CHARACTERIZATION OF AXONAL DEGENERATION MECHANISMS IN BORTEZOMIB-TREATED SENSORY NEURONS

Introduction: Bortezomib (BTZ) is a proteasome inhibitor used for the treatment of multiple myeloma. BTZ treatment results in axonal degeneration, producing a disabling painful toxic peripheral neuropathy in approximately 50% of patients. In the current study, we examined whether treatment with BTZ for 24 hours would affect microtubule(MTs) stability as well as axonal transport and oxidative phosphorylation in primary cultured dorsal root anglion (DRG) sensory neurons. Methods: MT stability was indirectly measured by western blotting and quantitative confocal microscopy of delta2- tubulin and tubulin acetylation, while axonal mitochondrial trafficking was evaluated by time-lapse confocal microscopy and kymograph analysis. To valuate the effects of BTZ on the generation and regulation of cellular bioenergetics, mitochondrial oxidative phosphorylation (OXPHOS) was assessed via western blot analysis of key molecular markers. Results: BTZ treatment of cultured DRG sensory neurons induced an approximately 2.5-fold increase of delta2 and acetylated tubulin levels, which occurred at the onset of axonal degeneration. Furthermore, DRG axonal mitochondrial motility and trafficking were decreased by BTZ; these changes occurred independently of the direction of mitochondrial trafficking. Finally, BTZ treated DRG neurons had differential protein expression of OXPHOS subunits compared to untreated DRG neurons. Conclusions: In summary, our results demonstrate that BTZ effects both MT stability and mitochondrial function in DRG neurons, supporting the idea that both loss of normal tubulin and mitochondria function together promote the development of BTZ mediated neuropathy. This work is supported by Fondazione Cariplo, Grant # 2019-1482