Induction Of Mitochondrial Oxidative Stress Research Articles

Detection of Induction of Mitochondrial Oxidative Stress by Nanoparticles in T Cells Using MitoSOX Red Dye.

The induction of oxidative stress by engineered nanomaterials has been associated with cytotoxic and inflammatory responses, damaging healthy cells and tissues. In contrast, when directed against cancer and autoinflammatory diseases, some nanomaterials inducing oxidative stress have also been reported as potential therapies for these disorders. Therefore, studying oxidative stress has become a popular tool not only in toxicology and immunotoxicology but in other areas of biology as well, including those related to developing novel therapies. Total oxidative stress may result from multiple cellular organelles. The protocol described herein allows for the analysis of oxidative stress in mitochondria.

Therapeutic Targeting of Ferroptosis Pathway in Combination with Mitochondrial Oxidative Stress Induction in Acute Myeloid Leukemia

Background: Targeting apoptosis pathways in cancer has been extensively studied including the recent breakthrough therapy using venetoclax in acute myeloid leukemia (AML). However, certain therapy-resistant subsets of AML still pose a clinical challenge, leading us to investigate therapeutic strategies to bypass apoptosis pathways. Recently, non-apoptotic regulated cell death modes have attracted attention. Ferroptosis is a form of regulated cell death characterized by reactive oxygen species (ROS) induction and iron-dependent lipid peroxidation. Leukemia cells exhibit increased oxidative stress, which is further enhanced by iron overload, but only limited studies have explored therapeutic potential of targeting ferroptosis in AML. With mitochondria being the center for ROS production and iron metabolism containing 20-50% of cellular iron, mitochondrial (mito-) regulation of ferroptosis remains unclear. Incorporating our recent knowledge on mito-protease ClpP, hyperactivation of which selectively kills cancer cells through mito-proteolysis and oxidative stress (Ishizawa et al. 2019 Cancer Cell), we here aim to study the therapeutic targeting of the ferroptosis pathway and potential combinatorial targeting of mitochondria in AML.Results: We first utilized the TCGA dataset to analyze gene expression of key components in one of the major anti-ferroptosis pathways cysteine-glutathione-GPX4 axis. AML patients with higher mRNA expression of SLC7A11 (cystine importer), GCLM (a subunit of rate-limiting glutathione synthetase) or GPX4 have significantly shorter overall survival (p < 0.05), suggesting potential prognostic impact of this pathway. Here we focused on GPX4, the most downstream molecule that reduces lipid hydroperoxide to block ferroptosis. Although GPX4 is essential for embryonic development in mice, acquired depletion with conditional knock out was reported to have no significant effect on the number and function of hematopoietic stem cells, suggesting the tolerability of GPX4-targeted therapy.We demonstrate that GPX4 inhibition with sub-micromolar concentrations of ML210, a covalent GPX4 inhibitor with proteome-wide specificity, or with shRNA-mediated knockdown induces prominent cell death in various AML cell lines. The anti-leukemia effects were associated with lipid peroxidation and were almost completely abrogated by a lipophilic antioxidant Liproxstatin-1 (Lip1). The effect of ML210 was also blocked by the iron chelator deferoxamine, indicating ferroptosis. We also demonstrate that GPX4 knockdown increases ROS prominently in mitochondria before cell death is induced, suggesting that mitochondria is involved in the anti-leukemia effects. Meanwhile, GPX4 is among the top 15 sensitization hits in a previously published genome-wide CRISPR screening of leukemia cells treated with potent ClpP agonists ONC201 and ONC212 (Jacques et al. 2020 Genetics), indicating the protective function of GPX4 against mito-oxidative stress. We then found that genetic or pharmacologic hyperactivation of ClpP upregulated GPX4 protein expression predominantly in mitochondria in AML cells, collectively suggesting that upregulation of mito-GPX4 is cell-protective against ClpP-mediated cell death. Indeed, combinatorial ClpP hyperactivation enhanced the anti-leukemia effects of GPX4 inhibition through increased induction of mito-ROS and lipid peroxidation. We further demonstrate that the synergistic cell killing is inhibited by Lip1, indicating that the dual targeting of ClpP and GPX4 induces lipid peroxidation-mediated cell death. Furthermore, the inhibition of lipid peroxidation simultaneously resulted in over 80% reduction of mito-ROS, unexpectedly. This suggests that most of the increased mito-ROS by this combination is associated with lipid peroxidation. Mechanisms of induction of mitochondrial lipid peroxides jointly engaged by ClpP and mito-GPX4, as well as its involvement in the induced oxidative cell death, have yet to be explored.Conclusion: Our data suggests the potential involvement of mitochondrial lipid peroxidation in the anti-leukemia effects of GPX4 inhibition, along with its therapeutic potential in conjunction with mito-oxidative stress induction though instability in mito-proteome. Further investigations are in progress to assess the molecular mechanisms and the in vivo efficacy of the combinatorial treatment. DisclosuresAndreeff: Oxford Biomedica UK: Research Funding; Aptose: Consultancy; Medicxi: Consultancy; Syndax: Consultancy; Glycomimetics: Consultancy; Senti-Bio: Consultancy; Novartis, Cancer UK; Leukemia & Lymphoma Society (LLS), German Research Council; NCI-RDCRN (Rare Disease Clin Network), CLL Foundation; Novartis: Membership on an entity's Board of Directors or advisory committees; Daiichi-Sankyo: Consultancy, Research Funding; ONO Pharmaceuticals: Research Funding; Amgen: Research Funding; Reata, Aptose, Eutropics, SentiBio; Chimerix, Oncolyze: Current holder of individual stocks in a privately-held company; Karyopharm: Research Funding; Breast Cancer Research Foundation: Research Funding; AstraZeneca: Research Funding.

