No abstract

Neuronal cells depend on mitochondrial oxidative phosphorylation for most of their energy needs and therefore are at a particular risk for oxidative stress. Mitochondria play an important role in energy production and oxidative stress-induced apoptosis. In the present study, we have demonstrated that external oxidative stress induces mitochondrial dysfunction leading to increased ROS generation and ultimately apoptotic cell death in neuronal cells. Furthermore, we have investigated the role of Coenzyme Q10 as a neuroprotective agent. Coenzyme Q10 is a component of the mitochondrial respiratory chain and a potent anti-oxidant. Our results indicate that total cellular ROS generation was inhibited by Coenzyme Q10. Further, pre-treatment with Coenzyme Q10 maintained mitochondrial membrane potential during oxidative stress and reduced the amount of mitochondrial ROS generation. Our study suggests that water-soluble Coenzyme Q10 acts by stabilizing the mitochondrial membrane when neuronal cells are subjected to oxidative stress. Therefore, Coenzyme Q10 has the potential to be used as a therapeutic intervention for neurodegenerative diseases.

Thioredoxin-interacting protein (TXNIP) is involved in oxidative stress and apoptosis in diabetic retinopathy. However, the role of TXNIP in the removal of damaged mitochondria (MT) via mitophagy, a process of macroautophagy, remains unexplored. Here we investigate the associated cellular and molecular mechanisms underlying mitophagy in retinal cells under diabetic conditions. For this, we maintained a rat Müller cell line (rMC1) under high-glucose (25 mM, HG) or low-glucose (5.5 mM, LG) condition for 5 days. Our data reveal that HG upregulates TXNIP in the cytosol as well as in the MT. Moreover, mitochondrial oxidative stress and membrane depolarization occur under prolonged hyperglycemia leading to fragmentation. These damaged MT are targeted to lysosome for mitophagic degradation, as is evident by co-localization of mitochondrial protein COXIV, a subunit of cytochrome c oxidase, with autophagosome marker LC3BII and the lysosomal membrane protein LAMP2A. In addition, under HG conditions, there is an accumulation of dynamin-related fission protein Drp1 and E3 ubiquitin ligase Parkin in damaged MT, suggesting their roles in mitochondrial fragmentation and ubiquitination, respectively, which is absent in LG conditions. Subsequently, ubiquitin receptors, optineurin and p62/sequestrome 1, bind to the damaged MT and target them to LC3BII autophagosomes. Conversely, TXNIP knockout via CRISPR/Cas9 and TXNIP gRNA prevents the HG-induced mitochondrial damage and mitophagy in rMC1. Last, TXNIP level is also significantly upregulated in the diabetic rat retina in vivo and induces radial glial fibrillary acidic protein expression, a marker for Müller glia activation, and the formation of LC3BII puncta, which are prevented by intravitreal injection of TXNIP siRNA. Therefore, TXNIP represents a potential target for preventing ocular complications of diabetes.

Thioredoxin-interacting protein (TXNIP) plays a critical role in oxidative stress, inflammation, apoptosis and the pathogenesis of diabetic retinopathy (DR). However, the role of TXNIP in high glucose-induced retinal pigment epithelium (RPE) dysfunction is still unknown. Here, we show that high glucose (HG; 25 mM,) significantly increases TXNIP expression at both the mRNA and protein levels when compared to low glucose (LG; 5.5 mM) in a human RPE cell line (ARPE-19) and primary human RPE (HRPE) cells. TXNIP upregulation is associated with mitochondrial membrane depolarization, fragmentation and mitophagic flux to lysosomes. We used confocal live-cell imaging of RPE cells expressing mt-Keima, a coral protein that emits green light in mitochondria (alkaline or neutral pH) and red light in the acidic lysosome, to measure mitophagic flux. We observed an elongated mitochondrial network of green mt-Keima under LG, which is fragmented in HG. Red mt-Keima accumulates in lysosomes as small punctate aggregations under LG in both ARPE-19 and HRPE cells, whereas they are significantly enlarged (two- to threefold) under HG. Lysosomal enlargement under HG is further illustrated by lysosomal membrane protein LAMP1-mCherry expression in both ARPE-19 and HRPE cells. Furthermore, HG causes lysosomal cathepsin L inactivation and pro-inflammatory caspase-1 activation in ARPE-19 cells. TXNIP knockdown by shRNA prevents mitochondrial fragmentation, mitophagic flux and lysosome enlargement under HG. In addition, antioxidant N-acetylcysteine (NAC) and Amlexanox (Amlx), an inhibitor of protein kinase TBK1 and of the mitophagic adaptors Optineurin (Optn) and Sequestosome 1 (p62/SQSTM1), prevent mitophagic flux and lysosome enlargement. These results suggest that TXNIP mediates several deleterious effects of high glucose on RPE, which may be implicated in the development of DR.

