Mitochondrial dysfunction induced by knockdown of mortalin is rescued by Parkin

Mutations in the parkin gene are the most common cause of autosomal recessive Parkinson’s disease (PD). As an E3-ubiquitin ligase, Parkin is associated with mitochondrial dynamics and mitophagy. Mortalin, a molecular chaperone, is located primarily in mitochondria, where it functions to maintain mitochondrial homeostasis and antagonize oxidative stress injury. A reduced expression level of mortalin has been observed in the affected brain regions of PD patients. Mortalin also interacts with a variety of PD-related proteins and plays an indispensible role in helping native protein refolding and importing proteins into the mitochondrial matrix. Thus, the main aims of the present study were to investigate mitochondrial dysfunction induced by knockdown of mortalin and to test whether Parkin overexpression could rescue this effect. We found that lentivirus-mediated knockdown of mortalin in HeLa cells resulted in a collapse of mitochondrial membrane potential, an abnormal accumulation of reactive oxygen species and apparent alterations in mitochondrial morphology under H₂O₂-induced stress conditions. Remarkably, Parkin overexpression rescued these mitochondrial abnormalities. In HeLa cells expressing Parkin, co-immunoprecipitation of endogenous mortalin and wild-type Parkin was detected when they were treated with carbonyl cyanide 3-chlorophenylhydrazone (CCCP). In conclusion, we indicate that the relatively decreased mortalin expression level and its impaired interaction with Parkin could affect its roles in mitochondrial function.     

Lipid accumulation and lysosomal pathways contribute to dysfunction and apoptosis of human endothelial cells caused by 7-oxysterols

Endothelial dysfunction and cell death play an important role in pathogenesis of atherosclerosis. 7-Oxysterols, the major cytotoxic component found in oxidized low-density lipoprotein, are toxic to endothelial cells. However, the pathways and molecular mechanism involved in the process remain incompletely understood. In this study, we first investigate whether 7β-hydroxycholesterol (7βOH) or 7-ketocholesterol (7keto) induces apoptosis of human endothelial cell line (HUVEC-CS). We then examine possible involved pathways by focusing on cellular lipid, lysosomal pathways, cellular oxidative stress and mitochondrial pathways. Our results for the first time showed that 7-oxysterols induced apoptotic cell death of HUVEC-CS after 24 h, which was preceded by early lipid accumulation (6 h) and lysosomal membrane permeabilization (6−12 h). Afterward, levels of reactive oxygen species, mitochondrial membrane permeabilization, and lysosomal cathepsin were increased assayed by immuno-cytochemistry and blotting. Notably, the exposure to 7βOH or 7keto induced expressions and secretion of isoforms of von Willebrand factor (VWF). We conclude that apoptosis of HUVEC-CS induced by 7βOH or 7keto mediates by early lysosomal lipid accumulation and oxidative lysosomal pathways, which results in induction and release of VWF. The results suggest the cell death induced by 7-oxysterols may contribute to endothelial dysfunction and atherothrombosis.     

Molecular basis of maillard amide-advanced glycation end product (AGE) formation in vivo

The Maillard reaction in vivo entails alteration of proteins or free amino acids by non-enzymatic glycation or glycoxidation. The resulting modifications are called advanced glycation end products (AGEs) and play a prominent role in various pathologies, including normoglycemic uremia. Recently, we established a new class of lysine amide modifications in vitro. Now, human plasma levels of the novel amide-AGEs N(6)-acetyl lysine, N(6)-formyl lysine, N(6)-lactoyl lysine, and N(6)-glycerinyl lysine were determined by means of LC-MS/MS. They were significantly higher in uremic patients undergoing hemodialysis than in healthy subjects. Model reactions with N(1)-t-butoxycarbonyl-lysine under physiological conditions confirmed 1-deoxy-d-erythro-hexo-2,3-diulose as an immediate precursor. Because formation of N(6)-formyl lysine from glucose responded considerably to the presence of oxygen, glucosone was identified as another precursor. Comparison of the in vivo results with the model experiments enabled us to elucidate possible formation pathways linked to Maillard chemistry. The results strongly suggest a major participation of non-enzymatic Maillard mechanisms on amide-AGE formation pathways in vivo, which, in the case of N(6)-acetyl lysine, parallels enzymatic processes.     

