SUN-479 compartmentalized regulation of adp-ribosylation by glucocorticoids

Abstract Disclosure: M. Deng: None. J. Ng: None. S. Heaselgrave: None. G.G. Lavery: None. C.L. Doig: None. Glucocorticoid excess induces profound muscle wasting, yet a complete understanding of causal mechanisms remains incomplete. ADP-ribosylating PARPs enzymatically cleave the metabolic cofactor NAD+ to post-translationally modify target proteins with an ADP-ribose moiety. ADP-ribosylation exists in either mono-ADP-ribose subunits (MAR) or poly-chains (PAR), with their signalling roles in skeletal muscle unclear. Previously, we showed a relationship between ADP-ribosylation and glucocorticoids that impact muscle cell turnover and differentiation. Given these findings and recent advances showing MAR and PAR can exist independently we set out to reveal their sub-cellular localisation in skeletal muscle and their response to the synthetic glucocorticoid dexamethasone. In whole murine muscle cells (C2C12s), we show dexamethasone(1μM), to inhibit PAR abundance (0.61-fold change ±0.13 sem, p<0.01; n=8) with a corresponding increase in NAD+ (20% increase, p<0.02; n=8). This reflects an inhibitory effect of glucocorticoids on PAR and the rate-limiting nature of NAD+. Interestingly, mono-ADP-ribose increases significantly (1.34 ± 0.20, p<0.001; n=6) in response to dexamethasone, suggesting glucocorticoids not only repress PAR formation but may stimulate PAR breakdown and liberation of mono ADP-ribose units. Examination of subcellular fractionations of myocytes reveals PAR is absent from cytoplasm despite an abundance of MAR in this compartment. This supports the notion that MAR can exist in cells as a distinct, independent signalling motif. PAR is found in microsomes (sarco/endoplasmic reticulum), mitochondria, and nuclei and it decreases with dexamethasone treatment (0.75 ± 0.08) (0.85 ± 0.07), (0.68 ± 0.1), respectively. MAR is significantly elevated in all fractionated organelles responding to dexamethasone treatment (nucleus: 1.61 ± 0.30; mitochondria: 2.37 ± 0.68; microsomes: 3.36 ± 1.18; cytoplasm: 3.28 ± 1.14, p<0.05; n=3). Examination of ex vivo skeletal muscle tissue from C57/BL6 mice fed glucocorticoid spiked drinking water reveals that corticosterone excess (100 mg/L) reduced PAR (0.79 ± 0.15, p<0.05; n=6) in skeletal muscle tissues. Based on the dexamethasone-induced elevation of MAR in microsomes, we examined transcriptional responses of PARPs 1-16 to glucocorticoids by QPCR and found microsomal PARP16 to be upregulated (1.82 fold change ± 0.18, p<0.05; n=3). Building on this, we propose that, like NAD+, variants of ADP-ribosylation (PAR and MAR) are compartmentalized, and dynamically controlled within the highly metabolic organ skeletal muscle. These findings also suggest that glucocorticoids impact the localisation and utilization of this potentially important post-translational modification. Finally, our work implicates the NAD+-PARP1-ADP-ribose signalling axis within muscle mass maintenance and glucocorticoid-driven myopathy. Presentation: Sunday, July 13, 2025