HIV TAT-mediated microglial senescence: Role of SIRT3-dependent mitochondrial oxidative stress

The advent of combined antiretroviral treatment (cART) as a treatment for HIV-1 infection has not only resulted in a dramatic decrease in the peripheral viral load but has also led to increased life expectancy of the infected individuals. Paradoxically, increased lifespan is accompanied with higher prevalence of age-related comorbidities, including HIV-associated neurocognitive disorders (HAND). Present study was aimed at exploring the role of HIV TAT protein in mediating microglial mitochondrial oxidative stress, ultimately resulting in neuroinflammation and microglial senescence. Our findings demonstrated that exposure of mouse primary microglial cells (mPMs) to HIV TAT protein resulted in a senescence-like phenotype, that was characterized by elevated expression of both p16 and p21 proteins, increased numbers of senescence-associated-β-galactosidase positive cells, augmented cell-cycle arrest, increased release of proinflammatory cytokines and decreased telomerase activity. Additionally, exposure of mPMs to HIV TAT also resulted downregulation of SIRT3 with a concomitant increase in mitochondrial oxidative stress. Dual luciferase reporter assay identified miR-505 as a novel target of SIRT3, which was upregulated in mPMs exposed to HIV TAT. Furthermore, transient transfection of mPMs with either the SIRT3 plasmid or miRNA-505 inhibitor upregulated the expression of SIRT3 and mitochondrial antioxidant enzymes, with a concomitant decrease in microglial senescence. These in vitro findings were also validated in the prefrontal cortices and striatum of HIV transgenic rats as well as cART-treated HIV-infected individuals. In summary, this study underscores a yet undiscovered novel mechanism(s) underlying HIV TAT-mediated induction of senescence phenotype in microglia, involving the miR-505-SIRT3 axis-mediated induction of mitochondrial oxidative stress.

Oxidative stress contributes to hepatocyte growth factor-dependent pro-senescence activity of ovarian cancer cells

The cancer-promoting activity of senescent peritoneal mesothelial cells (HPMCs) has already been well evidenced both in vitro and in vivo. Here we sought to determine if ovarian cancer cells may activate senescence in HPMCs. The study showed that conditioned medium (CM) from ovarian cancer cells (OVCAR-3, SKOV-3, A2780) inhibited growth and promoted the development of senescence phenotype (increased SA-β-Gal, γ-H2A.X, 53BP1, and decreased Cx43) in HPMCs. An analysis of tumors isolated from the peritoneum of patients with ovarian cancer revealed an abundance of senescent HPMCs in proximity to cancerous tissue. The presence of senescent HPMCs was incidental when fragments of peritoneum free from cancer were evaluated. An analysis of the cells' secretome followed by intervention studies with exogenous proteins and neutralizing antibodies revealed hepatocyte growth factor (HGF) as the mediator of the pro-senescence impact of the cancer cells. The activity of cancerous CM and HGF was associated with an induction of mitochondrial oxidative stress. Signaling pathways involved in the senescence of HPMCs elicited by the cancer-derived CM and HGF included p38 MAPK, AKT and NF-κB. HPMCs that senesced prematurely in response to the cancer-derived CM promoted adhesion of ovarian cancer cells, however this effect was effectively prevented by the cell protection against oxidative stress. Collectively, our findings indicate that ovarian cancer cells can elicit HGF-dependent senescence in HPMCs, which may contribute to the formation of a metastatic niche for these cells within the peritoneal cavity.

