Hypothesis

Aging in healthy neurons drives a gain of m6A modifications at specific 3'UTR sites of ubiquitin-proteasome system (UPS) and autophagy genes, enhancing their translation and sustaining proteostasis. In Alzheimer’s disease this adaptive m6A remodeling fails, causing selective hypomethylation and reduced expression of these proteostasis transcripts, which leads to accumulation of ubiquitinated substrates and protein aggregation.

Mechanistic Basis

Recent human dorsolateral prefrontal cortex data show an age‑associated increase in m6A on synaptic gene 3'UTRs, whereas AD brains lose this gain specifically on UPS and autophagy transcripts (Human brain m6A aging). We propose that METTL3/14 activity does not decline globally with age; instead, aging neurons undergo a redistribution of methyltransferase complexes toward specific RNA motifs enriched in the 3'UTRs of UPS genes (e.g., GGACU flanked by U-rich elements). This targeted methylation recruits YTHDF1/3, promoting ribosome loading and protein synthesis without altering overall mRNA stability. Loss of this site‑specific m6A reduces translation efficiency of key proteasome subunits (PSMB5, PSMC1) and autophagy receptors (SQSTM1, LC3B), tipping the balance toward aggregation‑prone species.

Testable Predictions

1. Quantitative MeRIP‑seq of sorted neurons from young, aged, and AD human brains will reveal a significant increase in m6A peak density at UPS/autophagy 3'UTRs in aged versus young, with a reversal in AD.
2. Ribosome profiling will show higher translation efficiency of m6A‑modified UPS transcripts in aged neurons compared with young, and reduced efficiency in AD neurons.
3. CRISPR‑dCas13‑METTL3 fusion targeted to the 3'UTR of PSMB5 will rescue its translation and reduce ubiquitin‑positive inclusions in iPSC‑derived neurons exposed to oligomeric Aβ.
4. Conversely, dCas13‑ALKBH5 mediated demethylation of the same site in young neurons will recapitulate AD‑like translation defects and increase protein aggregation.

Experimental Approach

• Isolate NeuN+ nuclei from post‑mortem dorsolateral prefrontal cortex (young, cognitively normal aged, AD) and perform MeRIP‑seq coupled with spike‑in normalization to quantify site‑specific m6A changes.
• Perform parallel Ribo‑seq on the same samples to correlate m6A density with translation rates.
• Generate human iPSC‑derived cortical neurons; introduce dCas13‑METTL3 or dCas13‑ALKBH5 guided to the PSMB5 3'UTR via sgRNA.
• Measure PSMB5 protein levels by Western blot, proteasome activity using fluorogenic substrates, and aggregate load by filter‑trap assay and immunofluorescence for p62 and ubiquitin.
• Rescue experiments will include METTL3 catalytic dead control and non‑targeting sgRNA to rule out off‑target effects.

If the hypothesis holds, restoring m6A at defined UPS transcripts will reestablish proteasome flux and attenuate aggregation, demonstrating that AD pathology stems from a failure of adaptive, site‑specific m6A remodeling rather than a generic loss of methyltransferase activity.