A NAD+-sensitive RNA-binding protein couples cellular metabolic state to m6A-dependent translation of autophagy genes to sustain neuronal proteostasis during aging

Hypothesis: In cognitively normal aging, increased m6A modification in the 3'UTRs of synaptic and autophagy-related transcripts is not merely a passive mark but actively promotes their translation through a NAD+-dependent RNA‑binding protein, RBP‑X, which senses the cellular NAD+/NADH ratio. When NAD+ levels are high (as occurs during mild metabolic stress or exercise), RBP‑X binds m6A‑modified RNAs with higher affinity, recruiting YTHDF1 and the eukaryotic initiation factor eIF3 to enhance ribosome loading. This boosts synthesis of autophagy regulators such as ULK1, LC3, and lysosomal proteases, thereby reinforcing proteostasis. In Alzheimer’s disease, chronic NAD+ depletion reduces RBP‑X binding affinity, uncoupling the rise in m6A from translational output despite elevated m6A levels, leading to deficient autophagy and accumulation of protein aggregates.

Testable predictions:

1. RBP‑X binding affinity is NAD+‑dependent. Perform electrophoretic mobility shift assays (EMSAs) using purified RBP‑X and synthetic 3'UTR RNAs bearing m6A; compare binding at physiological NAD+ (≈500 µM) versus low NAD+ (≈50 µM) conditions. Expect a ≥2‑fold decrease in Kd under low NAD+.
2. RBP‑X mediates the translational boost of m6A‑modified autophagy transcripts. Use CRISPRi to knock down RBP‑X in primary neurons from young mice, then measure ribosome profiling of m6A‑enriched autophagy mRNAs (e.g., Ulk1, Atg5, Lamp2). Prediction: RBP‑X loss reduces ribosome footprints on these transcripts by ≥30 % without altering total mRNA levels or m6A stoichiometry (verified by MeRIP‑seq).
3. Restoring NAD+ rescues autophagy in aged neurons via RBP‑X. Treat aged mouse hippocampal cultures with NAD+ precursor (NR) ± RBP‑X knockdown. Measure autophagic flux (LC3‑II turnover with bafilomycin A1) and protein aggregate load (p62, ubiquitin‑positive inclusions). Prediction: NR increases flux by ~40 % in control cells; this increase is abolished when RBP‑X is knocked down, confirming that NAD+ acts through RBP‑X.
4. In Alzheimer’s disease models, NAD+ supplementation fails to induce autophagy unless RBP‑X is overexpressed. In APP/PS1 neurons, administer NR with or without AAV‑mediated RBP‑X overexpression. Prediction: NR alone does not improve autophagic flux; NR + RBP‑X overexpression restores flux to youthful levels and reduces amyloid‑beta‑induced toxicity.

Falsifiable outcomes: If RBP‑X binding shows no NAD+ sensitivity, or if manipulating RBP‑X does not affect translation of m6A‑modified autophagy transcripts despite changes in m6A levels, the hypothesis would be refuted. Similarly, if NAD+ supplementation enhances autophagy independently of RBP‑X status, the proposed coupling mechanism would be invalid.

This hypothesis extends the existing data by proposing a metabolic sensor that translates the observed age‑related increase in m6A into functional proteostatic output, explains why the same m6A rise is pathogenic in Alzheimer’s (via NAD+ deficit), and offers a concrete, intervenable target (RBP‑X or NAD+ boosting) for restoring neuronal proteostasis in aging.