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Mitochondrial derived reactive oxygen species (mtROS) are known for their signaling qualities in both physiology and pathology. To elucidate mitochondrial complex I-dependent ROS-signaling after lipopolysaccharide (LPS)-stimulation THP-1 macrophages with a knockdown of the transmembrane protein TMEM126B were generated. TMEM knockdown cells (sh126B) showed a reduced assembly of complex I and attenuated mtROS production. In these cells we identified protein oxidization by mtROS upon LPS-treatment using the BIAM switch assay coupled to liquid chromatography and mass spectrometry. One of the identified targets of mtROS was succinate dehydrogenase (SDH) flavoprotein subunit A (SDHA). Oxidation of SDHA decreased its enzymatic activity and pharmacological inhibition of SDH in turn stabilized hypoxia inducible factor (HIF)-1α and caused the subsequent, sustained expression of interleukin-1β (IL-1β). Oxidation of SDHA in sh126B cells was attenuated, while pharmacological inhibition of SDH by atpenin A5 restored IL-1β expression in sh126B cells upon LPS-treatment. Conclusively, oxidation of SDH by mtROS links an altered metabolism, i.e. succinate accumulation to HIF-1-driven, inflammatory changes in macrophages.
Nucleoredoxin is a thioredoxin-like redoxin that has been recognized as redox modulator of WNT signaling. Using a Yeast-2-Hybrid screen, we identified calcium calmodulin kinase 2a, Camk2a, as a prominent prey in a brain library. Camk2a is crucial for nitric oxide dependent processes of neuronal plasticity of learning and memory. Therefore, the present study assessed functions of NXN in neuronal Nestin-NXN-/- deficient mice. The NXN-Camk2a interaction was confirmed by coimmunoprecipitation, and by colocalization in neuropil and dendritic spines. Functionally, Camk2a activity was reduced in NXN deficient neurons and restored with recombinant NXN. Proteomics revealed reduced oxidation in the hippocampus of Nestin-NXN-/- deficient mice, including Camk2a, further synaptic and mitochondrial proteins, and was associated with a reduction of mitochondrial respiration. Nestin-NXN-/- mice were healthy and behaved normally in behavioral tests of anxiety, activity and sociability. They had no cognitive deficits in touchscreen based learning & memory tasks, but omitted more trials showing a lower interest in the reward. They also engaged less in rewarding voluntary wheel running, and in exploratory behavior in IntelliCages. Accuracy was enhanced owing to the loss of exploration. The data suggested that NXN maintained the oxidative state of Camk2a and thereby its activity. In addition, it supported oxidation of other synaptic and mitochondrial proteins, and mitochondrial respiration. The loss of NXN-dependent pro-oxidative functions manifested in a loss of exploratory drive and reduced interest in reward in behaving mice.