Coiled-coil-helix-coiled-coil-helix domaincontaining 10 (CHCHD10) and CHCHD2 (MNRR1) are homologous proteins with 58% sequence identity and belong to the twin CX 9 C family of proteins that mediate cellular stress responses. Despite the identification of several neurodegeneration-associated mutations in the CHCHD10 gene, few studies have assessed its physiological role. Here, we investigated CHCHD10's function as a regulator of oxidative phosphorylation in the mitochondria and the nucleus. We show that CHCHD10 co-purifies with cytochrome c oxidase (COX) and up-regulates COX activity by serving as a scaffolding protein required for MNRR1 phosphorylation, mediated by ARG (ABL proto-oncogene 2, non-receptor tyrosine kinase [ABL2]). The CHCHD10 gene was maximally transcribed in cultured cells at 8% oxygen, unlike MNRR1, which was maximally expressed at 4%, suggesting a fine-tuned oxygen-sensing system that adapts to the varying oxygen concentrations in the human body under physiological conditions. We show that nuclear CHCHD10 protein down-regulates the expression of genes harboring the oxygenresponsive element (ORE) in their promoters by interacting with and augmenting the activity of the largely uncharacterized transcriptional repressor CXXC finger protein 5 (CXXC5). We further show that two genetic CHCHD10 disease variants, G66V and P80L, in the mitochondria exhibit faulty interactions with MNRR1 and COX, reducing respiration and increasing reactive oxygen species (ROS), and in the nucleus abrogating transcriptional repression of ORE-containing genes. Our results reveal that CHCHD10 positively regulates mitochondrial respiration and contributes to transcriptional repression of ORE-containing genes in the nucleus, and that genetic CHCHD10 variants are impaired in these activities.CHCHD10 is a member of the twin CX 9 C family of proteins. This family, of which a number of members have displayed roles in disease (1), consists of proteins that contain two pairs of cysteine residues separated by 9 amino acids. These 4 cysteines form 2 disulfide bonds that stabilize the prototypical Coiled-coil Helix Coiled-coil Helix Domain (CHCHD) that traps CHCHD10 and other twin CX 9 C family members in the mitochondrial intermembrane space (IMS). Other members of this family include CHCHD4 (also called Mia40), necessary for import and proper folding of twin CX 9 C proteins in the IMS (2), and CHCHD3, which acts with CHCHD6 to stabilize mitochondrial cristae (3, 4). Another recently characterized member of this family is MNRR1 (Mitochondrial Nuclear Retrograde Regulator 1; also called CHCHD2), a protein that is upregulated under hypoxic stress, and maximally so at 4% oxygen. 2Besides residing in the mitochondria, MNRR1 is also present in the nucleus. Mitochondrial MNRR1 interacts with cytochrome c oxidase (COX) and is required for optimal enzyme function. A stable knockdown of MNRR1 resembles a mitochondrial disease phenotype with decreased oxygen consumption, decreased cellular growth and mitochondrial membrane potential,...