Dietary iron loading negatively affects liver mitochondrial function.

Iron is an essential co-factor for several metabolic processes, including mitochondrial respiration, and mitochondria are the major sites of iron-utilization. Cellular iron homeostasis must be tightly regulated, as intracellular iron deficiency can lead to insufficient energy production, whereas iron overload triggers ROS (reactive oxygen species) formation via the Fenton reaction. So far little is known on how iron imbalances affect mitochondrial function in vivo and the impact of the genotype on that, we studied the effects of dietary iron loading on mitochondrial respiratory capacity in liver by comparing two genetically divergent mouse strains, namely C57BL/6N and FVB mice. Both mouse strains differed in their basal iron levels and their metabolic responses to iron loading as determined by expression of iron trafficking proteins (ferritin was increased in livers of animals receiving high iron diet) as well as tissue iron content (2-fold increase, FVB p = 0.0013; C57BL/6N p = 0.0022). Dietary iron exposure caused a significant impairment of mitochondrial oxidative phosphorylation, especially regarding OXPHOS capacity (FVB p = 0.0006; C57BL/6N p = 0.0087) and S-ETS capacity (FVB p = 0.0281; C57BL/6N p = 0.0159). These effects were more pronounced in C57BL/6N than in FVB mice and were paralleled by an iron mediated induction of oxidative stress in mitochondria. The increased susceptibility of C57BL6/N mice to iron loading may be due to reduced expression of anti-oxidant defense mechanisms and altered iron trafficking upon dietary challenge pointing to a role of genetic modifiers for cellular and mitochondrial iron trafficking. Finally, iron-mediated induction of mitochondrial oxidative stress and reduction of oxidative phosphorylation may underlie fatigue in subjects with iron loading diseases.

Drug-Induced Mitochondrial Toxicity.

The mitochondrial respiratory chain (MRC) and ATP synthase (complex V) play an essential role in cellular energy production by the process of oxidative phosphorylation. In addition to inborn errors of metabolism, as well as secondary causes from disease pathophysiology, an impairment of oxidative phosphorylation can result from drug toxicity. These 'off-target' pharmacological effects can occur from a direct inhibition of MRC enzyme activity, an induction of mitochondrial oxidative stress, an uncoupling of oxidative phosphorylation, an impairment of mitochondrial membrane structure or a disruption in the replication of mitochondrial DNA. The purpose of this review is to focus on the off-target mitochondrial toxicity associated with both commonly used pharmacotherapies and a topical 'weight loss' agent. The mechanisms of drug-induced mitochondrial impairment will be discussed together with putative therapeutic strategies to counteract the adverse effects of the pharmacotherapy.

Dicarbonyl Stress in the Absence of Hyperglycemia Increases Endothelial Inflammation and Atherogenesis Similar to That Observed in Diabetes

The deleterious effects of high glucose levels and enhanced metabolic flux on the vasculature are thought to be mediated by the generation of toxic metabolites, including reactive dicarbonyls like methylglyoxal (MG). In this article, we demonstrate that increasing plasma MG to levels observed in diabetic mice either using an exogenous source (1% in drinking water) or generated following inhibition, its primary clearance enzyme, glyoxalase-1 (with 50 mg/kg IP bromobenzyl-glutathione cyclopentyl diester every second day), was able to increase vascular adhesion and augment atherogenesis in euglycemic apolipoprotein E knockout mice to a similar magnitude as that observed in hyperglycemic mice with diabetes. The effects of MG appear partly mediated by activation of the receptor for advanced glycation end products (RAGE), as deletion of RAGE was able to reduce inflammation and atherogenesis associated with MG exposure. However, RAGE deletion did not completely prevent inflammation or vascular damage, possibly because the induction of mitochondrial oxidative stress by dicarbonyls also contributes to inflammation and atherogenesis. Such data would suggest that a synergistic combination of RAGE antagonism and antioxidants may offer the greatest utility for the prevention and management of diabetic vascular complications.

