NAD+ as a rheostat: SIRT1-mediated transcriptional restraint links metabolite decline to HSC fate choices in aging

Elle Acheampong (DPhil)· Mar 23, 2026

Mechanism: In aged HSCs, NAD+ decline due to CD38 activity reduces SIRT1, lifting repression on PU.1/C/EBPα and promoting myeloid differentiation. Readout: Readout: NAD+ restoration increases SIRT1, represses differentiation factors, and improves long-term repopulation capacity by 75%.

Hypothesis

NAD+ decline in aged hematopoietic stem cells (HSCs) is not merely a passive consequence of damage; it actively tunes a SIRT1‑dependent transcriptional program that limits self‑renewal and promotes a quiescent, differentiation‑biased state as a protective response to inflammatory stress.

Mechanistic Rationale

Recent work shows that inflammation‑driven CD38 upregulation depletes NAD+ 3, lowering SIRT1 activity and creating a pseudohypoxic state 1. While SIRT1 loss is often viewed as deleterious, SIRT1 also deacetylates and represses key pro‑differentiation transcription factors such as PU.1 and C/EBPα 4. When NAD+ falls, SIRT1 activity drops, lifting this repression and allowing differentiation programs to proceed. However, in the inflammatory milieu of aged bone marrow, transient SIRT1 inhibition can be advantageous: it reduces the energetic cost of maintaining a highly proliferative epigenome and forces HSCs into a low‑metabolism, stress‑resistant state 2. Thus, NAD+ decline functions as a rheostat that couples metabolite availability to SIRT1‑mediated restraint of lineage commitment, preserving the stem cell pool under stress.

Testable Predictions

In aged HSCs, pharmacologically raising NAD+ (e.g., with NR or CD38 inhibitor) will increase SIRT1 activity and thereby increase expression of PU.1 and C/EBPα, leading to accelerated myeloid differentiation and reduced long‑term repopulation capacity.
Genetic ablation of SIRT1 in young HSCs will mimic the aged transcriptional profile (high PU.1/C/EBPα, low self‑renewal genes) even when NAD+ levels are normal.
Combining NAD+ restoration with SIRT1 knockdown in aged mice will rescue the differentiation bias caused by NAD+ elevation alone, restoring balanced output.
Single‑cell ATAC‑seq will reveal that NAD+ elevation reduces chromatin accessibility at quiescence‑associated loci while increasing accessibility at myeloid enhancers, an effect abrogated by SIRT1 loss.

Experimental Approach

Isolate LT‑HSCs from young (3 mo) and old (24 mo) CD38‑wildtype and CD38‑knockout mice.
Treat ex vivo with NR (1 g kg⁻¹ day⁻¹) or vehicle for 48 h, measure NAD+ levels, SIRT1 activity (fluorometric deacetylase assay), and PU.1/C/EBPα protein by intracellular flow.
Perform competitive repopulation assays (1 × 10⁴ donor cells) to assess long‑term chimerism at 16 weeks.
In parallel, generate Vav‑Cre;Sirt1^fl/fl mice to delete SIRT1 specifically in hematopoietic cells and repeat NAD+ supplementation.
For mechanistic depth, conduct scRNA‑seq and scATAC‑seq on sorted HSCs from each condition to map transcriptional and chromatin changes.

Falsification: If NAD+ elevation does not increase PU.1/C/EBPα expression or does not impair repopulation capacity, or if SIRT1 loss fails to reproduce the aged differentiation bias, the hypothesis that NAD+ decline functions as a rheostat via SIRT1‑mediated transcriptional restraint would be refuted.

References

[1] https://doi.org/10.1016/j.cell.2013.11.037 [2] https://pmc.ncbi.nlm.nih.gov/articles/PMC12809054/ [3] https://doi.org/10.1016/j.bbrc.2019.03.199 [4] https://doi.org/10.1016/j.cmet.2018.03.016

Mechanistic Rationale

Testable Predictions

In aged HSCs, pharmacologically raising NAD+ (e.g., with NR or CD38 inhibitor) will increase SIRT1 activity and thereby increase expression of PU.1 and C/EBPα, leading to accelerated myeloid differentiation and reduced long‑term repopulation capacity.

Genetic ablation of SIRT1 in young HSCs will mimic the aged transcriptional profile (high PU.1/C/EBPα, low self‑renewal genes) even when NAD+ levels are normal.

Combining NAD+ restoration with SIRT1 knockdown in aged mice will rescue the differentiation bias caused by NAD+ elevation alone, restoring balanced output.

Single‑cell ATAC‑seq will reveal that NAD+ elevation reduces chromatin accessibility at quiescence‑associated loci while increasing accessibility at myeloid enhancers, an effect abrogated by SIRT1 loss.

Experimental Approach

Isolate LT‑HSCs from young (3 mo) and old (24 mo) CD38‑wildtype and CD38‑knockout mice.

Treat ex vivo with NR (1 g kg⁻¹ day⁻¹) or vehicle for 48 h, measure NAD+ levels, SIRT1 activity (fluorometric deacetylase assay), and PU.1/C/EBPα protein by intracellular flow.

Perform competitive repopulation assays (1 × 10⁴ donor cells) to assess long‑term chimerism at 16 weeks.

In parallel, generate Vav‑Cre;Sirt1^fl/fl mice to delete SIRT1 specifically in hematopoietic cells and repeat NAD+ supplementation.

For mechanistic depth, conduct scRNA‑seq and scATAC‑seq on sorted HSCs from each condition to map transcriptional and chromatin changes.