β-Amyloid peptides induce mitochondrial dysfunction and oxidative stress in astrocytes and death of neurons through activation of NADPH oxidase

β-Amyloid (βA) peptide is strongly implicated in the neurodegeneration underlying Alzheimer's disease, but the mechanisms of neurotoxicity remain controversial. This study establishes a central role for oxidative stress by the activation of NADPH oxidase in astrocytes as the cause of βA-induced neuronal death. βA causes a loss of mitochondrial potential in astrocytes but not in neurons. The mitochondrial response consists of Ca2+-dependent transient depolarizations superimposed on a slow collapse of potential. The slow response is both prevented by antioxidants and, remarkably, reversed by provision of glutamate and other mitochondrial substrates to complexes I and II. These findings suggest that the depolarization reflects oxidative damage to metabolic pathways upstream of mitochondrial respiration. Inhibition of NADPH oxidase by diphenylene iodonium or 4-hydroxy-3-methoxy-acetophenone blocks βA-induced reactive oxygen species generation, prevents the mitochondrial depolarization, prevents βA-induced glutathione depletion in both neurons and astrocytes, and protects neurons from cell death, placing the astrocyte NADPH oxidase as a primary target of βA-induced neurodegeneration

Changes in intracellular calcium and glutathione in astrocytes as the primary mechanism of amyloid neurotoxicity

Although the accumulation of the neurotoxic peptide {beta} amyloid ({beta}A) in the CNS is a hallmark of Alzheimer's disease, the mechanism of {beta}A neurotoxicity remains controversial. In cultures of mixed neurons and astrocytes, we found that both the full-length peptide {beta}A (1–42) and the neurotoxic fragment (25–35) caused sporadic cytoplasmic calcium [intracellular calcium ([Ca2+]c)] signals in astrocytes that continued for hours, whereas adjacent neurons were completely unaffected. Nevertheless, after 24 hr, although astrocyte cell death was marginally increased, ~50% of the neurons had died. The [Ca2+]c signal was entirely dependent on Ca2+ influx and was blocked by zinc and by clioquinol, a heavy-metal chelator that is neuroprotective in models of Alzheimer's disease. Neuronal death was associated with Ca2+-dependent glutathione depletion in both astrocytes and neurons. Thus, astrocytes appear to be the primary target of {beta}A, whereas the neurotoxicity reflects the neuronal dependence on astrocytes for antioxidant support

Expression and modulation of an NADPH oxidase in mammalian astrocytes

Amyloid β peptides generate oxidative stress in hippocampal astrocytes through a mechanism sensitive to inhibitors of the NADPH oxidase [diphenylene iodonium (DPI) and apocynin]. Seeking evidence for the expression and function of the enzyme in primary hippocampal astrocytes, we confirmed the expression of the subunits of the phagocyte NADPH oxidase by Western blot analysis and by immunofluorescence and coexpression with the astrocyte-specific marker glial fibrillary acidic protein both in cultures and in vivo. Functional assays using lucigenin luminescence, dihydroethidine, or dicarboxyfluorescein fluorescence to measure the production of reactive oxygen species (ROS) demonstrated DPI and apocynin-sensitive ROS generation in response to the phorbol ester PMA and to raised [Ca2+]c after application of ionomycin or P2u receptor activation. Stimulation by PMA but not Ca2+ was inhibited by the protein kinase C (PKC) inhibitors staurosporine and hispidin. Responses were absent in transgenic mice lacking gp91phox. Expression of gp91phox and p67phox was increased in reactive astrocytes, which showed increased rates of both resting and stimulated ROS generation. NADPH oxidase activity was modulated by intracellular pH, suppressed by intracellular alkalinization, and enhanced by acidification. The protonophore carbonyl cyanide p-trifluoromethoxyphenylhydrazone suppressed basal ROS generation but markedly increased PMA-stimulated ROS generation. This was independent of mitochondrial ROS production, because it was unaffected by mitochondrial depolarization with rotenone and oligomycin. Thus, the NADPH oxidase is expressed in astrocytes and is functional, activated by PKC and intracellular calcium, modulated by pHi, and upregulated by astrocyte activation. The astrocytic NADPH oxidase is likely to play important roles in CNS physiology and pathology

