NCX Inhibitors’ Pivotal Role in Neuroprotection Against Oxaliplatin-induced Peripheral Neurotoxicity and Necroptosis

Oxaliplatin (OHP) is a cornerstone drug for colorectal cancer treatment, but is hampered by OHP-induced Peripheral Neurotoxicity (OIPN), which comprises an acute and chronic form. Acute OIPN consists of a transient axonal hyperexcitability syndrome after each chemotherapy cycle, caused by functional alterations of Voltage-gated Sodium Channels (NaV). Chronic OIPN is, instead, a sensory neuropathy with a persistent axonal damage (AxD). Acute and chronic OIPN might be linked: acute OHP-related NaV dysfunction leads to an excessive intake of Na+, which might be compensated via the Sodium-Calcium exchanger (NCX) switching to its reverse-mode. To rebalance Na+, NCX exchanges the latter with Ca2+, potentially leading to Ca2+-related toxicity and AxD. We aimed to verify this hypothesis in an in vitro setting, with particular attention to NCX2. We performed multiple studies on primary Dorsal Root Ganglia neuronal culture of adult mice (male C57BL/6 mice; 8-10weeks) using primarily the Nanolive CX-A-3D holotomographic microscope, a state-of-art live-imaging platform. We tested potential neuroprotectants, such as SEA0400, a potent and selective NCX inhibitor, and then siRNAs to specifically target NCX2. OHP-exposed neurons (7.5-25µM, 48h) showed an overall neurite fragmentation and a high-reduced viability, preceded by autophagic stress and necroptosis, in a dose and time-dependent manner. The latter events can be efficiently counteracted by SEA0400 pre-treatment (1µM, 3h before OHP-incubation). Moreover, OHP-induced alterations can be prevented specifically silencing NCX2 expression via siRNAs (5nM, 24h pre-treatment before OHP-incubation). Overall, results confirm the pivotal role of NCX in AxD development and specifically the NCX2 isoform involvement. We demonstrated that OIPN-related AxD was efficaciously prevented targeting NCX family and specifically NCX2 isoform, paving the way to new treatment strategies to prevent AxD. This mechanism might be implicated even beyond OIPN, since upstream and downstream events, respectively axonal hyperexcitability and Ca2+-related toxicity via NCX, were hypothesized to be involved in other important neuropathies.