Modification of endoplasmic reticulum Ca2+ stores by select oxidants produces changes reminiscent of those in cells from patients with Alzheimer disease

Abnormalities in calcium homeostasis and oxidative processes occur in fibroblasts from patients with Alzheimer disease (AD) and in fibroblasts and neurons from transgenic mice bearing a presenilin-1 (PS-1) mutation. Bombesin-releasable endoplasmic reticulum Ca2+ stores (BRCS) are exaggerated in all of these cells. Our previous studies show that H2O2 exaggerates BRCS. The goal of the present study was to determine whether select reactive species exaggerate BRCS in cultured human fibroblasts and to determine if the ability of fibroblasts to handle these specific oxidant species is altered in cells from AD patients. Two fluorescent indicators were used to distinguish different reactive oxygen species (ROS): 6-carboxy-2',7'-dichlorodihydrofluorescein diacetate, di(acetoxymethyl ester) (c-DCF) and 4-amino-5-methylamino-2',7'-difluorofluorescein diacetate (DAF-FM). ROS were produced by a variety of oxidants, including tert-butyl-hydroxyperoxide (t-BHP), hypoxanthine/xanthine oxidase, S-nitroso-N-acetylpenicillamine, 3-morpholinosydnonimine, and sodium nitroprusside. Different oxidants selectively induced various ROS in distinct patterns. These oxidants also induced selective modification in [Ca2+]i and/or BRCS. Of the several oxidants tested, t-BHP was most specific for exaggerating BRCS without affecting basal [Ca2+]i and inducing only c-DCF-detectable ROS. On the other hand, the results show that NO that reacted with DAF-FM was not responsible for alterations in BRCS. Furthermore, the c-DCF-detectable ROS production induced by t-BHP was higher in fibroblasts from AD patients bearing a PS-1 mutation (n = 7) than in those from aged controls (n = 8). The higher production of c-DCF-detectable ROS may underlie the exaggeration of BRCS in fibroblasts from AD patients. Thus, these results are consistent with the hypothesis that abnormalities in selective cellular ROS cause AD-related changes in intracellular calcium regulation.     

Oxidative stress increases internal calcium stores and reduces a key mitochondrial enzyme

Fibroblasts from patients with genetic and non-genetic forms of Alzheimer's disease (AD) show many abnormalities including increased bombesin-releasable calcium stores (BRCS), diminished activities of the mitochondrial alpha-ketoglutarate dehydrogenase complex (KGDHC), and an altered ability to handle oxidative stress. The link between genetic mutations (and the unknown primary event in non-genetic forms) and these other cellular abnormalities is unknown. To determine whether oxidative stress could be a convergence point that produces the other AD-related changes, these experiments tested in fibroblasts the effects of H(2)O(2), in the presence or absence of select antioxidants, on BRCS and KGDHC. H(2)O(2) concentrations that elevated carboxy-dichlorofluorescein (c-H(2)DCF)-detectable ROS increased BRCS and decreased KGDHC activity. These changes are in the same direction as those in fibroblasts from AD patients. Acute treatments with the antioxidants Trolox, or DMSO decreased c-H(2)DCF-detectable ROS by about 90%, but exaggerated the H(2)O(2)-induced increases in BRCS by about 4-fold and did not alter the reduction in KGDHC. Chronic pretreatments with Trolox more than doubled the BRCS, tripled KGDHC activities, and reduced the effects of H(2)O(2). Pretreatment with DMSO or N-acetyl cysteine diminished the BRCS and either had no effect, or exaggerated the H(2)O(2)-induced changes in these variables. The results demonstrate that BRCS and KGDHC are more sensitive to H(2)O(2) derived species than c-H(2)DCF, and that oxidized derivatives of the antioxidants exaggerate the actions of H(2)O(2). The findings support the hypothesis that select abnormalities in oxidative processes are a critical part of a cascade that leads to the cellular abnormalities in cells from AD patients.     

Ionic and signal transduction alterations in Alzheimer’s disease

Several lines of, evidence indicate that Alzheimer’s disease (AD) has systemic expression. Systemic changes are manifested as alterations in a number of molecular and cellular processes. Although, these alterations appear to have little or no consequence in peripheral systems, their parallel expression in the central nervous system (CNS) could account for the principal clinical manifestations of the disease. Recent research seems to indicate that alterations in ion channels, calcium homeostasis, and protein kinase C (PKC) can be linked and thereby constitute a model of pathophysiological relevance. Considering the difficulties of studying dynamic pathophysiological processes in the disease-ridden postmortem AD brain, peripheral tissues such as fibroblasts provide a suitable model to study molecular and cellular aspects of the disease.     

