Hypothesis

Aged corticotropin‑releasing factor (CRF) neurons in the central amygdala actively suppress autophagy through a convergent mTORC1‑Rubicon‑DNMT2 axis, leading to accumulation of damaged mitochondria and protein aggregates that impair fear extinction.

Mechanistic Model

1. mTORC1 hyperactivation persists in aged CRF neurons, keeping ULK1 phosphorylated and TFEB cytoplasmic, thereby blocking autophagy initiation and lysosomal gene expression (1).
2. Rubicon upregulation binds BECN1 and inhibits Vps34 lipid kinase activity, preventing autophagosome nucleation specifically in these neurons (1).
3. Epigenetic silencing via DNMT2‑mediated promoter hypermethylation reduces transcription of Atg5 and LC3B, further diminishing autophagic flux (2).

Together, these mechanisms create a self‑reinforcing loop: impaired autophagy fails to clear ROS‑damaged mitochondria, which activates NF‑κB and sustains mTORC1 signaling, while accumulated p62 aggregates sequester upstream inhibitors of mTORC1, reinforcing the suppressive state.

Testable Predictions

• In aged mice, CRF‑neuron‑specific removal of Rubicon or pharmacological inhibition of DNMT2 will restore autophagic flux (measured by LC3‑II turnover and p62 degradation) and rescue fear extinction without altering baseline anxiety.
• Chemogenetic activation of TFEB in aged CRF neurons will bypass mTORC1‑mediated cytoplasmic retention, increase lysosomal gene expression, and improve extinction retention.
• Conversely, inducing mTORC1 hyperactivity in young CRF neurons will recapitulate the aged autophagic suppression phenotype and produce extinction deficits.

Experimental Approach

1. Use CRF‑Cre mice crossed with floxed Rubicon or DNMT2 alleles; administer tamoxifen at 18 months to delete genes selectively in CRF neurons.
2. Measure autophagy flux in amygdala slices via western blot for LC3‑II/I and p62 after bafilomycin A1 treatment.
3. Assess fear extinction using auditory fear conditioning; compare freezing rates across trials.
4. Perform RNA‑seq on sorted CRF neurons to verify TFEB target gene expression and mitochondrial stress signatures.
5. Rescue experiments: AAV‑TFEB (nuclear‑localized) or AAV‑constitutively active ULK1 delivered to amygdala of aged wild‑type mice.

Implications

If validated, this hypothesis reframes age‑related anxiety not as a passive decline but as an active neuronal strategy that sacrifices cellular renewal to maintain excitability. It opens therapeutic avenues targeting autophagy regulators specifically within stress circuits, potentially preserving cognitive flexibility without globally altering mTORC1 signaling.

References

[1] https://pmc.ncbi.nlm.nih.gov/articles/PMC11352966/ [2] https://pmc.ncbi.nlm.nih.gov/articles/PMC4889231/ [3] https://www.frontiersin.org/journals/neuroscience/articles/10.3389/fnins.2023.1152503/full