Effect of washing on the bioefficacy of insecticide-treated nets (ITNs) and long-lasting insecticidal nets (LLINs) against main malaria vector Anopheles stephensi by three bioassay methods

Background & objectives: The use of pyrethoid impregnated bednets is one of the main malaria vector control strategies worldwide. The objective of the present study was to evaluate the bioefficacy of bednets impregnated with various pyrethroids after repeated washings. Methods: The effectiveness of bednets impregnated with permethrin, deltamethrin, bifenthrin, etofenprox and long-lasting bednets like OlysetNet® and PermaNet® which were provided by WHOPES was evaluated. The tests were carried out according to the WHO-recommended methods. Malaria vector, Anopheles stephensi was exposed to impregnated bednets for 3 min and the mortality was measured after 24 h recovery period. Knockdown was measured as well.Results: Results of three methods of bioassay tests showed that between two LLINs, PermaNet® was more efficient than OlysetNet®. Results of ITNs exhibited that deltamethrin and permethrin were more effective than etofenprox and bifenthrin as impregnants.Interpretation & conclusion: Findings of this study will be useful for WHO, local authorities and people who wish to use different pyrethroid-impregnated bednets for malaria vector control

Flirting in little space: the ER/mitochondria Ca2+ liaison.

Mitochondria have long been known to accumulate Ca2+; the apparent inconsistency between the low affinity of mitochondrial Ca2+ uptake mechanisms, the low concentration of global Ca2+ signals observed in cytoplasm, and the efficiency in intact cells of mitochondrial Ca2+ uptake led to the formulation of the "hotspot hypothesis." This hypothesis proposes that mitochondria preferentially accumulate Ca2+ at microdomains of elevated Ca2+ concentration ([Ca2+]) that exist near endoplasmic reticulum (ER) Ca2+ release sites and other Ca2+ channels. Physiological Ca2+ signals may affect mitochondrial function--both by stimulating key metabolic enzymes and, under some conditions, by promoting apoptosis. Mitochondria in turn may affect both Ca2+ release from the ER and capacitative Ca2+ entry across the plasma membrane, thereby shaping the size and duration of the intracellular Ca2+ signal. Interactions between mitochondria and the ER are critically dependent on the spatial localization of mitochondria within the cell. The molecular mechanisms that define the organization of mitochondria with regard to the ER and other Ca2+ sources, and the extent to which mitochondrial function varies among different cell types, are open questions whose answers remain to be determined

Chloride intracellular channel 1 (CLIC1): Sensor and effector during oxidative stress

Oxidative stress, characterized by overproduction of reactive oxygen species (ROS), is a major feature of several pathological states. Indeed, many cancers and neurodegenerative diseases are accompanied by altered redox balance, which results from dysregulation of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase. In this review, we consider the role of the intracellular chloride channel 1 (CLIC1) in microglial cells during oxidative stress. Following microglial activation, CLIC1 translocates from the cytosol to the plasma membrane where it promotes a chloride conductance. The resultant anionic current balances the excess charge extruded by the active NADPH oxidase, supporting the generation of superoxide by the enzyme. In this scenario, CLIC1 could be considered to act as both a second messenger and an executor

Chloride intracellular channel 1 (CLIC1): Sensor and effector during oxidative stress

Oxidative stress, characterized by overproduction of reactive oxygen species (ROS), is a major feature of several pathological states. Indeed, many cancers and neurodegenerative diseases are accompanied by altered redox balance, which results from dysregulation of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase. In this review, we consider the role of the intracellular chloride channel 1 (CLIC1) in microglial cells during oxidative stress. Following microglial activation, CLIC1 translocates from the cytosol to the plasma membrane where it promotes a chloride conductance. The resultant anionic current balances the excess charge extruded by the active NADPH oxidase, supporting the generation of superoxide by the enzyme. In this scenario, CLIC1 could be considered to act as both a second messenger and an executor