Liang-Jun Yan

Multiple organ pathology, metabolic abnormalities and impaired homeostasis of reactive oxygen species in Epas1-/- mice

Hypoxia-inducible factor (HIF) transcription factors respond to multiple environmental stressors, including hypoxia and hypoglycemia. We report that mice lacking the HIF family member HIF-2α (encoded by Epas1) have a syndrome of multiple-organ pathology, biochemical abnormalities and altered gene expression patterns. Histological and ultrastructural analyses showed retinopathy, hepatic steatosis, cardiac hypertrophy, skeletal myopathy, hypocellular bone marrow, azoospermia and mitochondrial abnormalities in these mice. Serum and urine metabolite studies showed hypoglycemia, lactic acidosis, altered Krebs cycle function and dysregulated fatty acid oxidation. Biochemical assays showed enhanced generation of reactive oxygen species (ROS), whereas molecular analyses indicated reduced expression of genes encoding the primary antioxidant enzymes (AOEs). Transfection analyses showed that HIF-2α could efficiently transactivate the promoters of the primary AOEs. Prenatal or postnatal treatment of Epas1−/− mice with a superoxide dismutase (SOD) mimetic reversed several aspects of the null phenotype. We propose a rheostat role for HIF-2α that allows for the maintenance of ROS as well as mitochondrial homeostasis.

Human αB-Crystallin Mutation Causes Oxido-Reductive Stress and Protein Aggregation Cardiomyopathy in Mice

The autosomal dominant mutation in the human alphaB-crystallin gene inducing a R120G amino acid exchange causes a multisystem, protein aggregation disease including cardiomyopathy. The pathogenesis of cardiomyopathy in this mutant (hR120GCryAB) is poorly understood. Here, we show that transgenic mice overexpressing cardiac-specific hR120GCryAB recapitulate the cardiomyopathy in humans and find that the mice are under reductive stress. The myopathic hearts show an increased recycling of oxidized glutathione (GSSG) to reduced glutathione (GSH), which is due to the augmented expression and enzymatic activities of glucose-6-phosphate dehydrogenase (G6PD), glutathione reductase, and glutathione peroxidase. The intercross of hR120GCryAB cardiomyopathic animals with mice with reduced G6PD levels rescues the progeny from cardiac hypertrophy and protein aggregation. These findings demonstrate that dysregulation of G6PD activity is necessary and sufficient for maladaptive reductive stress and suggest a novel therapeutic target for abrogating R120GCryAB cardiomyopathy and heart failure in humans.

Mouse heat shock transcription factor 1 deficiency alters cardiac redox homeostasis and increases mitochondrial oxidative damage

In this study, using heat shock factor 1 (Hsf1) knockout mice as a model, we tested the hypothesis that HSF1‐dependent regulation of heat shock proteins (Hsps) is required to maintain redox state and attenuate oxidative damage in the normal heart. Here we report that, in mice, HSF1 deficiency reduces cardiac expression of Hsp25, αB‐crystallin and Hsp70, but not Hsp60 and Hsp90. Consistent with the downregulation of Hsp25, for example, a significantly lower glutathione (GSH)/glutathione disulfate (GSSG) ratio was associated with the decreased activity, but not protein content, of glucose 6‐phosphate dehydrogenase. Con sequently, superoxide was generated at a higher rate, and several mitochondrial proteins, including adenine nucleotide translocase 1 (ANT1), were more oxidized by HSF1 deficiency in vivo. Oxidative damage to ANT1 protein, a structural component of the mitochondrial permeability transition pore (MPTP), decreases its catalytic activity and increases MPTP opening, respectively. Taken together, our results indicate for the first time that constitutive expression of HSP chaperones requires HSF1 activity, and that such HSF1‐dependent requirements are directly and functionally linked to maintain redox homeostasis and antioxidative defenses at normal (37°C) temperature.

UV-irradiation depletes antioxidants and causes oxidative damage in a model of human skin.

The degree to which antioxidant loss occurs in human skin after UV irradiation is unknown, as is the cascade of events that might occur. We have, therefore, evaluated a tissue culture model of human skin for its usefulness for studying oxidative injury by UV-irradiation. Human skin equivalents, a tissue culture model, were irradiated using a full solar UV spectrum (UVA and UVB, 280-400 nm) (0 to 16.8 J/cm2, 0-12 minimal erythemal dose, MED), then incubated from 1 to 24 h. Ubiquinol was the most UV-light sensitive antioxidant and was depleted by 2.1 J/cm2 (1.5 MED, p < .004); ubiquinone decreased with 4.2 J/cm2 (3 MED, p < .0007). A linear decrease in alpha-tocopherol occurred--approximately 1.7 pmol tocopherol/cm2 surface were destroyed per J/cm2 UV-light. Urate was depleted by irradiation with 8.4 J/cm2 (6 MED), while ascorbate was depleted by 16.8 J/cm2 (12 MED). Cellular protein carbonyls and lactic dehydrogenase (LDH) leakage into the medium were only increased at 1 h incubation following exposure to 16.8 J/cm2 (12 MED). At 24 h incubation, PGE2 was increased in the medium of cells exposed to UV-irradiation at 0.35 J/cm2 (0.25 MED) compared with sham-exposed cells (p < .04); higher UV exposures lead to significant increases in both PGE2 (p < .001) and LDH (p < .001) in the medium. In conclusion, human skin equivalents respond to suberythemal levels of UV-irradiation by increasing production of PGE2; higher levels of UV-irradiation (at least 1 MED) were needed to deplete cellular antioxidants and induce immediately detectable oxidative damage.

