Hypothesis

In normally aging neurons, METTL14 protein levels remain unchanged in the soma but decrease selectively in distal axons, leading to reduced m6A on autophagy‑related transcripts (e.g., UBR1, FOXO3, ATG5) locally and impaired axonal autophagy. This spatial deficit, not a global loss, explains why bulk tissue assays miss the change and why proteostasis failure first appears in synapses.

Rationale

• Existing data show METTL14 loss in senescent fibroblasts and HGPS cells [2] and link METTL14‑mediated m6A on UBR1 to autophagy inhibition in spinal cord injury [1]
• m6A modification positively regulates FOXO3, ATG5, ATG7 expression across contexts [3]
• Alzheimer’s data reveal a normal age‑associated increase in m6A that is lost in pathology [4], suggesting compartment‑specific dynamics
• Neurons exhibit strong polarity; RNA‑binding proteins and granule formation can sequester writers away from axons [6] Thus, aging may trigger axonal sequestration or degradation of METTL14, uncoupling somatic m6A levels from axonal m6A deposition.

Testable Predictions

1. Subcellular fractionation of young vs aged mouse cortical neurons will show unchanged METTL14 in somatic fractions but a significant decrease in axonal fractions (Western blot, immunofluorescence)
2. m6A‑RIP‑seq of axonal RNA will reveal loss of m6A peaks on UBR1, FOXO3, ATG5, ATG7 in aged axons, while somatic RNA shows no change
3. Functional readout: axonal autophagosome turnover (LC3‑II/p62 dynamics in microfluidic chambers) will be reduced in aged neurons and rescued by axonal‑targeted METTL14 overexpression (AAV‑synaptophysin‑METTL14)
4. Proteostatic consequence: aged neurons expressing axonal METTL14 will exhibit lower accumulation of ubiquitinated proteins and synaptic debris compared with controls
5. Falsification: if axonal METTL14 levels are unchanged with age, or axonal m6A on autophagy transcripts does not differ, the hypothesis is refuted

Experimental Approach

• Use Thy1‑YFP mice to isolate axons via laser capture or microfluidic separation
• Validate fractionation with compartment markers (MAP2 for soma, Tau‑1 for axon)
• Perform Western blot for METTL14, METTL3, and demethylases FTO/ALKBH5
• Conduct m6A‑RNA immunoprecipitation followed by sequencing on pooled axonal and somatic fractions
• Assess autophagy flux with tandem mCherry‑GFP‑LC3 reporters in axons
• Overexpress METTL14 using an axon‑targeted AAV (Synapsin‑1 promoter plus axonal localization signal) and measure outcomes
• Include young (3 mo) and aged (24 mo) cohorts, both sexes, n≥5 per group

Implications

Confirming this hypothesis would shift the focus from global writer decline to localized epitranscriptomic remodeling as a driver of age‑related proteostatic collapse, suggesting therapeutic strategies that restore axonal m6A deposition rather than boosting overall METTL14 levels.