Hypothesis\n\nAging neurons that fall below a critical activity threshold expose phosphatidylserine (PS) on their surface, marking them for microglial phagocytosis via TREM2‑dependent recognition. It's unlikely that protein aggregation alone accounts for the selective loss of weakly firing neurons. This process operates parallel to, and can be uncoupled from, the proteostasis decline described in recent work (Age-related slowing of brain protein clearance and Neuronal loss patterns in the aging brain). Thus, neuronal loss in aging is not solely a passive consequence of protein aggregation but also an active, activity‑sensing pruning mechanism.\n\n## Mechanistic Basis\n\n- Activity sensor: Neuronal firing regulates the balance between flippase and scramblase activity in the plasma membrane. Low firing reduces ATP‑dependent flippase function, allowing PS to accumulate outward (Nervous system activity might influence human longevity). We can't ignore that mitochondrial decline with age further curtails ATP, worsening the imbalance.\n\n- Eat‑me signal: Exposed PS binds microglial TREM2, triggering phagocytic uptake (Microglia use selective autophagy to clear neuron-released proteins). Complement C1q may amplify this signal but is not required for the basal activity‑dependent route.\n\n- Proteostasis intersect: When proteasome capacity falls (Age-related slowing of brain protein clearance shows doubled protein half‑lives), stressed neurons are more likely to reduce firing, thereby increasing PS exposure. However, PS exposure can occur even when proteasome function is chemically rescued, indicating a separable pathway.\n\n## Predictions\n\n1. Blocking microglial TREM2 in aged mice will reduce neuron loss without altering levels of insoluble synaptic proteins.\n\n2. Chemogenetic activation of hippocampal neurons in old animals will decrease PS exposure and preserve neuron numbers, even when proteasome activity is inhibited pharmacologically. We don't expect this activation to affect baseline protein aggregation.\n\n3. Microglia lacking TREM2 will fail to clear PS‑positive neurons but will still accumulate phagocytosed protein aggregates, showing a dissociation between protein waste removal and neuron removal.\n\n4. REST overexpression, known to suppress excitotoxicity (Nervous system activity might influence human longevity), will lower PS exposure by maintaining higher basal activity, linking the longevity pathway to the pruning axis. It's plausible that REST also modulates flippase expression directly.\n\n## Experimental Approaches\n\n- Use in vivo two‑photon imaging of PS‑binding annexin V‑fluorophore in young vs. old mice, combined with cell‑type specific RNA‑seq to quantify flippase/scrmblase expression.\n\n- Apply chemogenetic DREADDs to drive excitatory or inhibitory firing in defined cortical layers, then quantify neuron numbers and microglial phagocytosis markers. It's helpful to include a control group receiving inert ligand.\n\n- Employ TREM2‑conditional knockout mice crossed with a PS‑reporter line; assess cognitive behavior and histology. We should also measure soluble oligomeric species to confirm proteostasis status.\n\n- Treat aged mice with low‑dose proteasome activator (e.g., IU1) to test whether rescuing proteostasis alone prevents PS elevation. It's possible that this treatment lowers PS only when neuronal firing is simultaneously boosted.\n\n## Potential Implications\n\nIf activity‑dependent tagging contributes significantly to age‑related neuronal loss, interventions that sustain neuronal firing or block microglial recognition could preserve cognition without needing to reverse protein aggregation. This reframes the therapeutic goal from merely boosting proteostasis to also modulating neuron‑glia communication based on metabolic state. It's becoming clear that the brain’s energy budget influences not just which proteins accumulate, but which cells are earmarked for removal.\n\nAll cited works support the premise that proteostasis failure and microglial compensation are central to aging (Age-related slowing of brain protein clearance, Microglia use selective autophagy to clear neuron-released proteins, Neuronal loss patterns in the aging brain), while neuronal activity influences longevity (Nervous system activity might influence human longevity) and microglial rejuvenation rescues function (Replacing aged microglia with young ones reverses cognitive deficits). The proposed mechanism adds a testable, activity‑regulated layer to this framework.