SOD1 (copper/zinc superoxide dismutase) deficiency drives amyloid β protein oligomerization and memory loss in mouse model of Alzheimer disease

Oxidative stress is closely linked to the pathogenesis of neurodegeneration. Soluble amyloid β (Aβ) oligomers cause cognitive impairment and synaptic dysfunction in Alzheimer disease (AD). However, the relationship between oligomers, oxidative stress, and their localization during disease progression is uncertain. Our previous study demonstrated that mice deficient in cytoplasmic copper/zinc superoxide dismutase (CuZn-SOD, SOD1) have features of drusen formation, a hallmark of age-related macular degeneration (Imamura, Y., Noda, S., Hashizume, K., Shinoda, K., Yamaguchi, M., Uchiyama, S., Shimizu, T., Mizushima, Y., Shirasawa, T., and Tsubota, K. (2006) Proc. Natl. Acad. Sci. U.S.A. 103, 11282-11287). Amyloid assembly has been implicated as a common mechanism of plaque and drusen formation. Here, we show that Sod1 deficiency in an amyloid precursor protein-overexpressing mouse model (AD mouse, Tg2576) accelerated Aβ oligomerization and memory impairment as compared with control AD mouse and that these phenomena were basically mediated by oxidative damage. The increased plaque and neuronal inflammation were accompanied by the generation of N(ε)-carboxymethyl lysine in advanced glycation end products, a rapid marker of oxidative damage, induced by Sod1 gene-dependent reduction. The Sod1 deletion also caused Tau phosphorylation and the lower levels of synaptophysin. Furthermore, the levels of SOD1 were significantly decreased in human AD patients rather than non-AD age-matched individuals, but mitochondrial SOD (Mn-SOD, SOD2) and extracellular SOD (CuZn-SOD, SOD3) were not. These findings suggest that cytoplasmic superoxide radical plays a critical role in the pathogenesis of AD. Activation of Sod1 may be a therapeutic strategy for the inhibition of AD progression.     

Poly(ADP-ribose) polymerase 1 (PARP-1) binds to 8-oxoguanine-DNA glycosylase (OGG1)

Human 8-oxoguanine-DNA glycosylase (OGG1) plays a major role in the base excision repair pathway by removing 8-oxoguanine base lesions generated by reactive oxygen species. Here we report a novel interaction between OGG1 and Poly(ADP-ribose) polymerase 1 (PARP-1), a DNA-damage sensor protein involved in DNA repair and many other cellular processes. We found that OGG1 binds directly to PARP-1 through the N-terminal region of OGG1, and this interaction is enhanced by oxidative stress. Furthermore, OGG1 binds to PARP-1 through its BRCA1 C-terminal (BRCT) domain. OGG1 stimulated the poly(ADP-ribosyl)ation activity of PARP-1, whereas decreased poly(ADP-ribose) levels were observed in OGG1(-/-) cells compared with wild-type cells in response to DNA damage. Importantly, activated PARP-1 inhibits OGG1. Although the OGG1 polymorphic variant proteins R229Q and S326C bind to PARP-1, these proteins were defective in activating PARP-1. Furthermore, OGG1(-/-) cells were more sensitive to PARP inhibitors alone or in combination with a DNA-damaging agent. These findings indicate that OGG1 binding to PARP-1 plays a functional role in the repair of oxidative DNA damage.     

Androgens exacerbate motor asymmetry in male rats with unilateral 6-hydroxydopamine lesion

Parkinson's disease (PD) is a progressive neurodegenerative disorder characterized by dopamine neuron loss in the nigrostriatal pathway that shows greater incidence in men than women. The mechanisms underlying this gender bias remain elusive, although one possibility is that androgens may increase dopamine neuronal vulnerability to oxidative stress. Motor impairment can be modeled in rats receiving a unilateral injection of 6-hydroxydopamine (6-OHDA), a neurotoxin producing nigrostriatal degeneration. To investigate the role of androgens in PD, we compared young (2 months) and aged (24 months) male rats receiving gonadectomy (GDX) and their corresponding intact controls. One month after GDX, rats were unilaterally injected with 6-OHDA, and their motor impairment and asymmetry were assessed 2 weeks later using the cylinder test and the amphetamine-induced rotation test. Plasma samples were also collected to assess the concentration of testosterone and advanced oxidation protein products, a product of oxidative stress. GDX decreased lesion-induced asymmetry along with oxidative stress and increased amphetamine-induced rotations. These results show that GDX improves motor behaviors by decreasing motor asymmetry in 6-OHDA-treated rats, an effect that may be ascribed to increased release of striatal dopamine and decreased oxidative stress. Collectively, the data support the hypothesis that androgens may underlie the gender bias observed in PD.     

Effects of age, gender and time on receptor expression and anti-Aspergillus functions of human phagocytes

Phagocytes play a central role in immune defense. Their dysfunction predisposes to infections. This study determined the expression level of nine receptors involved in Aspergillus immune response as well as the values of phagocytosis and production of radical oxygen species after Aspergillus stimulation, in a healthy adult population.The expression values of the CD11b, CD11c, CD14, CD18, CD35, CD181, CD182, CD282 and CD284 receptors on peripheral human monocytes and granulocytes was established. A heterogenous expression of the CD282 on granulocytes was observed as CD181, CD182 and CD284 on monocytes. Similarly, we observed considerable variation in the expression of these receptors over time. Only CD282 on granulocytes varied with sex. No variation with age was observed. Adherence of Aspergillus conidia to phagocytes was dependent of individual, sex, age and time. A better characterization of these innate immunity parameters is necessary to develop in the future an immunologic surveillance strategy for transplant recipients.