Hypothesis

Age‑related decline in neuronal phosphatidylcholine (PC) synthesis, driven by reduced choline availability and impaired CDP‑choline pathway activity, leads to aberrant remodeling of mitochondrial cardiolipin (CL) membranes. This CL remodeling increases mitochondrial ROS production, activates the NLRP3 inflammasome, and triggers synaptic loss independently of acetylcholine depletion.

Mechanistic Rationale

1. Choline is a precursor for PC via the CDP‑choline (Kennedy) pathway and for phosphatidylethanolamine N‑methyltransferase (PEMT)‑derived PC. With aging, choline transporter efficiency falls (2), lowering hepatic and brain PC synthesis.
2. Reduced PC limits the substrate for CL remodeling; CL becomes enriched in monolysocardiolipin and oxidized species, impairing electron‑transport chain efficiency and elevating mitoROS (4).
3. Elevated mitoROS stimulates NLRP3 inflammasome activation, causing IL‑1β release and synaptic pruning (6).
4. Parallelly, choline supports one‑carbon metabolism, maintaining SAM‑dependent methylation that sustains SIRT1 expression; SIRT1 deacetylates PGC‑1α, promoting mitochondrial biogenesis. Cholinedeficiency thus doubly hurts mitochondrial health.
5. This mechanism predicts cognitive decline even when acetylcholine levels are pharmacologically maintained.

Predictions and Experimental Design

• Prediction 1: In aged ApoE4 mice, chronic low choline diet will decrease brain PC and increase oxidized CL species, accompanied by heightened mitoROS and NLRP3 activation, prior to measurable ACh decline.
• Prediction 2: Supplementation with CDP‑choline (500 mg/kg/day) will restore PC, normalize CL composition, reduce mitoROS/NLRP3 signaling, and improve spatial memory, effects blocked by mitochondrial ROS scavenger (MitoTEMPO) only if ROS is downstream.
• Prediction 3: Neuronal‑specific overexpression of PEMT will rescue CL defects and memory despite low choline, indicating PC synthesis suffices.

Approach: Use lipidomics (PC, CL species), mitoROS probes (MitoSOX), inflammasome assays (caspase‑1 cleavage), and behavior (Morris water maze). Include groups: young control, aged control, aged low‑choline, aged low‑choline + CDP‑choline, aged low‑choline + CDP‑choline + MitoTEMPO, aged PEMT‑overexpression.

Falsifiability: If CDP‑choline fails to correct CL remodeling or mitoROS, or if memory improves without CL normalization, the hypothesis is weakened. Conversely, if PEMT overexpression rescues memory without altering choline levels, it supports the PC‑centric mechanism.

Broader Implications

Linking choline‑dependent phospholipid synthesis to mitochondrial membrane integrity offers a unifying explanation for why choline deficits correlate with NfL elevation (1) and anxiety‑related cortical choline loss (6). It also suggests that combining CDP‑choline with mitochondria‑targeted antioxidants could be more effective than choline alone in early‑intervention trials targeting NfL as a biomarker.