We have previously shown that deletion of GOA1 (growth and oxidant adaptation) of Candida albicans results in a loss of mitochondrial membrane potential, ATP synthesis, increased sensitivity to oxidants and killing by human neutrophils, and avirulence in a systemic model of candidiasis. We established that translocation of Goa1p to mitochondria occurred during peroxide stress. In this report, we show that the goa1⌬ (GOA31), compared to the wild type (WT) and a gene-reconstituted (GOA32) strain, exhibits sensitivity to inhibitors of the classical respiratory chain (CRC), including especially rotenone (complex I [CI]) and salicylhydroxamic acid (SHAM), an inhibitor of the alternative oxidase pathway (AOX), while potassium cyanide (KCN; CIV) causes a partial inhibition of respiration. In the presence of SHAM, however, GOA31 has an enhanced respiration, which we attribute to the parallel respiratory (PAR) pathway and alternative NADH dehydrogenases. Interestingly, deletion of GOA1 also results in a decrease in transcription of the alternative oxidase gene AOX1 in untreated cells as well as negligible AOX1 and AOX2 transcription in peroxide-treated cells. To explain the rotenone sensitivity, we measured enzyme activities of complexes I to IV (CI to CIV) and observed a major loss of CI activity in GOA31 but not in control strains. Enzymatic data of CI were supported by blue native polyacrylamide gel electrophoresis (BN-PAGE) experiments which demonstrated less CI protein and reduced enzyme activity. The consequence of a defective CI in GOA31 is an increase in reactive oxidant species (ROS), loss of chronological aging, and programmed cell death ([PCD] apoptosis) in vitro compared to control strains. The increase in PCD was indicated by an increase in caspase activity and DNA fragmentation in GOA31. Thus, GOA1 is required for a functional CI and partially for the AOX pathway; loss of GOA1 compromises cell survival. Further, the loss of chronological aging is new to studies of Candida species and may offer an insight into therapies to control these pathogens. Our observation of increased ROS production associated with a defective CI and PCD is reminiscent of mitochondrial studies of patients with some types of neurodegenerative diseases where CI and/or CIII dysfunctions lead to increased ROS and apoptosis.

Glutathione (GSH) plays critical roles in the inflammatory response by acting as the master substrate for antioxidant enzymes and an important anti-inflammatory agent. In the early phase of the inflammatory response of macrophages, GSH content is decreased due to the down regulation of the catalytic subunit of glutamate cysteine ligase (GCLC). In the current study we investigated the underlying mechanism for this phenomenon. In human THP1-differentiated macrophages, GCLC mRNA had a half-life of 4 h under basal conditions, and it was significantly reduced to less than 2 h upon exposure to lipopolysaccharide (LPS), suggesting an increased decay of GCLC mRNA in the inflammatory response. The half-life of GCLC protein was >10 h under basal conditions, and upon LPS exposure the degradation rate of GCLC protein was significantly increased. The pan-caspase inhibitor Z-VAD-FMK but not the proteasome inhibitor MG132, prevented the down regulation of GCLC protein caused by LPS. Both caspase inhibitor Z-LEVD-FMK and siRNA of caspase-5 abrogated LPS-induced degradation of GCLC protein. In addition, supplement with γ-GC, the GCLC product, efficiently restored GSH content and suppressed the induction of NF-κB activity by LPS. In conclusion, these data suggest that GCLC down-regulation in the inflammatory response of macrophages is mediated through both increased mRNA decay and caspase-5-mediated GCLC protein degradation, and γ-GC is an efficient agent to restore GSH and regulate the inflammatory response.

BackgroundExposure to urban air pollution particles is strongly associated with higher risks of accelerated cognitive decline, cerebral atrophy, and dementia in multiple population studies. Among possible mechanisms is the decrease of neurotrophins, shown for BDNF in human exposures, which have critical roles in regulating adult neurogenesis and synaptic plasticity.MethodC57BL/6 mice were exposed to nano‐sized particulate matter (nPM, batch nPM2016a) from urban traffic air pollution for 8 weeks and the mRNAs of neurotrophins and receptors in mouse brain were measured by qPCR assay and neural stem cells measured by immunohistochemistry.ResultnPM exposure altered mRNA levels of neurotrophin genes (Ngf, Bdnf, Ntf‐3 and Ntf‐4/5) with brain region‐specificity. In cerebral cortex (CX), Ngf and Ntf‐3 were decreased (17% and 29% respectively), Ntf‐4/5 increased (78%), and Bdnf unchanged. In hippocampus (HP), Bdnf and Ntf‐4/5 were decreased (40% and 38% respectively) while Ngf and NTF‐3 unchanged. In olfactory bulb (OB), only Bdnf was decreased (10%). The mRNAs of neurotrophin receptors (Trka, Trkb, Trkc and p75Ntr) in CX, HP and OB were less responsive to nPM exposure, and only shown by OB with 22% decrease of p75Ntr and 14% decrease of Vgr (VGF nerve growth factor inducible). Exposure to nPM increased the quiescence of neural stem cells in hippocampal SGZ by IHC assay (Control 69.3% VS nPM 76.6%, P = 0.02), but did not alter the total number of neural stem cells. .ConclusionChronic air pollution exposure altered neurotrophin signaling with factor‐ and brain region‐specificity, and increased neural stem cell quiescence.