%0 Generic
%A Raith, Fabio
%C Heidelberg
%D 2022
%F heidok:31254
%R 10.11588/heidok.00031254
%T Development of fluorogenic substrates for NAD(P)‑Snifits enables the investigation of nicotinamide adenine dinucleotide metabolism in primary neurons
%U https://archiv.ub.uni-heidelberg.de/volltextserver/31254/
%X The members of the cofactor family of nicotinamide adenine dinucleotides are involved in many biological processes. New insights about their role in health and disease have highlighted the need to measure these metabolites in live cells. Fluorescent biosensors have been proven to be valuable tools for metabolite measurements with subcellular resolution. Among them, the NAD(P)‑Snifits have been developed to measure free NAD⁺ levels and NADPH/NADP⁺ ratios, respectively. The applicability of these FRET‑based biosensors in physiological relevant model systems is limited by the permeability of the tetramethylrhodamine (TMR)‑based FRET donor substrate and its SNAP‑tag labelling.  In this work, fluorogenic FRET donor substrates were developed by manipulating the spirocyclization equilibrium of TMR. Their permeability and cellular SNAP‑tag labelling outperformed early versions of these substrates, especially when they were derivatized with an optimized SPR inhibitor. Systematic characterization selected two FRET donor substrates that could also label the NAD(P)‑Snifits in cultured primary neurons. Consequently, free subcellular NAD⁺ levels and NADPH/NADP⁺ ratios could be measured for the first time in primary neurons. These improvements allowed the comparison of free subcellular NAD⁺ levels and NADPH/NADP⁺ ratios between U2OS cells and primary neurons by FLIM‑FRET measurements. Significant differences were revealed in free mitochondrial NAD⁺ levels and NADPH/NADP⁺ ratios, which could be the result of differences in oxidative phosphorylation activity and have not been described before.  In short, this work provides new tools for the quantification of NAD(P) metabolites in physiological relevant model system and thus help to gain new insights into their  metabolism.