In recent years, increased attention has been paid to the role of mitochondrial alterations in studies of traumatic brain injury (TBI). Mitochondria participate in the regulation of neuroinflammation after TBI. Disrupted mitochondrial respiration and impaired bioenergetics cause energy deficiency and reactive oxygen species (ROS) accumulation, facilitating the activation of pro-inflammatory signaling pathways in microglia, the innate immune cells of the brain. To robustly evaluate brain bioenergetics after TBI, we optimized a new Seahorse respirometry-based protocol, CRABS-ROC (Complex Respirometry Assay Bypassing Substrate Restricted Oxygen Consumption). This protocol not only unmasks the full electron transport chain (ETC) Complex I-dependent respiratory capacity by bypassing substrate restrictions but also measures Complex-II- and IV-dependent respiration in the same experiment, on a variety of sample types. Using CRABS-ROC, we assessed ETC complex respiratory capacities in a rat pediatric TBI model, revealing sex-specific Complex I and IV deficiencies in males at an individual ETC complex level.
To restore impaired mitochondrial respiration in pro-inflammatory microglial cells, a Coenzyme Q10 (CoQ) short-chain analogue idebenone was employed. Idebenone supplies electron flux through ETC Complex III, bypassing a disruption at Complex I. To achieve this activity, idebenone first requires a cytoplasmic reduction to its active form, idebenol. Previously, it was shown that the enzyme NAD(P)H quinone oxidoreductase 1 (NQO1) reduces idebenone in astrocytes. Here, we identified a different enzyme, inducible nitric oxide synthase (iNOS), which catalyzes most of the idebenone reduction in microglia.
Restoring impaired microglial respiratory function is predicted to inhibit pro-inflammatory signaling and mitigate secondary brain injury. Therefore, we also investigated ETC complex degradation mechanisms in microglia. We first observed that among representative subunits of each ETC complex, only the Complex III subunit UQCRC2 and the Complex V subunit ATPA5 remained unchanged in microglial cells stimulated to a pro-inflammatory state by lipopolysaccharide (LPS) and interferon-gamma. Using iNOS-deficient Nos2 knockout microglial cells, we found that nitric oxide (NO) regulates the degradation of the Complex II subunit SDHB and the Complex IV subunit MTCO1, but not the Complex I subunit NDUFB8. These findings indicate that multiple mechanisms contribute to ETC impairment in activated, pro inflammatory microglia. A positive feedback loop between iNOS and activated caspase-1 protease was also identified, revealing a new system regulating the degradation of ETC complexes. These findings complement existing knowledge on pro-inflammatory signaling amplification.