Recently, after years of "sales restrictions," the U.S. FDA announced that it will no longer restrict the sale of NMN before July 31, 2025. NMN, which has long been mired in controversy, has finally gotten a reprieve—but the battle is far from over.
The restrictions on NMN were not just about debating whether it is a "supplement" or "drug." Critical questions remain: Is its content mislabeled? Is it sufficiently safe? To completely escape the "ban list," more in-depth research on NMN is essential.
At this crucial juncture for NMN, Chinese researcher Prof. Quliyang Yu and his team delivered exciting news: They developed a protein probe that can real-time monitor dynamic changes of NMN in living cells. With this tool, scientists say they no longer have to guess how cells utilize NMN—it is a powerful weapon for NMN research [1].
1. The Emergence of the NMN Protein Probe
While methods to detect NMN and its related metabolites have existed in previous NAD+ research, the new probe stands out by simultaneously meeting multiple key requirements: anti-interference, high sensitivity, subcellular localization capability, and real-time monitoring.
This probe is NMoRI—a genetically encoded protein probe iteratively optimized and specifically designed for intracellular NMN detection.
Figure Note: Schematic diagram of NMoRI.
NMoRI is a protein structure highly sensitive and specific to NMN. Its two ends are linked to a blue-light-emitting luciferase (cpNLuc) and a red fluorescent protein (RFP) that resonates with cpNLuc to emit red light. The BRET (Bioluminescence Resonance Energy Transfer) efficiency between cpNLuc and RFP can be monitored via spectroscopy. In short, if the target substance (NMN) changes the relative position of cpNLuc and RFP at the probe’s ends, its concentration can be measured by spectral changes.
- Before binding to NMN: cpNLuc and RFP are far apart, resulting in low BRET signals.
- After NMN binds to NMoRI: The probe’s conformation changes, bringing cpNLuc and RFP closer. This increases energy transfer efficiency and enhances BRET signals—showing NMN concentration-dependent BRET changes.
The core technology of NMoRI lies in quantifying intracellular NMN concentration by monitoring BRET signal intensity.
Figure Note: NMN binding induces conformational changes in NMoRI, shortening the distance between the N-terminal and C-terminal of the protein scaffold.
What makes NMoRI even more valuable is its high specificity for NMN: It shows no significant response to other NMN analogs, and is barely affected by interference factors like autofluorescence or pH. As NMoRI "declares": "I only respond to NMN—mistaking other molecules is impossible!"
Figure Note: NMoRI has high specificity for NMN and no significant response to other analogs (e.g., NAD+, AMP, ADP, ATP).
2. Uncovering the Mystery of NMN Uptake and Distribution
To test NMoRI’s performance, researchers conducted experiments on the human embryonic kidney cell line HEK 293T. The results showed that NMoRI perfectly revealed the dynamic trajectory of NMN uptake under different NAD+ precursors (NAM, NMN, NMNH, NR, NRH):
- For NMN: The intracellular BRET signal of NMoRI increased rapidly within 4 minutes in a concentration-dependent manner—proving NMN is quickly taken up by cells.
- For other precursors: NR and NRH, which can be rapidly converted into NMN intracellularly, also triggered a fast BRET signal increase. However, NA and NAM showed no significant response within 10 minutes—likely due to differences in cell uptake efficiency and precursor-to-NMN conversion rates.
Figure Note: BRET signal intensity of NMoRI in HEK 293T cells exposed to different concentrations of NAD+ precursors.
Further studies showed that when CD73 (an enzyme that converts extracellular NMN to NR) and ENTs (nucleoside transporters)—key players in the conversion process—were inhibited:
- The signals of NR and NRH uptake and conversion disappeared.
- The NMN uptake signal remained significant.
Through NMoRI, researchers confirmed that cells use multiple mechanisms to take up NMN—solving the long-standing question of "which precursor is more effective."
Figure Note: Left: Main transport pathways of NAD+ precursors; Right: Effects of inhibiting CD73 and ENTs on NR/NRH conversion to NMN.
Another key question: How to track NMN distribution in subcellular structures? The answer is simple—attach organelle-targeting peptides (precision locators) to NMoRI.
After establishing a calibration curve (to correlate probe signals with actual NMN concentrations) for HEK 293T cells expressing subcellular protein probes, researchers found:
- Under normal conditions, NMN levels in HEK 293T cells were 3.07 μM (cytoplasm), 3.65 μM (nucleus), and 6.16 μM (mitochondria).
- Most NAD+ precursors significantly increased NMN levels in these subcellular compartments.
Figure Note: Top: Establishment of calibration curves for HEK 293T cells; Bottom: NAD+ precursors significantly increased NMN levels in subcellular structures.
By real-time tracking NMN’s uptake trajectory and accurately quantifying its subcellular levels, NMoRI proves itself a "powerhouse" in exploring how cells utilize NMN.
3. The Delicate Balance of NMN/NAD+ Matters
Ultimately, whether studying NMN uptake or quantifying its levels, the focus remains on NAD+—a molecule closely linked to aging and lifespan. However, "more is better" does not apply to NMN. Growing evidence shows that an increased NMN/NAD+ ratio may activate the SARM1 protein, triggering neurological diseases.
With NMoRI, researchers made a surprising discovery: Some potent NAD+-boosting precursors (e.g., NMNH, NRH) significantly increased the NMN/NAD+ ratio and activated SARM1 (elevated cADPR levels are a marker of SARM1 activation). This effect was more pronounced in cells with high SARM1 expression, such as SH-SY5Y cells.
Figure Note: Effects of different NAD+ precursors on the NMN/NAD+ ratio and cADPR levels (cADPR increase was more obvious in SH-SY5Y cells).
Beyond NAD+ precursors, other molecules also play key roles in maintaining NMN/NAD+ balance:
- NMNATs (NAD+ synthases): Responsible for converting NMN to NAD+ and regulating adipocyte differentiation. NMoRI signals showed that knocking down NMNATs significantly increased cytoplasmic NMN levels, while overexpressing NMNATs decreased NMN levels.
- NudtX hydrolases and peroxisomal membrane proteins (e.g., Pxmp2).
Figure Note: Effects of altered NMNATs expression on intracellular NMN, NAD+, and NMN/NAD+ ratio.
In this context, NMoRI’s value becomes even more prominent: It helps research teams precisely control intracellular NMN levels, maximizing anti-aging benefits while ensuring safety. As the role of NMN/NAD+ imbalance in SARM1 activation gains more attention, NMoRI will undoubtedly turn this attention into actionable research progress.
For anyone interested in NMN, the future of "precision anti-aging with NMN" may just depend on this well-honed tool.
References
[1] Chen, L., Wang, P., Huang, G., Cheng, W., Liu, K., & Yu, Q. (2025). Quantitative dynamics of intracellular NMN by genetically encoded biosensor. Biosensors and Bioelectronics, 267, 116842. https://doi.org/10.1016/j.bios.2024.116842