Nitrosative stress and redox-cycling agents synergize to cause mitochondrial dysfunction and cell death in endothelial cells

Nitric oxide production by the endothelium is required for normal vascular homeostasis; however, in conditions of oxidative stress, interactions of nitric oxide with reactive oxygen species (ROS) are thought to underlie endothelial dysfunction. Beyond canonical nitric oxide signaling pathways, nitric oxide production results in the post-translational modification of protein thiols, termed S-nitrosation. The potential interplay between S-nitrosation and ROS remains poorly understood and is the focus of the current study. The effects of the S-nitrosating agent S-nitrosocysteine (CysNO) in combination with redox-cycling agents was examined in bovine aortic endothelial cells (BAEC). CysNO significantly impairs mitochondrial function and depletes the NADH/NAD+ pool; however, these changes do not result in cell death. When faced with the additional stressor of a redox-cycling agent used to generate ROS, further loss of NAD+ occurs, and cellular ATP pools are depleted. Cellular S-nitrosothiols also accumulate, and cell death is triggered. These data demonstrate that CysNO sensitizes endothelial cells to redox-cycling agent-dependent mitochondrial dysfunction and cell death and identify attenuated degradation of S-nitrosothiols as one potential mechanism for the enhanced cytotoxicity

Reply to Gurgul-Convey and Lenzen: Cytokines, Nitric Oxide, and β-Cells

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Effect of Nitric Oxide on Naphthoquinone Toxicity in Endothelial Cells: Role of Bioenergetic Dysfunction and Poly(ADP-ribose) Polymerase Activation

When produced at physiological levels, reactive oxygen species (ROS) can act as signaling molecules to regulate normal vascular function. Produced under pathological conditions, ROS can contribute to the oxidative damage of cellular components (e.g., DNA and proteins) and trigger cell death. Moreover, the reaction of superoxide with nitric oxide (NO) produces the strong oxidant peroxynitrite and decreases NO bioavailability, both of which may contribute to activation of cell death pathways. The effects of ROS generated from the 1,4-naphthoquinones alone and in combination with NO on the activation status of poly­(ADP-ribose) polymerase (PARP) and cell viability were examined. Treatment with redox cycling quinones activates PARP, and this stimulatory effect is attenuated in the presence of NO. Mitochondria play a central role in cell death signaling pathways and are a target of oxidants. We show that simultaneous exposure of endothelial cells to NO and ROS results in mitochondrial dysfunction, ATP and NAD⁺ depletion, and cell death. Alone, NO and ROS have only minor effects on cellular bioenergetics. Further, PARP inhibition does not attenuate reduced cell viability or mitochondrial dysfunction. These results show that concomitant exposure to NO and ROS impairs energy metabolism and triggers PARP-independent cell death. While superoxide-mediated PARP activation is attenuated in the presence of NO, PARP inhibition does not modify the loss of mitochondrial function or adenine and pyridine nucleotide pools and subsequent bioenergetic dysfunction. These findings suggest that the mechanisms by which ROS and NO induce endothelial cell death are closely linked to the maintenance of mitochondrial function and not overactivation of PARP

Decreased Sirtuin Deacetylase Activity in LRRK2 G2019S iPSC-Derived Dopaminergic Neurons

Summary: Mitochondrial changes have long been implicated in the pathogenesis of Parkinson's disease (PD). The glycine to serine mutation (G2019S) in leucine-rich repeat kinase 2 (LRRK2) is the most common genetic cause for PD and has been shown to impair mitochondrial function and morphology in multiple model systems. We analyzed mitochondrial function in LRRK2 G2019S induced pluripotent stem cell (iPSC)-derived neurons to determine whether the G2019S mutation elicits similar mitochondrial deficits among central and peripheral nervous system neuron subtypes. LRRK2 G2019S iPSC-derived dopaminergic neuron cultures displayed unique abnormalities in mitochondrial distribution and trafficking, which corresponded to reduced sirtuin deacetylase activity and nicotinamide adenine dinucleotide levels despite increased sirtuin levels. These data indicate that mitochondrial deficits in the context of LRRK2 G2019S are not a global phenomenon and point to distinct sirtuin and bioenergetic deficiencies intrinsic to dopaminergic neurons, which may underlie dopaminergic neuron loss in PD. : In this article, Ebert and colleagues show that iPSC-derived dopaminergic neurons expressing the LRRK2 G2019S mutation exhibit mitochondrial abnormalities, reduced sirtuin activity, and low endogenous NAD+ levels compared with other neuronal subtypes generated from the same patient samples. Therefore, the distinct sirtuin and bioenergetic deficiencies intrinsic to dopaminergic neurons may underlie dopaminergic neuron loss in Parkinson's disease. Keywords: Parkinson's disease, induced pluripotent stem cells, NAD+, mitochondri