Mitochondrial Oxidative Stress-Induced Epigenetic Modif ications in Pancreatic Epithelial Cells

Emerging studies have linked prooxidative carbamate compound exposures with various human pathologies including pancreatic cancer. In these studies, our aim was to examine mitochondrial oxidative stress-mediated aberrant chromatin responses in human pancreatic ductal epithelial cells. Posttranslational histone modifications, promoter DNA methylation, and micro-RNA (miRNA) expression patterns were evaluated following induction of mitochondrial oxidative stress by N-succinimidyl N-methylcarbamate exposure. In treated cells, perturbation in mitochondrial machinery led to hypermethylation of p16 and smad4 gene promoters and downregulation of respective gene products. Posttranslational histone modifications that include hypoacetylation of acetylated histone (AcH) 3 and AcH4, hypermethylation of monomethylated histone 3 at lysine 9 and trimethylated histone 4 at lysine 20 ubiquitinated histone (uH) 2A/uH2B, and increased phosphorylation of H2AX and H3 were observed in the treated cells. Altered expression of miRNAs denoted possible location of corresponding genes at oxidatively damaged fragile sites. Collectively, our results provide a direct role of mitochondrial oxidative stress-mediated epigenetic imbalance to perturbed genomic integrity in oxygen radical-induced pancreatic injury. Further, identification and characterization of molecular switches that affect these epigenomic signatures and targets thereof will be imperative to understand the complex role of redox-regulatory network in pancreatic milieu.

Chemotherapeutic induction of mitochondrial oxidative stress activates GSK-3α/β and Bax, leading to permeability transition pore opening and tumor cell death

Survival of tumor cells is favored by mitochondrial changes that make death induction more difficult in a variety of stress conditions, such as exposure to chemotherapeutics. These changes are not fully characterized in tumor mitochondria, and include unbalance of the redox equilibrium, inhibition of permeability transition pore (PTP) opening through kinase signaling pathways and modulation of members of the Bcl-2 protein family. Here we show that a novel chemotherapeutic, the Gold(III)-dithiocarbamato complex AUL12, induces oxidative stress and tumor cell death both favoring PTP opening and activating the pro-apoptotic protein Bax of the Bcl-2 family. AUL12 inhibits the respiratory complex I and causes a rapid burst of mitochondrial superoxide levels, leading to activation of the mitochondrial fraction of GSK-3α/β and to the ensuing phosphorylation of the mitochondrial chaperone cyclophilin D, which in turn facilitates PTP opening. In addition, following AUL12 treatment, Bax interacts with active GSK-3α/β and translocates onto mitochondria, where it contributes to PTP induction and tumor cell death. These findings provide evidence that targeting the redox equilibrium maintained by mitochondria in tumor cells allows to hit crucial mechanisms that shield neoplasms from the toxicity of many anti-tumor strategies, and identify AUL12 as a promising chemotherapeutic compound.

Mitochondrial oxidative stress initiates visual loss in sympathetic ophthalmia

The visual loss that occurs with sympathetic ophthalmia (SO) in the absence of recognizable retinal damage and inflammatory cell infiltration is an enigma. Experimental autoimmune uveoretinitis (EAU) is an animal model used to study human endogenous uveitis. Both innate and adaptive immune responses have been well studied in the photoreceptor damage mechanism of EAU. In our studies, in the early phase of EAU, proinflammatory molecules such as tumor necrosis factor (TNF)-α and inducible nitric oxide synthase (iNOS) and the subsequent mitochondrial DNA damage, mitochondrial protein alteration, and mitochondrial dysfunction by oxidative stress were observed before retinal inflammatory cell infiltration. Our recent study shows the importance of Toll-like receptors (TLRs) in the production of proinflammatory molecules and the induction of mitochondrial oxidative stress. Thus, the innate immune responses occur first with the activation of TLRs; this activation upregulates proinflammatory molecules, leading to mitochondrial oxidative stress before retinal inflammatory cell infiltration and the subsequent adaptive immune responses. Like EAU, SO also results in photoreceptor mitochondrial oxidative damage without retinal inflammatory cell infiltration. Such damage was associated with TNF-α, TNF-α receptors, and iNOS expression in the photoreceptors, suggesting that this molecular mechanism without retinal inflammatory cell infiltration may initiate photoreceptor damage in SO.

