Hypothesis

The variability in autophagy enhancement observed upon disrupting the BCL-2/Beclin-1 interaction across aging stem cell populations is determined by the expression level of NAF-1, which stabilizes the BCL-2/Beclin-1 complex at the ER and modulates downstream Vps34 activity.

Mechanistic Rationale

NAF-1 (nutrient-deprivation autophagy factor-1) binds BCL-2 and promotes its association with Beclin-1, thereby suppressing autophagy initiation [2]. Recent work shows that NAF-1 also interacts with the IP3R calcium channel, influencing ER Ca2+ release that is required for optimal Vps34 lipid kinase activity [3]. In aged hematopoietic stem cells (HSCs), approximately 30 % retain high autophagy, suggesting a subpopulation that may bypass NAF-1‑mediated inhibition. We propose that these autophagy‑competent HSCs express low NAF-1, rendering their BCL-2/Beclin-1 interaction less stable and more susceptible to disruption by BH3‑only proteins or post‑translational modifications. Conversely, NAF‑high HSCs maintain a robust BCL-2/Beclin-1 block, requiring stronger perturbations to achieve comparable autophagy flux.

In neural stem cells (NSCs), the Becn1F121A/F121A knock‑in model shows enhanced autophagy and neurogenesis [1]. We predict that NAF-1 expression in NSCs is dynamically regulated during aging, with higher NAF-1 correlating with the observed decline in autophagy flux. Manipulating NAF-1 levels should therefore amplify or diminish the healthspan benefits seen in the knock‑in mice.

Testable Predictions

1. Correlation – Single‑cell RNA‑flow cytometry of young versus aged HSCs and NSCs will reveal an inverse relationship between NAF-1 protein abundance and basal autophagy flux (measured by LC3‑II/I ratio and SQSTM1/p62 turnover).
2. Causality – Overexpression of NAF-1 in Becn1F121A/F121A NSCs will attenuate the increase in autophagy flux and rescue the age‑related decline in neurogenesis, whereas NAF-1 knockdown in wild‑type aged HSCs will mimic the autophagy‑enhanced phenotype of the knock‑in model.
3. Pharmacological Sensitivity – Treatment with a BCL-2 BH3 mimetic (e.g., ABT‑737) will increase autophagy flux only in NAF‑low stem cells; NAF‑high cells will require higher drug concentrations or combined NAF-1 inhibition to achieve similar effects.
4. Calcium Link – NAF-1 overexpression will dampen ER Ca2+ release upon IP3 stimulation, reducing Vps34 activity and autophagosome formation; this effect will be rescued by IP3R agonists or by expressing a NAF-1 mutant deficient in IP3R binding.

Experimental Approach

• Generate inducible NAF-1 overexpression and CRISPR‑mediated knockout lines in both HSCs (Vav‑Cre) and NSCs (Nestin‑Cre).
• Assess autophagy flux using tandem mCherry‑GFP‑LC3 reporters and SQSTM1/p62 degradation with/without Bafilomycin A1.
• Measure functional outcomes: competitive repopulation assays for HSCs, neurosphere formation and BrdU incorporation for NSCs, and lifespan/healthspan monitoring in mice.
• Validate mechanistic links via co‑immunoprecipitation of BCL‑2/Beclin‑1, NAF‑1, and IP3R, coupled with ER Ca2+ imaging using Cameleon sensors.

Potential Impact

Confirming NAF‑1 as a rheostat for BCL‑2/Beclin‑1‑dependent autophagy would explain cell‑type‑specific responses to aging‑related autophagy modulation and identify NAF‑1 as a therapeutic target to fine‑tune autophagy enhancement without broadly compromising BCL‑2’s anti‑apoptotic functions.