PROTEASOME INHIBITORS: IN VITRO MORPHO- FUNCTIONAL CHANGES

Chemotherapy has significantly increased patient survival rates, but it has also led to a rise in chemotherapy-induced peripheral neuropathy (CIPN), which can severely impact quality of life and may lead to treatment discontinuation. Among chemotherapy drugs, 20S proteasome inhibitors like bortezomib (BTZ) and carfilzomib (CFZ), approved by the U.S. Food and Drug Administration for treating multiple myeloma and some other liquid tumors, have several limitations, including CIPN development. This study aims to investigate the effects of BTZ and CFZ on the cytoskeleton and mitochondria in primary cultures of dorsal root ganglion (DRG) sensory neurons isolated from adult mice. Neurons were treated with BTZ (10 nM) and CFZ (60 nM) for 10 and 24 h. Mitochondrial activity and functionality were assessed using the Seahorse Assay, mitochondrial membrane potential and mitochondrial trafficking measurements, while mitochondrial morphology was analysed using Mitochondrial Network Analysis (MiNA). Then, we examined the drug-induced changes in multiple proteins involved in the cytoskeleton and mitochondrial dynamics using immunoblotting. Our data show that both BTZ and CFZ impact mitochondrial membrane potential at 10 h post-treatment while mitochondrial respiration is affected by both drugs only after 24 h. Regarding mitochondrial trafficking, both BTZ and CFZ appear to affect anterograde transport as early as 12 h post-treatment, but only BTZ causes a significant increase in the number of stationary mitochondria at 24 h. Moreover, BTZ can alter microtubule dynamics by promoting more stable tubulin isoforms, as evidenced by increased MAP2 expression and stable tubulin modifications, such as acetylated and Δ2-tubulin, as early as 10 hours after treatment. Taken together, these results suggest that changes in mitochondrial morphology might represent a common mechanism of cellular toxicity, while the neurotoxic effects of BTZ might be linked to specific cytoskeletal alterations, resulting in significant axonal transport impairment. A deeper understanding of these pathways could help to identify potential therapeutic targets for BTZ-induced CIPN. This work is supported by Fondazione Cariplo, Grant #2019- 1482