Background. Oxidative stress and reactive oxygen species (ROS) are important in the pathogenesis of amyotrophic lateral sclerosis (ALS). Hypochlorous acid (HOCl) is a powerful oxidant of the reactive oxygen species (ROS) family. HOCl’s role in the progress of ALS remains unclear due to the lack of an effective HOCl detection method. Cumulative evidence supports oxidative damage incurred by mutant hSOD1 contributing to motor neuron death; however, whether HOCl as well as its catalytic enzyme myeloperoxidase (MPO) function in the cell death of SOD1G93A ALS remains elusive. Methods. The hSOD1WT and hSOD1G93A NSC-34 cell and SOD1G93A ALS mouse models were employed. With a novel fluorescent HOCl probe, HKOCl-3, we detected the expressions of HOCl and its catalytic enzyme, MPO, in the above models in vitro and in vivo. The regulation of MPO/HOCl by hSOD1G93A mutation and cell deaths by MPO/HOCl were also assayed, including apoptosis, ferroptosis, and autophagy. Results. Our results showed that hSOD1G93A mutation promoted the activation of the MPO/HOCl pathway in SOD1G93A ALS cell models. The activation of MPO/HOCl pathways facilitated apoptosis and ferroptosis through increasing the Bax/Bcl-2 ratio and expression of caspase-3 or inhibiting the expressions of GPX4 and NQO1 and thus leading to irreversible lipid peroxidation. Overexpressed FSP1, a glutathione-independent suppressor, could ameliorate ferroptosis. In vivo, we demonstrated that the activation of the MPO/HOCl pathway occurred differently in motor neurons of the motor cortices, brain stems, and spinal cords in male and female SOD1G93A transgenic mice. In addition, inhibiting MPO improved the motor performance of SOD1G93A transgenic mice, as demonstrated by the rotarod test. Conclusions. We concluded that aggregation of mutant hSOD1 proteins contributed to activation of the MPO/HOCl pathway, triggering apoptosis and ferroptosis in motor neuronal deaths and exerting impaired motor performance.
Background
Chronic morphine exposure induces immunosuppression in the peripheral and central nervous system, resulting in susceptibility of patients to invading pathogens. Mitophagy is a crucial regulator of inflammation, and dysregulated mitophagy may cause immunosuppression, but whether mitophagy is linked with morphine-induced immunosuppression in the brain remains unknown. NLRX1 is the only mitochondrially localized NOD family receptor protein which serves as a critical regulator in immunity and mitophagy activation, but it remains an enigma how NLRX1 functions in the crosstalk between microglial inflammatory defense and mitophagy in the presence of morphine.
Methods
Primary microglia and astrocytes, BV2 and MA cell lines were utilized. Mice were stimulated with repeated morphine treatment to mimic chronic morphine exposure, and activation of mitophagy, lysosomal functions, and inflammation were assayed in specific brain regions and immune organs with or without NLRX1-silencing.
Results
Morphine induced microglial mitophagy in a LC3 (microtubule-associated proteins light chain 3)-dependent manner, which was mediated by NLRX1. Contrastingly, morphine impaired lysosomal functions, including generation, acidification and mitophagosome–lysosome fusion, thus leading to insufficient mitophagy activation in microglia. NLRX1-silencing inhibited mitophagy activity and rescued lysosomal functions including generation and acidification in microglia. The NLRX1-mediated incomplete mitophagy in microglial cells contributed to immunosuppression and vulnerability towards pathogenic challenge after morphine treatment. In vivo, NLRX1-mediated microglial mitophagy activation by morphine was mainly located in the murine brain cortex, striatum, and cerebellum, where NLRX1 functioned as a negative immune regulator and facilitated septic shock. Collectively, microglial immune responses to septic shock were amenable to NLRX1 silencing in the brain with morphine treatment.
Conclusion
Morphine activated insufficient mitophagy in microglia which was regulated by NLRX1, ultimately leading to host immunosuppression and susceptible conditions in the brain.
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