Hypothesis

Aging‑related sleep fragmentation impairs lysosomal autophagic flux in central amygdala (CeA) CRF‑expressing neurons, leading to the accumulation of CRF receptor 1 (CRFR1) protein. This buildup sustains heightened CRF neuron excitability, blocks fear extinction, and drives age‑dependent anxiety‑like phenotypes.

Mechanistic Rationale

Sleep promotes autophagic clearance of membrane proteins through glymphatic‑linked lysosomal activation. In young mice, spontaneous sleep cycles trigger periodic bursts of lysosomal acidification and cathepsin activity in CeA CRF+ neurons, facilitating CRFR1 turnover. With age, fragmented sleep reduces noradrenergic tone during REM, diminishing cAMP‑PKA signaling that normally activates mTORC1 inhibition and initiates autophagosome formation. Consequently, CRFR1 escapes degradation, accumulates at the plasma membrane, and enhances Gs‑mediated cAMP production. Elevated cAMP potentiates NMDA‑dependent calcium influx, further increasing CRF neuron firing and impairing GABAergic inhibition—both mechanisms shown to disrupt fear extinction [5][6].

Novel Insight

We propose that the critical node is the sleep‑regulated interaction between orexin‑A receptors on CeA CRF+ neurons and the lysosomal v‑ATPase complex. Orexin‑A, whose release peaks during wakefulness, suppresses v‑ATPase assembly via PKC‑dependent phosphorylation of the ATP6V1 subunit. During consolidated sleep, low orexin levels permit v‑ATPase re‑assembly, lysosomal acidification, and CRFR1 degradation. Age‑related loss of orexinergic tone disrupts this cyclic switch, trapping lysosomes in a semi‑active state that degrades bulk cytosol but fails to efficiently clear transmembrane receptors like CRFR1.

Testable Predictions

1. In aged mice, sleep fragmentation will correlate with increased CRFR1 immunoreactivity and decreased lysosomal LAMP1 colocalization in CeA CRF+ neurons compared with age‑matched mice with consolidated sleep.
2. Pharmacological enhancement of lysosomal acidification (e.g., with low‑dose chloroquine withdrawal or AMPK activator AICAR) during the sleep window will restore CRFR1 turnover and rescue fear extinction deficits in sleep‑fragmented aged mice.
3. Chemogenetic inhibition of orexinergic projections from the lateral hypothalamus to the CeA during the dark phase will mimic the effects of sleep fragmentation on CRFR1 accumulation and extinction impairment, whereas optogenetic activation of these projections during fragmented sleep will ameliorate the phenotype.
4. Viral‑mediated expression of a pH‑sensitive CRFR1‑GFP reporter will show slower fluorescence decay kinetics in CeA CRF+ neurons of aged, sleep‑fragmented mice, indicating reduced receptor turnover.

Experimental Approach

• Use aged (18‑month) C57BL/6J mice equipped with EEG/EMG to quantify sleep fragmentation.
• Perform immunofluorescence for CRFR1, LAMP1, and p‑S6 (mTOR activity) in CeA CRF+ neurons identified via CRF‑Cre;Rosa‑tdTomato lineage labeling.
• Measure fear extinction using conditioned freezing paradigms; correlate extinction retention with sleep metrics and CRFR1 levels.
• Apply lysosomal modulators (AICAR, V-ATPase enhancer) via intracerebroventricular delivery timed to the sleep phase.
• Employ DREADDs to inhibit orexin‑lateral hypothalamus → CeA projections and assess CRFR1 dynamics and behavior.
• Validate findings with the pH‑sensitive CRFR1‑GFP reporter and lysosomal pH probes (LysoSensor).

If lysosomal autophagic pruning of CRFR1 is indeed sleep‑dependent, rescuing this pathway should normalize fear extinction despite persistent sleep disruption, establishing a mechanistic bridge between sleep quality, proteostatic fidelity, and emotional memory regulation in aging.