No abstract

The pathogenesis of Alzheimer's disease (AD), a slowly-developing age-related neurodegenerative disorder, is a result of the action of multiple factors including deregulation of Ca 2+ homeostasis, mitochondrial dysfunction, and dysproteostasis. Interaction of these factors in astrocytes, principal homeostatic cells in the central nervous system, is still poorly understood. Here we report that in immortalized hippocampal astrocytes from 3xTg-AD mice (3Tg-iAstro cells) bioenergetics is impaired, including reduced glycolysis and mitochondrial oxygen consumption, and increased production of reactive oxygen species. Shotgun proteomics analysis of mitochondria-ER-enriched fraction showed no alterations in the expression of mitochondrial and OxPhos proteins, while those related to the ER functions and protein synthesis were deregulated. Using ER-and mitochondria-targeted aequorin-based Ca 2+ probe we show that, in 3Tg-iAstro cells, ER was overloaded with Ca 2+ while Ca 2+ uptake by mitochondria upon ATP stimulation was reduced. This was accompanied by the increase in short distance (≈8-10 nm) contact area between mitochondria and ER, upregulation of ER-stress/unfolded protein response genes Atf4, Atf6 and Herp, and reduction of global protein synthesis rate. We suggest that familial AD mutations in 3Tg-iAstro cells induce mitochondria-ER interaction changes that deregulate astrocytic bioenergetics, Ca 2+ homeostasis and proteostasis. These factors may interact, creating a pathogenic loop compromising homeostatic and defensive functions of astroglial cells predisposing neurons to dysfunction.

As in other organs, oxidative stress-induced injury and cell death may result from free oxygen radical-dependent mechanisms and alterations in signal transduction pathways leading to apoptosis. Among the new suggested therapies for injuries caused by oxidative stress, the use of levosimendan has been reported to be quite promising. In the present study, we aimed to examine the protective effects of levosimendan against liver oxidative stress in anesthetized rats and to analyze the involvement of mitochondrial adenosine triphosphate-dependent potassium (mitoK ATP ) channels and nitric oxide (NO). In 50 anesthetized rats, liver ischemia/reperfusion (I/R) was performed via nontraumatic portal occlusion. In some animals, levosimendan was infused into the portal vein at the onset of reperfusion, whereas other rats received the vehicle only. Moreover, in some rats, levosimendan was given after the intraportal administration of L-Nx-nitro-arginine methyl ester (L-NAME) or 5-hydroxydecanoate (5HD). The portal vein blood flow was measured, and blood samples were taken for the determination of transaminases, thiobarbituric acid reactive substances (TBARS), and reduced glutathione (GSH); liver biopsy samples were used for B cell lymphoma 2-associated X protein, caspase-9, Akt, and endothelial nitric oxide synthase (eNOS) activation through western blotting. Also, caspase-3 activity was measured. In rats, I/R caused an increase in apoptotic markers, transaminases, and TBARS and a decrease in GSH and Akt activation. Levosimendan administration was able to counteract oxidative damage and apoptosis in a dose-dependent way and to increase GSH, Akt, and eNOS activation. All effects of levosimendan were abolished by pretreatment with L-NAME and 5HD. In conclusion, the results of the present study show that levosimendan can exert protection against ischemic liver damage through mechanisms related to NO production and mitoK ATP channel function. These data provide interesting perspectives into the use of levosimendan in hepatic surgery and transplantation. Liver Transpl 20:361-375, 2014. V C 2013 AASLD.Received August 1, 2013; accepted November 9, 2013.Oxidative stress has been implicated as a mechanism of liver injury due to ischemia/reperfusion (I/R) and can occur in a variety of clinical contexts, including hepatectomy, transplantation, hypovolemic shock, cardiogenic shock, and low-output syndrome.1-5 The precise mechanisms of I/R-induced liver injury have Abbreviations: 5HD, 5-hydroxydecanoate; ALT, alanine aminotransferase; AST, aspartate aminotransferase; Bax, B cell lymphoma 2-associated X protein; eNOS, endothelial nitric oxide synthase; GSH, reduced glutathione; H&E, hematoxylin and eosin; I/R, ischemia/reperfusion; L-NAME, L-Nx-nitro-arginine methyl ester; MDA, malondialdehyde; mitoK ATP , mitochondrial adenosine triphosphate-dependent potassium; NO, nitric oxide; p-Akt, phosphorylated Akt; p-Bax, phosphorylated B cell lymphoma 2-associated X protein; p-caspase-9, phosphorylated caspase-9; p-eNOS, phosphorylated endotheli...

Background/Aims: the anti-vascular endothelial growth factors (VEGF), Aflibercept and Ranibizumab, are used for the treatment of macular degeneration. Here we examined the involvement of nitric oxide (NO), mitochondria function and of apoptosis/autophagy in their antioxidant effects in human retinal pigment epithelium cells (RPE). Methods: RPE were exposed to Ranibizumab/Aflibercept in the absence or presence of NO synthase (NOS) inhibitor and of autophagy activator/blocker, rapamicyn/3-methyladenine. Specific kits were used for cell viability, NO and reactive oxygen species detection and mitochondrial membrane potential measurement, whereas Western Blot was performed for apoptosis/ autophagy markers and other kinases detection. Results: In RPE cultured in physiological conditions, Aflibercept/Ranibizumab increased NO release in a dose and time-dependent way. Opposite results were obtained in RPE pretreated with hydrogen peroxide. Moreover, both the anti-VEGF agents were able to prevent the fall of cell viability and of mitochondrial membrane potential. Those effects were reduced by the NOS inhibitor and 3-methyladenine and were potentiated by rapamycin. Finally, Aflibercept and Ranibizumab counteracted the changes of apoptosis/autophagy markers, NOS, Phosphatidylinositol-3-Kinase/Protein Kinase B and Extracellular signal–regulated kinases 1/2 caused by peroxidation. Conclusion: Aflibercept and Ranibizumab protect RPE against peroxidation through the modulation of NO release, apoptosis and autophagy.