ER-resident NAF-1 amplifies BCL-2/Beclin-1 inhibition in aging macrophages, linking ER stress to inflammasome activation

Hypothesis

Aging-associated ER stress upregulates NAF-1, which strengthens BCL-2 binding to Beclin-1 specifically at the ER membrane of macrophages. This heightened interaction suppresses autophagy, leading to mitochondrial ROS accumulation and NLRP3 inflammasome activation. Consequently, chronic inflammasome signaling drives age‑related tissue damage. Disrupting the NAF-1–BCL-2 interaction in myeloid cells should restore autophagy, dampen inflammasome activity, and extend healthspan.

Rationale

It's known that the BCL-2/Beclin-1 complex is a well‑characterized brake on autophagy that intensifies with age 23220384. Genetic release of Beclin-1 via the Becn1 F121A mutation improves autophagy, lifespan and organ function 29849149. NAF-1 has been shown to enhance BCL-2’s inhibitory function specifically at the ER 3304572. Aging triggers ER stress, which can increase NAF-1 expression 3901098. In macrophages, ER stress–induced ROS promotes NLRP3 inflammasome activation 23220384. Thus, NAF-1 may act as a stress‑dependent amplifier of the BCL-2/Beclin-1 brake, coupling ER stress to defective autophagy and inflammasome signaling.

Predictions

1. NAF-1 protein levels will be elevated in ER fractions of peritoneal macrophages from old (≥24 mo) mice compared with young (3 mo) mice.
2. Co‑immunoprecipitation will show increased BCL-2/Beclin-1 binding in ER extracts from aged macrophages, correlating with NAF-1 abundance.
3. We're expecting that myeloid‑specific NAF-1 knockout (Naf1^fl/fl LysM‑Cre) will restore basal autophagy (measured by LC3‑II turnover and p62 degradation) in peritoneal macrophages from old mice.
4. These mice will exhibit reduced mitochondrial ROS, lower NLRP3 inflammasome activation (decreased cleaved caspase‑1 and IL‑1β), and ameliorated age‑related renal fibrosis and cardiac hypertrophy.
5. Lifespan analysis will reveal a modest but significant extension of median survival in Naf1^fl/fl LysM‑Cre mice versus controls.

Experimental Approach

• Generate Naf1^fl/fl mice and cross with LysM‑Cre to delete NAF-1 in monocytes/macrophages.
• Isolate peritoneal macrophages from young and old WT and knockout mice.
• Perform subcellular fractionation to obtain ER membranes; immunoblot for NAF-1, BCL-2, Beclin-1, and assess BCL-2/Beclin-1 co‑IP.
• Measure autophagy flux with bafilomycin A1 treatment and LC3‑II/p62 immunoblotting.
• Assess mitochondrial ROS using MitoSOX flow cytometry and NLRP3 inflammasome activation via Western blot for cleaved caspase‑1 and ELISA for IL‑1β.
• Histologically evaluate kidney (Masson’s trichrome) and heart (Wheat germ agglutinin) for fibrosis/hypertrophy.
• Cohort survival study (n ≥ 30 per sex) monitoring natural death.

Potential Implications

If validated, this hypothesis would identify NAF-1 as a myeloid‑specific checkpoint that couples ER stress to autophagy suppression and inflammasome activation during aging. Targeting NAF-1 (e.g., with peptide disruptors or small molecules) could rejuvenate macrophage autophagy without globally inhibiting BCL-2/Bax apoptosis, offering a precision strategy to mitigate inflammaging while preserving cell death regulation.