Laura Canevari

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 Alzheimers 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.

β-Amyloid inhibits integrated mitochondrial respiration and key enzyme activities

Disrupted energy metabolism, in particular reduced activity of cytochrome oxidase (EC 1.9.3.1), α‐ketoglutarate dehydrogenase (EC 1.2.4.2) and pyruvate dehydrogenase (EC 1.2.4.1) have been reported in post‐mortem Alzheimers disease brain. β‐Amyloid is strongly implicated in Alzheimers pathology and can be formed intracellularly in neurones. We have investigated the possibility that β‐amyloid itself disrupts mitochondrial function. Isolated rat brain mitochondria have been incubated with the β‐amyloid alone or together with nitric oxide, which is known to be elevated in Alzheimers brain. Mitochondrial respiration, electron transport chain complex activities, α‐ketoglutarate dehydrogenase activity and pyruvate dehydrogenase activity have been measured. β‐Amyloid caused a significant reduction in state 3 and state 4 mitochondrial respiration that was further diminished by the addition of nitric oxide. Cytochrome oxidase, α‐ketoglutarate dehydrogenase and pyruvate dehydrogenase activities were inhibited by β‐amyloid. The Km of cytochrome oxidase for reduced cytochrome c was raised by β‐amyloid. We conclude that β‐amyloid can directly disrupt mitochondrial function, inhibits key enzymes and may contribute to the deficiency of energy metabolism seen in Alzheimers disease.

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.

β-Amyloid fragment 25-35 causes mitochondrial dysfunction in primary cortical neurons

β-Amyloid deposition and compromised energy metabolism both occur in vulnerable brain regions in Alzheimers disease. It is not known whether β-amyloid is the cause of impairment of energy metabolism, nor whether impaired energy metabolism is specific to neurons. Our results, using primary neuronal cultures, show that 24-h incubation with Aβ25–35 caused a generalized decrease in the specific activity of mitochondrial enzymes per milligram of cellular protein, induced mitochondrial swelling, and decreased total mitochondrial number. Incubation with Aβ25–35 decreased ATP concentration to 58% of control in neurons and 71% of control in astrocytes. Levels of reduced glutathione were also lowered by Aβ25–35 in both neurons (from 5.1 to 2.9 nmol/mg protein) and astrocytes (from 25.2 to 14.9 nmol/mg protein). We conclude that 24-h treatment with extracellular Aβ25–35 causes mitochondrial dysfunction in both astrocytes and neurons, the latter being more seriously affected. In astrocytes mitochondrial impairment was confined to complex I inhibition, whereas in neurons a generalized loss of mitochondria was seen.

Toxicity of amyloid beta peptide: tales of calcium, mitochondria, and oxidative stress.

Alzheimers disease (AD) is characterized by the accumulation of amyloid-β (Aβ) peptides. Although the disease undoubtedly reflects the interaction of complex multifactorial processes, Aβ itself is toxic to neurons in vitro and the load of Aβ in vivo correlates well with the degree of cognitive impairment. There has therefore been considerable interest in the mechanism(s) of Aβ neurotoxicity. We here review the basic biology of Aβ processing and consider some of the major areas of focus of this research. It is clear that both AD and Aβ toxicity are characterized by oxidative stress, alterations in the activity of enzymes of intermediary metabolism, and mitochondrial dysfunction, especially impaired activity of cytochrome c oxidase. Studies in vitro also show alterations in cellular calcium signaling. We consider the mechanisms proposed to mediate cell injury and explore evidence to indicate which of these many changes in function are primary and which secondary.

β‐Amyloid fragment 25–35 selectively decreases complex IV activity in isolated mitochondria

Defects in mitochondrial oxidative metabolism, in particular decreased activity of cytochrome c oxidase, have been demonstrated in Alzheimers disease, and after the expression of the amyloid precursor protein (APP) in cultured cells, suggesting that mitochondria might be involved in β‐amyloid toxicity. Recent evidence suggests that the proteolysis of APP to generate β‐amyloid is at least in part intracellular, preceding the deposition of extracellular fibrils. We have therefore investigated the effect of incubation of isolated rat brain mitochondria with the β‐amyloid fragment 25–35 (100 μM) on the activities of the mitochondrial respiratory chain complexes I, II–III, IV (cytochrome c oxidase) and citrate synthase. The peptide caused a rapid, dose‐dependent decrease in the activity of complex IV, while it had no effect on the activities on any of the other enzymes tested. The reverse sequence peptide (35–25) had no effect on any of the activities measured. We conclude that inhibition of mitochondrial complex IV might be a contributing factor to the pathogenesis of Alzheimers disease.

