Hypothesis

During normal brain aging, METTL14 protein declines selectively in excitatory neurons, causing a neuron‑specific loss of m6A on transcripts encoding synaptic proteins and lysosomal autophagy regulators. This dual loss simultaneously weakens synaptic translation and unleashes YTHDF2‑driven decay of lysosomal mRNAs, producing a feed‑forward loop that impairs proteostasis and accelerates AD‑related pathology.

Mechanistic Rationale

• Global m6A drops 20‑30% in aged cortex [1], yet bulk measurements mask cell‑type differences.
• In Parkinson’s models METTL14 falls 40‑60% [2], suggesting vulnerability of the writer complex to stress.
• m6A can either promote decay (via YTHDF2) or enhance translation (via YTHDC1/eIF3) depending on cell maturity and transcript context [5].
• In spinal cord injury, METTL14‑mediated m6A on UBR1 3′UTR reduces UBR1 and boosts autophagy [3]; in ALS motor neurons, YTHDF2‑mediated decay of ULK1/ATG5/7 disrupts autophagy [4].

We propose that aging neurons experience a switch: reduced METTL14 lessens m6A on synaptic mRNAs (PSD‑95, synapsin), lowering their translation, while the same loss diminishes protective m6A on lysosomal transcripts, exposing them to YTHDF2‑dependent decay. The resulting autophagy deficit amplifies accumulation of damaged proteins, further suppressing METTL14 expression through oxidative stress, completing a vicious cycle.

Testable Predictions

1. Neuron‑specific METTL14 decline: Immunofluorescence and western blot of sorted NeuN+ neurons from young (3 mo) and aged (24 mo) mouse cortex will show a ≥35% reduction in METTL14 protein, whereas glial fractions remain unchanged.
2. Dual transcript targeting: MeRIP‑seq from aged neuronal RNA will reveal decreased m6A peaks on synaptic gene exons (e.g., Dlg4, Syn1) and increased m6A loss on lysosomal mRNAs (e.g., Lamp2, Ctsd), with concomitant YTHDF2 binding gains on the latter.
3. Functional link: Overexpressing METTL14 in aged neurons via AAV will rescue m6A on both synaptic and lysosomal transcripts, restore PSD‑95/synapsin synthesis, reduce LC3II/LC3I ratio normalization, and decrease p62 accumulation.
4. Causality in vivo: Inducing METTL14 knockdown in adult excitatory neurons (Camk2a‑CreERT2) will recapitulate the aging phenotype: reduced synaptic protein synthesis, autophagy flux impairment, and accelerated amyloid‑β accumulation in an APP/PS1 background.

Experimental Approach

• Cell isolation: Use FACS to separate NeuN+/CD11b− neurons from microglia/astrocytes in mouse cortex.
• Protein quantification: Quantitative western blot with fluorescent secondary antibodies; normalize to total protein stain.
• m6A mapping: Perform MeRIP‑seq and eCLIP for YTHDF2 on isolated neuronal RNA; validate peaks by qPCR‑MeRIP.
• Translational assay: Puromycin‑based SUnSET assay on sorted neurons to measure nascent synaptic protein synthesis.
• Autophagy flux: Treat slices with bafilomycin A1 and quantify LC3II turnover by western blot; monitor p62 levels.
• Rescue experiments: AAV‑Camk2a‑METTL14‑IRES‑GFP injected into aged mice; assess behavioral cognition (Morris water maze) and pathology after 3 months.

If neuronal METTL14 loss drives both synaptic weakening and lysosomal mRNA decay, restoring METTL14 should simultaneously improve translation and autophagy, breaking the cycle that links m6A decline to neurodegeneration. Failure to observe these effects would falsify the hypothesis and suggest that aging‑related m6A changes act primarily through non‑neuronal or extracellular mechanisms.