Mitochondrial activity tunes cellular resilience to excitotoxicity

The capsaicin receptor, TRPV1, mediates the detection of noxious chemical and thermal stimuli by nociceptors, primary sensory neurons of the pain pathway. Overactivation of TRPV1 leads to cellular damage or death through calcium entry and excitotoxicity. We have exploited this phenomenon to conduct a systematic analysis of excitotoxicity through a genome-wide CRISPRi screen, thereby revealing a comprehensive network of regulatory pathways. We show that decreased expression of mitochondrial electron transport chain (ETC) components protects against capsaicin-induced toxicity and other challenges by mitigating both calcium imbalance and the generation of mitochondrial reactive oxygen species via distinct pathways. Moreover, we confirm the regulatory roles of the ETC in sensory neurons through gain-of-function and loss-of-function experiments. Interestingly, TRPV1+ sensory neurons maintain lower expression of ETC components and can better tolerate excitotoxicity and oxidative stress compared to other sensory neuron subtypes, implicating ETC tuning as an intrinsic cellular strategy that protects nociceptors against excitotoxicity. For the CRISPRi screen, we engineered a TRPV1+ K562 clonal cell line expressing the required CRISPRi machinery. These cells were transduced with a well-validated genome-scale single-guide RNA (sgRNA) library that targets 18905 genes with 5 sgRNAs/gene and 1895 non-targeting (NT) sgRNA controls (hCRISPRi_v2, Horlbeck et al., 2016). Samples were collected from two biological replicates at the outset of the experiment (T0) and from either untreated or capsaicin treated cells at day 14, the experimental endpoint . For comparative transcriptomics, RNA was extracted from triplicated samples (TRPV1+ K562 cells) that were pretreated with either vehicle (Veh) or Piericidin A (PierA) for 3 days then treated with either vehicle (Veh) or capsaicin (Cap) for 4 hours. cDNA libraries were prepared from RNA and sequenced.