑inhibitory signals, leading to C1q tagging and microglial phagocytosis. This process mirrors developmental synaptic pruning but operates on the soma level, driven by activity‑dependent release of adenosine triphosphate (ATP) that normally suppresses C1q deposition.

Rationale

• C1q accumulates near synapses in the aging hippocampus (1), yet its role in soma elimination has only been shown in disease or development (2, 3).
• Neuronal activity promotes DNA repair and sustains a protective transcriptional state (5); reduced activity therefore diminishes this defense.
• Metabolically active neurons release ATP into the extracellular space, which activates microglial P2Y12 receptors and inhibits complement activation (6 discusses oxidative stress as a baseline, supporting the idea that loss of ATP shifts the balance toward complement tagging).
• As neurons age, mitochondrial efficiency declines, lowering ATP release and shifting the balance toward C1q tagging.

Predictions

1. In aged mouse hippocampus, neurons with low intracellular calcium flux (a proxy for activity) will show higher C1q deposition on their soma compared to hyperactive neighbors.
2. Blocking ATP release (e.g., with pannexin inhibitors) in young mice will increase C1q‑mediated neuronal loss, mimicking the aged phenotype.
3. Enhancing neuronal ATP release via chemogenetic stimulation will reduce C1q tagging and preserve neuron numbers in old mice.
4. CR3 deficiency will blunt the activity‑dependent loss, rescuing soma numbers despite low metabolic output.

Experimental Approach

• Use in vivo two‑photon imaging of GCaMP6f to monitor calcium activity in hippocampal neurons of young (3 mo) and old (24 mo) mice.
• Apply immunolabeling for C1q and neuronal markers (NeuN) to quantify soma‑bound complement.
• Manipulate ATP release with pharmacological agents (probenecid to block pannexin‑1 channels) or chemogenetic actuators (hM3Dq) targeting excitatory neurons.
• Assess neuronal counts stereologically and microglial phagocytic activity via CD68 staining.
• Repeat experiments in CR3‑knockout background to test dependence on complement phagocytosis.

Falsifiability

If aged neurons show no correlation between low activity and somatic C1q tagging, or if altering ATP release fails to change C1q deposition or neuronal survival, the hypothesis is refuted. Likewise, if CR3 loss does not protect low‑activity neurons from elimination, the proposed complement‑mediated mechanism is invalid.