Overexpression of Amyloid-β Protein Precursor Induces Mitochondrial Oxidative Stress and Activates the Intrinsic Apoptotic Cascade

Aberrant processing of amyloid-β protein precursor (AβPP) into amyloid-β (Aβ) fragments underlies the formation of senile plaques in Alzheimer's disease (AD). Moreover, Aβ fragments, particularly Aβ(42), exert direct toxic effects within neurons including the induction of mitochondrial oxidative stress (MOS). Interestingly, individuals with Down syndrome (DS) frequently develop early onset AD as a major co-morbid phenotype. One hypothesis for AD associated with DS involves the overexpression of wild type (WT) AβPP protein, due to its location on chromosome 21. However, the mechanism by which the overexpression of WT AβPP might trigger MOS and induce cell death is presently unclear. Here we show that transient overexpression of DsRed2-tagged AβPP (WT) in CHO cells induces caspase-3 activation and nuclear fragmentation indicative of apoptosis. AβPP localizes to the mitochondrial fraction of transfected CHO cells and induces glutathione-sensitive opening of the mitochondrial permeability transition pore (mPTP) and cytochrome c release. MOS and intrinsic apoptosis induced by AβPP are significantly inhibited by co-expression of Bcl-2 or treatment with either glutathione or a pan-caspase inhibitor. The mPTP inhibitor, cyclosporin A, also significantly attenuates AβPP-induced apoptosis. AβPP-induced apoptosis is unaffected by a β-secretase inhibitor and is independent of detectable Aβ(42); however, a γ-secretase inhibitor significantly protects against AβPP overexpression, suggesting a possible role of the AβPP intracellular domain in cell death. These data indicate that overexpression of WT AβPP is sufficient to induce MOS and intrinsic apoptosis, suggesting a novel pro-oxidant role for AβPP at mitochondria which may be relevant in AD and DS disease pathologies.

Oxidative photoreceptor cell damage in autoimmune uveitis

Uveitis comprises an extensive array of intraocular inflammatory diseases and often results in irreversible visual loss. Experimental autoimmune uveitis (EAU) is an animal model used to study human uveitis. Both innate and adaptive immune responses are known to mediate retinal damage in EAU. The innate immune response occurs first with activation of toll-like receptors which upregulate inflammatory cytokines, leading to oxidative stress; subsequently, the adaptive immune response results in inflammatory cytokine upregulation and mitochondrial oxidative stress. In early EAU, mitochondrial DNA is damaged before inflammatory cellular infiltration and alters mitochondrial protein levels and the functions of mitochondria in AU. Our recent study confirms the importance of TLR4 in the generation of inflammatory cytokines, initiation of oxidative DNA damage, and induction of mitochondrial oxidative stress. Like EAU, sympathetic ophthalmia also results in photoreceptor mitochondrial oxidative damage. Agents that prevent mitochondrial oxidative stress and photoreceptor apoptosis may help prevent retinal damage and preserve vision in uveitis.

2-Methoxyestradiol induces endoreduplication through the induction of mitochondrial oxidative stress and the activation of MAPK signaling pathways

2-Methoxyestradiol (2ME2) is a normal physiological metabolite of 17β-estradiol with anti-proliferative and anti-angiogenic activities. The purpose of this study is to elucidate the mechanism whereby 2ME2 induces endoreduplication of the well-differentiated nasopharyngeal carcinoma (NPC) cells. We report here that 2ME2 induces G2/M phase cell cycle arrest followed by endoreduplication of the well-differentiated HK-1 cells. The increase in chromosome number was confirmed by cytogenetic study. Analysis of stress signaling pathways revealed the phosphorylation activation of ERK, JNK and p38 MAPKs at various times after 2ME2 treatment. Pre-treatment of 2ME2-treated HK-1 cells with JNK inhibitor (SP600125), ERK inhibitor (PD98059) and p38 MAPK inhibitor (SB203580) resulted in the reduction of endoreduplicating cells. Furthermore, the increase in the phosphorylation of JNK was accompanied by an increase in the reactive oxygen species. In addition, endoreduplication was observed in cells after treatment with superoxide donor, 2,3-dimethoxy-1,4-naphoquinone (DMNQ). Confocal microscopic analysis also revealed the increase in mitochondrial superoxide anion in 2ME2-treated HK-1 cells. Pre-treatment of HK-1 cells with superoxide dismutase mimetic 2,2,6,6-tetramethylpiperidine-1-oxyl (TEMPO) or overexpressing the mitochondrial enzyme MnSOD resulted in the reduction of phosphorylation of JNK and the formation of endoreduplicating cells. Furthermore, the tubulin filaments in cytoplasm remain intact in 2ME2-treated HK-1 cells after pre-treatment of TEMPO. Our results suggest that 2ME2 induces endoreduplication through the induction of oxidative stress and the activation of MAPK signal pathways. The biological significance of drug-induced endoreduplication will also be discussed.