Heat shock factor 1 and heat shock proteins: Critical partners in protection against acute cell injury

Objective Life-threatening conditions cause severe changes in the organization and conformation of macromolecules, creating urgent requirements for protein repair to ensure survival. As molecular chaperones, heat shock proteins (HSP) that have specialized functions in protein folding are now well established to restore homeostasis in cells and organisms. Augmentation of HSP synthesis is tightly regulated by stress-inducible heat shock factors (HSF), which are part of a transcriptional signaling cascade with both positive (e.g., HSP) and negative (e.g., proinflammatory cytokines) properties. In this review, we discuss the biological roles and mechanisms of HSP-mediated protection in pathophysiologic conditions (ischemia, sepsis, and preeclampsia) and the regulation for stress-dependent HSP synthesis and speculate about future applications for harnessing HSF and HSP partners as cytoprotective agents. Data Sources Reactive oxygen species are major pathogenic factors in cell death pathways (e.g., necrosis, apoptosis), in part, because of proteotoxic effects. In intact organisms, forced overexpression of HSP per se affords effective counterbalance against ischemia challenges (e.g., heart and brain) and systemic conditions (e.g., sepsis). Besides stressful conditions, gene-targeting studies have uncovered new functions for heat shock transcription factors (e.g., maintenance of intrauterine pregnancy) in mammals. In parallel, pharmacologic studies using small molecules are paving the way for future prospects to exploit the beneficial properties of HSP, albeit an important but presently elusive goal. Conclusions Together, HSF and HSP partners are attractive targets in therapeutic strategies designed to stimulate endogenous protective mechanisms against deleterious consequences of oxidative stress. With further technological advances, it is anticipated that the spotlight on HSP, alone or in combination with other stress response pathways, could, ultimately, reduce injury and accelerate functional recovery of susceptible organs in living organisms including humans.

Alternative Mitochondrial Electron Transfer as a Novel Strategy for Neuroprotection

Neuroprotective strategies, including free radical scavengers, ion channel modulators, and anti-inflammatory agents, have been extensively explored in the last 2 decades for the treatment of neurological diseases. Unfortunately, none of the neuroprotectants has been proved effective in clinical trails. In the current study, we demonstrated that methylene blue (MB) functions as an alternative electron carrier, which accepts electrons from NADH and transfers them to cytochrome c and bypasses complex I/III blockage. A de novo synthesized MB derivative, with the redox center disabled by N-acetylation, had no effect on mitochondrial complex activities. MB increases cellular oxygen consumption rates and reduces anaerobic glycolysis in cultured neuronal cells. MB is protective against various insults in vitro at low nanomolar concentrations. Our data indicate that MB has a unique mechanism and is fundamentally different from traditional antioxidants. We examined the effects of MB in two animal models of neurological diseases. MB dramatically attenuates behavioral, neurochemical, and neuropathological impairment in a Parkinson disease model. Rotenone caused severe dopamine depletion in the striatum, which was almost completely rescued by MB. MB rescued the effects of rotenone on mitochondrial complex I-III inhibition and free radical overproduction. Rotenone induced a severe loss of nigral dopaminergic neurons, which was dramatically attenuated by MB. In addition, MB significantly reduced cerebral ischemia reperfusion damage in a transient focal cerebral ischemia model. The present study indicates that rerouting mitochondrial electron transfer by MB or similar molecules provides a novel strategy for neuroprotection against both chronic and acute neurological diseases involving mitochondrial dysfunction.

Pyruvate protects mitochondria from oxidative stress in human neuroblastoma SK-N-SH cells.

