Transient signaling of free ADP-ribose monitored with an intracellular biosensor

Goyal, S.; Nguyen, V.; Lyons, S. N.; Dangerfield, T. L.; Yang, W.; Fields, C.; Chan, H.-R.; Mahendravarman, Y.; Cantu, L.; Rubin, N.; Deloney, K.; Johnson, K. A.; Cambronne, X. A.

doi:10.64898/2026.02.06.704453

bioRxiv · 10.64898/2026.02.06.704453

Transient signaling of free ADP-ribose monitored with an intracellular biosensor

Goyal, S.·Nguyen, V.·Lyons, S. N.·Dangerfield, T. L.·Yang, W.·Fields, C.·Chan, H.-R.·Mahendravarman, Y.·Cantu, L.·Rubin, N.·Deloney, K.·Johnson, K. A.·Cambronne, X. A.

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Abstract

We have developed a biosensor enabling the dynamic, compartmentalized, and longitudinal measurements of intracellular ADP-ribose (ADPR) in live cells. Free ADPR is a critical signaling metabolite derived from nicotinamide adenine dinucleotide (NAD+). As an agonist for Transient Receptor Potential Melastatin 2 (TRPM2), ADPR levels can regulate immune responses during infection, as well as nociception and adjustment of core body temperature. The study of ADPR signaling has been limited, however, by a lack of methods to measure this metabolite in situ. Using the biosensor and its paired non-responsive control, we determine that intracellular ADPR accumulation was transient and tunable. We found that basal concentrations were in the nanomolar range and could be stimulated [~]30-fold to activate TRPM2. We identified that TRPM2 activation, measured by calcium influx, required an intracellular ADPR threshold concentration between 2 - 4 {micro}M at physiological temperature. We observed that the timing of the ADPR rise coincided with TRPM2 activation, thus providing support for ADPR fluctuations being a critically regulated aspect for channel activation. Notably, transient fluctuations of ADPR were not accurately reflected by measurements of intracellular NAD+ loss or calcium levels. Significance StatementWe have developed a unique real-time biosensor for free ADP-ribose that is tuned to physiological concentrations and capable of intracellular measurements in individual cells. Using a calibrated system we determined that concentrations and timing of induced intracellular ADPR aligned with the thermosensitive TRPM2 activity. The data support ADPR as a critical component whose intracellular levels are regulated to control TRPM2 channel opening in cells.

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Goyal, S., Nguyen, V., Lyons, S. N., Dangerfield, T. L., Yang, W., Fields, C., Chan, H.-R., Mahendravarman, Y., Cantu, L., Rubin, N., Deloney, K., Johnson, K. A., Cambronne, X. A.. 2026-02-08. Transient signaling of free ADP-ribose monitored with an intracellular biosensor. https://doi.org/10.64898/2026.02.06.704453

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