Aminoethyl nitrate – the novel super nitrate?

Long-term use of most organic nitrates is limited by development of tolerance, induction of oxidative stress and endothelial dysfunction. In this issue of the BJP, Schuhmacher et al. characterized a novel class of organic nitrates with amino moieties (aminoalkyl nitrates). Aminoethyl nitrate was identified as a novel organic mononitrate with high potency but devoid of induction of mitochondrial oxidative stress. Cross-tolerance to nitroglycerin or the endothelium-dependent agonist acetylcholine after in vivo treatment was not observed. Like all nitrates, aminoethyl nitrate induced vasorelaxation by activation of soluble guanylate cyclase. Thus, in contrast to the prevailing view, high potency in an organic nitrate is not necessarily accompanied by induction of oxidative stress or endothelial dysfunction. This work from Daiber's group is an important step forward in the understanding of nitrate bioactivation, tolerance phenomena and towards the development of better organic nitrates for clinical use.

Dyslipidemia-Induced Neuropathy in Mice

OBJECTIVENeuropathy is a frequent and severe complication of diabetes. Multiple metabolic defects in type 2 diabetic patients result in oxidative injury of dorsal root ganglia (DRG) neurons. Our previous work focused on hyperglycemia clearly demonstrates induction of mitochondrial oxidative stress and acute injury in DRG neurons; however, this mechanism is not the only factor that produces neuropathy in vivo. Dyslipidemia also correlates with the development of neuropathy, even in pre-diabetic patients. This study was designed to explore the contribution of dyslipidemia in neuropathy.RESEARCH DESIGN AND METHODSMice (n = 10) were fed a control (10% kcal %fat) or high-fat (45% kcal %fat) diet to explore the impact of plasma lipids on the development of neuropathy. We also examined oxidized lipid–mediated injury in cultured DRG neurons from adult rat using oxidized LDLs (oxLDLs).RESULTSMice on a high-fat diet have increased oxLDLs and systemic and nerve oxidative stress. They develop nerve conduction velocity (NCV) and sensory deficits prior to impaired glucose tolerance. In vitro, oxLDLs lead to severe DRG neuron oxidative stress via interaction with the receptor lectin-like oxLDL receptor (LOX)-1 and subsequent NAD(P)H oxidase activity. Oxidative stress resulting from oxLDLs and high glucose is additive.CONCLUSIONSMultiple metabolic defects in type 2 diabetes directly injure DRG neurons through different mechanisms that all result in oxidative stress. Dyslipidemia leads to high levels of oxLDLs that may injure DRG neurons via LOX-1 and contribute to the development of diabetic neuropathy.

Nanoparticle-delivered biosensor for reactive oxygen species in diabetes

The cell’s own antioxidant response element (ARE) can be used to evaluate the complications of diabetes mellitus. The hypothesis that a synthetic ARE could be used as a genetic switch, or biosensor, to turn on and off therapeutic genes is tested herein. Mitochondrial oxidative stress (MOS) has been hypothesized as one of the earliest insults in diabetes. Fluorescent probes used to monitor MOS revealed that the addition of glucose at physiological levels to cultures of endothelial cells was able to induce MOS above normal levels and in a dose-dependant manner. Additional data showed that increased glucose levels activated the ARE-GFP in a dose-dependant manner. These data support the hypothesis that the induction of MOS is more sensitive to hyperglycemia than the induction of the ARE. Delivery of an ARE-GFP construct with nanoparticles to the eye was successful using sub-retinal injection. This ARE-GFP/nanoparticle construct was functional and reported the activation of the ARE in diabetic rat retinal pigment epithelium (RPE). These data support the use of nanoparticle-delivered biosensors for monitoring the oxidative status of tissues in vivo.

