Hypothesis

In aged neurons, elevated N6‑methyladenosine (m6A) marks on the 3′UTRs of core autophagy genes (e.g., ATG5, ATG7, TFEB, LC3, p62/SQSTM1, ULK1) recruit the YTHDF2 reader, leading to accelerated transcript decay and active suppression of autophagy. This suppression is not a passive loss of function but a protective response that limits autophagic flux when lysosomal capacity declines, thereby preventing catastrophic self‑digestion of a fragile proteome. In Alzheimer’s disease, the age‑associated m6A increase is lost, releasing this brake and contributing to dysregulated autophagy that exacerbates pathology.

Mechanistic Rationale

Aging is linked to increased nuclear METTL3/14 activity or altered subcellular localization that favors methylation of transcripts with specific sequence contexts enriched in autophagy mRNAs. The modified 3′UTRs create high‑affinity docking sites for YTHDF2, which interacts with the CCR4‑NOT deadenylase complex, shortening poly(A) tails and promoting exonucleolytic decay. Simultaneously, stress‑induced phosphorylation of YTHDF2 reduces its nuclear export, trapping it in the cytoplasm where it can act on target mRNAs. This model explains why global m6A levels rise in normal cognition while autophagy‑related proteins decline, and why AD—characterized by METTL3/14 down‑regulation—shows both loss of the age‑related m6A gain and aberrant autophagy activation.

Testable Predictions

1. Neurons from old (≥20 mo) mice will show significantly higher m6A enrichment at the 3′UTRs of ATG5, ATG7, TFEB, LC3, p62, and ULK1 compared with young (3 mo) counterparts, as measured by MeRIP‑qPCR.
2. Knocking down METTL3 or overexpressing a catalytically dead METTL3 in aged neurons will reduce m6A on these autophagy transcripts, increase their mRNA stability, and restore autophagic flux (LC3‑II/I ratio, p62 turnover) without affecting global protein synthesis.
3. Conversely, neuronal overexpression of YTHDF2 in young mice will recapitulate the aged phenotype: decreased autophagy transcript levels, reduced autophagosome formation, and accumulation of ubiquitin‑positive inclusions.
4. In AD mouse models (e.g., 5xFAD), the age‑associated m6A increase on autophagy genes will be attenuated, and rescuing METTL3 activity will reinstate YTHDF2‑mediated decay, normalizing autophagy flux and improving cognitive performance.

Experimental Approach

• Perform MeRIP‑seq on FACS‑sorted cortical neurons from young and old mice; validate peaks on autophagy genes with targeted MeRIP‑qPCR.
• Use AAV‑mediated shRNA or CRISPRi to knock down METTL3 specifically in hippocampal neurons of aged mice; assess mRNA half‑life (actinomycin D chase), protein levels, and autophagy markers via immunoblotting and live‑cell LC3‑RFP‑GFP‑Lysosomal assay.
• Over‑express YTHDF2 via AAV in young mice; repeat autophagy flux assays and evaluate behavior (open‑field, Morris water maze) to detect premature aging‑like deficits.
• Cross METTL3 rescue (AAV‑METTL3) into 5xFAD mice; measure m6A levels on autophagy transcripts, autophagic activity, plaque burden, and memory.
• Include controls: scrambled shRNA, GFP‑only AAV, and pharmacologic inhibition of YTHDF2 (e.g., CHT25) to confirm specificity.

Potential Implications

If validated, this hypothesis redefines autophagy decline in aging as an actively regulated, m6A‑dependent safeguard rather than mere wear‑and‑tear. It suggests that modulating the m6A‑YTHDF2 axis could restore autophagic homeostasis in neurodegenerative disease without triggering excessive self‑digestion, offering a precision‑therapy avenue that respects the cell’s adapted state.