The purpose of this study is to test the effects of whole-body animal exposure to airborne particulate matter (PM) with an aerodynamic diameter of <10 μm (PM10) in the mouse cornea and in vitro. C57BL/6 mice were exposed to control or 500 µg/m3 PM10 for 2 weeks. In vivo, reduced glutathione (GSH) and malondialdehyde (MDA) were analyzed. RT-PCR and ELISA evaluated levels of nuclear factor erythroid 2-related factor 2 (Nrf2) signaling and inflammatory markers. SKQ1, a novel mitochondrial antioxidant, was applied topically and GSH, MDA and Nrf2 levels were tested. In vitro, cells were treated with PM10 ± SKQ1 and cell viability, MDA, mitochondrial ROS, ATP and Nrf2 protein were tested. In vivo, PM10 vs. control exposure significantly reduced GSH, corneal thickness and increased MDA levels. PM10-exposed corneas showed significantly higher mRNA levels for downstream targets, pro-inflammatory molecules and reduced Nrf2 protein. In PM10-exposed corneas, SKQ1 restored GSH and Nrf2 levels and lowered MDA. In vitro, PM10 reduced cell viability, Nrf2 protein, and ATP, and increased MDA, and mitochondrial ROS; while SKQ1 reversed these effects. Whole-body PM10 exposure triggers oxidative stress, disrupting the Nrf2 pathway. SKQ1 reverses these deleterious effects in vivo and in vitro, suggesting applicability to humans.

The central role of energy metabolism in cellular activities is becoming widely recognized. However, there are many gaps in our knowledge of the mechanisms by which mitochondria evaluate their status and call upon the nucleus to make adjustments. Recently, a protein family consisting of twin CX9C proteins has been shown to play a role in human pathophysiology. We focus here on two family members, the isoforms CHCHD2 (renamed MNRR1) and CHCHD10. The better studied isoform, MNRR1, has the unusual property of functioning in both the mitochondria and the nucleus and of having a different function in each. In the mitochondria, it functions by binding to cytochrome c oxidase (COX), which stimulates respiration. Its binding to COX is promoted by tyrosine-99 phosphorylation, carried out by ABL2 kinase (ARG). In the nucleus, MNRR1 binds to a novel promoter element in COX4I2 and itself, increasing transcription at 4% oxygen. We discuss mutations in both MNRR1 and CHCHD10 found in a number of chronic, mostly neurodegenerative, diseases. Finally, we propose a model of a graded response to hypoxic and oxidative stresses, mediated under different oxygen tensions by CHCHD10, MNRR1, and HIF1, which operate at intermediate and very low oxygen concentrations, respectively.

To determine the effects of airborne particulate matter (PM) <2.5 μm in vitro and on the normal and Pseudomonas aeruginosa (PA)-infected cornea. METHODS. An MTT viability assay tested the effects of PM 2.5 on mouse corneal epithelial cells (MCEC) and human corneal epithelial cells (HCET). MCEC were tested for reactive oxygen species using a 2 ,7-dichlorodihydrofluorescein assay; RT-PCR determined mRNA levels of inflammatory and oxidative stress markers in MCEC (HMGB1, toll-like receptor 2, IL-1β, CXCL2, GPX1, GPX2, GR1, superoxide dismutase 2, and heme oxygenase 1) and HCET (high mobility group box 1, CXCL2, and IL-1β). C57BL/6 mice also were infected and after 6 hours, the PM 2.5 was topically applied. Disease was graded by clinical score and evaluated by histology, plate count, myeloperoxidase assay, RT-PCR, ELISA, and Western blot. RESULTS. After PM 2.5 (25-200 μg/mL), 80% to 90% of MCEC and HCET were viable and PM exposure increased reactive oxygen species in MCEC and mRNA expression levels for inflammatory and oxidative stress markers in mouse and human cells. In vivo, the cornea of PA+PM 2.5 exposed mice exhibited earlier perforation over PA alone (confirmed histologically). In cornea, plate counts were increased after PA+PM 2.5 , whereas myeloperoxidase activity was significantly increased after PA+PM 2.5 over other groups. The mRNA levels for several proinflammatory and oxidative stress markers were increased in the cornea in the PA+PM 2.5 over other groups; protein levels were elevated for high mobility group box 1, but not toll-like receptor 4 or glutathione reductase 1. Uninfected corneas treated with PM 2.5 did not differ from normal. CONCLUSIONS. PM 2.5 triggers reactive oxygen species, upregulates mRNA levels of oxidative stress, inflammatory markers, and high mobility group box 1 protein, contributing to perforation in PA-infected corneas.