Threshold Effects in Synaptosomal and Nonsynaptic Mitochondria from Hippocampal CA1 and Paramedian Neocortex Brain Regions

Abstract: After a brief period of global ischemia, the hippocampal CA1 region is more susceptible to irreversible damage than the paramedian neocortex. To test whether primary differences in bioenergetic parameters may be present between these regions, respiration rates and respiratory control activities were measured. In synaptosomal and nonsynaptic mitochondria isolated from the hippocampal CA1 region, state 3 respiration rates and complex IV activities were significantly lower than those present in synaptosomal and nonsynaptic mitochondria from the paramedian neocortex. These results suggest that mitochondria from the CA1 hippocampal area differ in some properties of metabolism compared with the neocortex area, which may render them more susceptible to a toxic insult such as that of ischemia. In addition, when complex I and IV activities were titrated with specific inhibitors, thresholds in ATP synthesis and oxygen respiration became apparent. Complex I and IV activities were decreased by 60% in nonsynaptic mitochondria from the hippocampal CA1 region and paramedian neocortex before oxidative phosphorylation was severely compromised; however, in synaptosomes from these regions, complex I activities had a threshold of 25%, indicating heterogenous behaviour for brain mitochondria. Reduced complex I thresholds in mitochondria, in association with other constitutive defects in energy metabolism, may induce a decreased ATP supply in the synaptic region. The implications of these findings are discussed in relation to delayed neuronal death and processes of neurodegeneration.

GTP cyclohydrolase I gene transfer augments intracellular tetrahydrobiopterin in human endothelial cells: effects on nitric oxide synthase activity, protein levels and dimerisation

OBJECTIVES Tetrahydrobiopterin (BH4) is an essential cofactor for endothelial nitric oxide synthase (eNOS) activity. BH4 levels are regulated by de novo biosynthesis; the rate-limiting enzyme is GTP cyclohydrolase I (GTPCH). BH4 activates and promotes homodimerisation of purified eNOS protein, but the intracellular mechanisms underlying BH4-mediated eNOS regulation in endothelial cells remain less clear. We aimed to investigate the role of BH4 levels in intracellular eNOS regulation, by targeting the BH4 synthetic pathway as a novel strategy to modulate intracellular BH4 levels. METHODS We constructed a recombinant adenovirus, AdGCH, encoding human GTPCH. We infected human endothelial cells with AdGCH, investigated the changes in intracellular biopterin levels, and determined the effects on eNOS enzymatic activity, protein levels and dimerisation. RESULTS GTPCH gene transfer in EAhy926 endothelial cells increased BH4 >10-fold compared with controls (cells alone or control adenovirus infection), and greatly enhanced NO production in a dose-dependent, eNOS-specific manner. We found that eNOS was principally monomeric in control cells, whereas GTPCH gene transfer resulted in a striking increase in eNOS homodimerisation. Furthermore, the total amounts of both native eNOS protein and a recombinant eNOS-GFP fusion protein were significantly increased following GTPCH gene transfer. CONCLUSIONS These findings suggest that GTPCH gene transfer is a valid approach to increase BH4 levels in human endothelial cells, and provide new evidence for the relative importance of different mechanisms underlying BH4-mediated eNOS regulation in intact human endothelial cells. Additionally, these observations suggest that GTPCH may be a rational target to augment endothelial BH4 and normalise eNOS activity in endothelial dysfunction states.

Alzheimer’s Disease and Cholesterol: The Fat Connection

Since the discovery of the significance of the cholesterol-carrying apolipoprotein E and cholesterolaemia as major risk factors for Alzheimer’s Disease (AD) there has been a mounting interest in the role of this lipid as a possible pathogenic agent. In this review we analyse the current evidence linking cholesterol metabolism and regulation in the CNS with the known mechanisms underlying the development of Alzheimer’s Disease. Cholesterol is known to affect amyloid-β generation and toxicity, although it must be considered that the results studies using the statin class of drugs to lower plasma cholesterol may be affected by other effects associated with these drugs. Finally, we report some of our results pointing at the interplay between neurons and astrocytes and NADPH oxidase activation as a new candidate mechanism linking cholesterol and AD pathology.

Activity of Mitochondrial Respiratory Chain Enzymes After Transient Focal Ischemia in the Rat

Previous results demonstrated that after 2-hour middle cerebral artery occlusion (MCAO) in the rat, 1- to 2-hour recirculation temporarily restored the bioenergetic state and mitochondrial function, but secondary deterioration took place after 4 hours. The authors measured the activity of mitochondrial respiratory chain complexes, citrate synthase, and glutamate dehydrogenase as possible targets of secondary damage. Focal and penumbral tissues were sampled in the control condition, after 2 hours of MCAO, and after 1, 2, or 4 hours of postischemic recirculation; two groups were treated with α-phenyl-N-tert-butyl-nitrone (PBN). Complex IV activity transiently decreased after MCAO, but after recirculation all measured activities returned to control values.