Oxidative stress is implicated in neurodegenerative diseases including stroke, Alzheimers disease and Parkinsons disease, and has been extensively studied as a potential target for therapeutic intervention. Pyruvate, a natural metabolic intermediate and energy substrate, exerts antioxidant effects in brain and other tissues susceptible to oxidative stress. We tested the protective effects of pyruvate on hydrogen peroxide (H(2)O(2)) toxicity in human neuroblastoma SK-N-SH cells and the mechanisms underlying its protection. Hydrogen peroxide insult resulted in 85% cell death, but co-treatment with pyruvate dose-dependently attenuated cell death. At concentrations of >or=1 mM, pyruvate totally blocked the cytotoxic effects of H(2)O(2). Pyruvate exerted its protective effects even when its administration was delayed up to 2 h after H(2)O(2) insult. As a scavenger of reactive oxygen species (ROS), pyruvate dose-dependently attenuated H(2)O(2)-induced ROS formation, assessed from 2,7-dichlorofluorescein diacetate fluorescence. Furthermore, pyruvate suppressed superoxide production by submitochondrial particles, and attenuated oxidative stress-induced collapse of the mitochondrial membrane potential. Collectively, these results suggest that pyruvate protects neuronal cells through its antioxidant actions on mitochondria.

Prevention of flight activity prolongs the life span of the housefly, Musca domestica, and attenuates the age-associated oxidative damamge to specific mitochondrial proteins

The purpose of this study was to explore the mechanisms by which oxidative stress affects the aging process. The hypothesis that the rate of accumulation of oxidative damage to specific mitochondrial proteins is linked to the life expectancy of animals was tested in the housefly. The rate of oxygen consumption and life expectancy of the flies were experimentally altered by confining the flies in small jars, where they were unable to fly. Prevention of flight activity decreased the rate of oxygen utilization of flies and almost tripled their life span as compared to those permitted to fly. Rate of mitochondrial H(2)O(2) generation at various ages was lower in the low activity flies than in the high activity flies. Oxidative damage to mitochondrial proteins, adenine nucelotide translocase, and aconitase, detected as carbonyl modifications, was attenuated; and the loss in their functional activity occurring with age was retarded in the long-lived low activity flies as compared to the short-lived high activity flies. The two proteins were previously identified to be the only mitochondrial proteins exhibiting age-related increases in carbonylation. Results support the hypothesis that accrual of oxidative damage to specific protein targets and the consequent loss of their function may constitute a mechanism by which oxidative stress controls the aging process.

Pathogenesis of Chronic Hyperglycemia: From Reductive Stress to Oxidative Stress

Chronic overnutrition creates chronic hyperglycemia that can gradually induce insulin resistance and insulin secretion impairment. These disorders, if not intervened, will eventually be followed by appearance of frank diabetes. The mechanisms of this chronic pathogenic process are complex but have been suggested to involve production of reactive oxygen species (ROS) and oxidative stress. In this review, I highlight evidence that reductive stress imposed by overflux of NADH through the mitochondrial electron transport chain is the source of oxidative stress, which is based on establishments that more NADH recycling by mitochondrial complex I leads to more electron leakage and thus more ROS production. The elevated levels of both NADH and ROS can inhibit and inactivate glyceraldehyde 3-phosphate dehydrogenase (GAPDH), respectively, resulting in blockage of the glycolytic pathway and accumulation of glycerol 3-phospate and its prior metabolites along the pathway. This accumulation then initiates all those alternative glucose metabolic pathways such as the polyol pathway and the advanced glycation pathways that otherwise are minor and insignificant under euglycemic conditions. Importantly, all these alternative pathways lead to ROS production, thus aggravating cellular oxidative stress. Therefore, reductive stress followed by oxidative stress comprises a major mechanism of hyperglycemia-induced metabolic syndrome.

Roles of AMP-activated Protein Kinase in Alzheimer’s Disease

AMP-activated protein kinase (AMPK), a master regulator of cellular energy homeostasis and a central player in glucose and lipid metabolism, is potentially implicated in the pathogenesis of Alzheimer’s disease (AD). AMPK activity decreases in AD brain, indicating decreased mitochondrial biogenesis and function. Emerging evidence demonstrates that AMPK activation is a potential target for improving perturbed brain energy metabolism that is involved in the pathogenesis of AD. The roles of AMPK in the pathogenesis of AD include β-amyloid protein (Aβ) generation and tau phosphorylation. In particular, AMPK may regulate Aβ generation through modulating neuronal cholesterol and sphingomyelin levels and through regulating APP distribution in the lipid rafts. AMPK is activated by phosphorylation of Thr-172 by LKB1 complex in response to increase in the AMP/ATP ratio and by calmodulin-dependent protein kinase kinase-beta in response to elevated Ca2+ levels, which contributes to regulating Aβ generation. AMPK is a physiological tau kinase and can increase the phosphorylation of tau at Ser-262. AMPK can also directly phosphorylate tau at Thr-231 and Ser-396/404. Furthermore, AMPK activation decreases mTOR signaling activity to facilitate autophagy and promotes lysosomal degradation of Aβ. However, AMPK activation has non-neuroprotective property and may lead to detrimental outcomes, including Aβ generation and tau phosphorylation. Therefore, it is still unclear whether AMPK could serve a potential therapeutic target for AD, and hence, further studies will be needed to clarify the role of AMPK in AD.