Induction of mitochondrial oxidative stress in astrocytes by nitric oxide precedes disruption of energy metabolism

Inhibition of the mitochondrial electron transport chain (ETC) ultimately limits ATP production and depletes cellular ATP. However, the individual complexes of the ETC in brain mitochondria need to be inhibited by approximately 50% before causing significant depression of ATP synthesis. Moreover, the ETC is the key site for the production of intracellular reactive oxygen species (ROS) and inhibition of one or more of the complexes of the ETC may increase the rate of mitochondrial ROS generation. We asked whether partial inhibition of the ETC, to a degree insufficient to perturb oxidative phosphorylation, might nonetheless induce ROS production. Chronic increase in mitochondrial ROS might then cause oxidative damage to the ETC sufficient to produce prolonged changes in ETC function and so compound the defect. We show that the exposure of astrocytes in culture to low concentrations of nitric oxide (NO) induces an increased rate of O2*- generation that outlasts the presence of NO. No effect was seen on oxygen consumption, lactate or ATP content over the 4-6 h that the cells were exposed to NO. These data suggest that partial ETC inhibition by NO may initially cause oxidative stress rather than ATP depletion, and this may subsequently induce irreversible changes in ETC function providing the basis for a cycle of damage.

Constitutive activation of rac1 results in mitochondrial oxidative stress and induces premature endothelial cell senescence.

Oxidative stress has been implicated in cellular senescence and vascular aging. We determined the role and mechanism of the small GTPase rac1 in vascular endothelial cell senescence. Adenoviral-mediated expression of the constitutively active allele of rac1 (rac1V12) in human umbilical vein endothelial cells resulted in mitochondrial oxidative stress with induction of biochemical, molecular, and morphological features of senescence. Suppression of mitochondrial oxidative stress abrogated rac1-induced premature senescence. Rac1V12 expression also resulted in an increase in endothelial ceramide levels. Moreover, premature endothelial cell senescence induced by an exogenous cell-permeable ceramide analog was not suppressed by inhibiting endogenous rac1 signaling. Finally, in human umbilical vein endothelial cells that had undergone replicative senescence, rac1 was not activated, and expression of the dominant-negative rac1 allele (rac1N17) did not suppress mitochondrial oxidative stress. These findings paint a picture in which the constitutive activation of rac1, via the generation of ceramide, results in mitochondrial oxidative stress and premature endothelial cell senescence. However, they speak against a role for endogenous rac1 activation in the induction of mitochondrial oxidative stress associated with replicative senescence of endothelial cells.

Impairment of Glucose and Glutamate Transport and Induction of Mitochondrial Oxidative Stress and Dysfunction in Synaptosomes by Amyloid β‐Peptide: Role of the Lipid Peroxidation Product 4‐Hydroxynonenal

Deposits of amyloid beta-peptide (A beta), reduced glucose uptake into brain cells, oxidative damage to cellular proteins and lipids, and excitotoxic mechanisms have all been suggested to play roles in the neurodegenerative process in Alzheimer's disease. Synapse loss is closely correlated with cognitive impairments in Alzheimer's disease, suggesting that the synapse may be the site at which degenerative mechanisms are initiated and propagated. We report that A beta causes oxyradical-mediated impairment of glucose transport, glutamate transport, and mitochondrial function in rat neocortical synaptosomes. A beta induced membrane lipid peroxidation in synaptosomes that occurred within 1 h of exposure; significant decreases in glucose transport occurred within 1 h of exposure to A beta and decreased further with time. The lipid peroxidation product 4-hydroxynonenal conjugated to synaptosomal proteins and impaired glucose transport; several antioxidants prevented A beta-induced impairment of glucose transport, indicating that lipid peroxidation was causally linked to this adverse action of A beta. FeSO4 (an initiator of lipid peroxidation), A beta, and 4-hydroxynonenal each induced accumulation of mitochondrial reactive oxygen species, caused concentration-dependent decreases in 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide reduction, and reduced cellular ATP levels significantly. A beta also impaired glutamate transport, an effect blocked by antioxidants. These data suggest that A beta induces membrane lipid peroxidation, which results in impairment of the function of membrane glucose and glutamate transporters, altered mitochondrial function, and a deficit in ATP levels; 4-hydroxynonenal appears to be a mediator of these actions of A beta. These data suggest that oxidative stress occurring at synapses may contribute to the reduced glucose uptake and synaptic degeneration that occurs in Alzheimer's disease patients. They further suggest a sequence of events whereby oxidative stress promotes excitotoxic synaptic degeneration and neuronal cell death in a variety of different neurodegenerative disorders.