Bortezomib-induced peripheral neuropathy: an up-close look at endoplasmic reticulum-mitochondria interaction

Background Bortezomib-induced peripheral neuropathy (BIPN) is a condition developed by up to 60-70% of patients undergoing anti-cancer treatment with the first generation proteasome inhibitor bortezomib (BTZ). BIPN manifests both acutely and chronically as a set of sensory-related symptoms severely affecting patients’ quality of life and frequently lead to chemotherapy dose reduction or treatment withdrawal. Up to date, we lack effective therapeutic approaches against BIPN, mainly due to insufficient understanding of its pathogenesis. Therefore, our first aim was to unveil BIPN molecular basis, with a closer focus on those processes occurring at mitochondria-endoplasmic reticulum (ER) contact sites (MERCS). Secondarily, we proposed to test whether allopregnanolone (ALLO), a neuroactive steroid with already proven neuroprotective effects, could mitigate BTZ-induced toxicity by exerting its action specifically at ER-mitochondria interface, where both ALLO synthesis and metabolism are orchestrated. Methods We performed a set of functional and biochemical assays on MSC80, an immortalized model of murine Schwann cells, complemented by different imaging techniques: Ca2+ imaging, confocal and transmission electron microscopy (TEM). After preliminary dose-finding assays, BTZ was used at 10nM, while ALLO at 100nM, both for 24 hours. Results In MSC80, BTZ induces a drastic rearrangement of the mitochondrial network, with a contemporary clusterization of mitochondria in the perinuclear region. These alterations are accompanied by modifications in the expression levels of the main proteins involved in the regulation of mitochondrial dynamics, a set of processes aimed at adapting mitochondrial structure to the energetic requirements of the cell. Then, as we tested both by dedicated split-GFP contact sites sensors (SPLICS) and furtherly validated via TEM, BTZ induces a dramatic loss (~50%) of MERCS at different distances and an overall reduction in the juxtaposition between ER and mitochondrial membranes. Intriguingly, all the abovementioned alterations are significantly prevented when MSC80 are co-treated with ALLO in addition to BTZ. However, ALLO doesn’t seem to display the same protective effect over more functional outcomes affected by BTZ such as mitochondrial membrane potential and Ca2+ homeostasis in the mitochondrial matrix. Additionally, we found that BTZ also influences the expression levels of enzymes involved in the steroidogenic pathway, suggesting a never-explored link with the synthesis/metabolism of neuroactive steroids. Conclusions Collectively, our in vitro data introduce MERCS as new relevant targets for BTZ-induced toxicity over the peripheral nervous system and suggest that ALLO may represent a promising candidate for tackling BIPN, paving the way for further applications both in vitro and in vivo.