A Boolean probabilistic model of metabolic adaptation to oxygen in relation to iron homeostasis and oxidative stress

Background  

In aerobically grown cells, iron homeostasis and oxidative stress are tightly linked processes implicated in a growing number of diseases. The deregulation of iron homeostasis due to gene defects or environmental stresses leads to a wide range of diseases with consequences for cellular metabolism that remain poorly understood. The modelling of iron homeostasis in relation to the main features of metabolism, energy production and oxidative stress may provide new clues to the ways in which changes in biological processes in a normal cell lead to disease.     

Antioxidants in Down syndrome

Individuals with Down syndrome (DS) have high levels of oxidative stress throughout the lifespan. Mouse models of DS share some structural and functional abnormalities that parallel findings seen in the human phenotype. Several of the mouse models show evidence of cellular oxidative stress and have provided a platform for antioxidant intervention. Genes that are overexpressed on chromosome 21 are associated with oxidative stress and neuronal apoptosis. The lack of balance in the metabolism of free radicals generated during processes related to oxidative stress may have a direct role in producing the neuropathology of DS including the tendency to Alzheimer disease (AD). Mitochondria are often a target for oxidative stress and are considered to be a trigger for the onset of the AD process in DS. Biomarkers for oxidative stress have been described in DS and in AD in the general population. However, intervention trials using standard antioxidant supplements or diets have failed to produce uniform therapeutic effect. This chapter will examine the biological role of oxidative stress in DS and its relationship to abnormalities in both development and aging within the disorder. This article is part of a Special Issue entitled: Antioxidants and Antioxidant Treatment in Disease.     

Antioxidants in Down syndrome

Individuals with Down syndrome (DS) have high levels of oxidative stress throughout the lifespan. Mouse models of DS share some structural and functional abnormalities that parallel findings seen in the human phenotype. Several of the mouse models show evidence of cellular oxidative stress and have provided a platform for antioxidant intervention. Genes that are overexpressed on chromosome 21 are associated with oxidative stress and neuronal apoptosis. The lack of balance in the metabolism of free radicals generated during processes related to oxidative stress may have a direct role in producing the neuropathology of DS including the tendency to Alzheimer disease (AD). Mitochondria are often a target for oxidative stress and are considered to be a trigger for the onset of the AD process in DS. Biomarkers for oxidative stress have been described in DS and in AD in the general population. However, intervention trials using standard antioxidant supplements or diets have failed to produce uniform therapeutic effect. This chapter will examine the biological role of oxidative stress in DS and its relationship to abnormalities in both development and aging within the disorder. This article is part of a Special Issue entitled: Antioxidants and Antioxidant Treatment in Disease.     

Mitochondria as a primary target for vascular hypoperfusion and oxidative stress in Alzheimer's disease

It has been widely accepted that vascular hypoperfusion induces oxidative stress and the outcome of this misbalance is brain energy failure. This abnormality leads to neuronal death which manifests as cognitive impairment and the development of brain pathology as in Alzheimer's disease (AD). It has been demonstrated that the AD brain is characterized by impairments in energy metabolism. We theorize that hypoperfusion induced mitochondrial failure plays a key role in the generation of reactive oxygen species, resulting in oxidative damage to brain cellular compartments, especially in the vascular endothelium and in selective population of neurons with high metabolic activity in the AD brain. All of these abnormalities have been found to occur before classic AD pathology inducing neuronal degeneration and amyloid deposition during the progression of AD. Therefore, expanding investigations into both the mechanisms behind amyloid beta (Abeta) deposition and the possible accelerating effects of environmental factors such as chronic hypoxia/reperfusion may open a new avenue for effective treatments of AD. Future studies examining the importance of mitochondrial pathobiology in brain cellular compartments provide insight not only into the better understanding of the neurodegenerative and/or cerebrovascular disease but also provide targets for treating these conditions.     

Toxicity of Amyloid β Peptide: Tales of Calcium,Mitochondria, and Oxidative Stress

Alzheimer's disease (AD) is characterized by the accumulation of amyloid-beta (Abeta) peptides. Although the disease undoubtedly reflects the interaction of complex multifactorial processes, Abeta itself is toxic to neurons in vitro and the load of Abeta in vivo correlates well with the degree of cognitive impairment. There has therefore been considerable interest in the mechanism(s) of Abeta neurotoxicity. We here review the basic biology of Abeta processing and consider some of the major areas of focus of this research. It is clear that both AD and Abeta 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.     

Aberrant subcellular neuronal calcium regulation in aging and Alzheimer's disease

In this mini-review/opinion article we describe evidence that multiple cellular and molecular alterations in Alzheimer's disease (AD) pathogenesis involve perturbed cellular calcium regulation, and that alterations in synaptic calcium handling may be early and pivotal events in the disease process. With advancing age neurons encounter increased oxidative stress and impaired energy metabolism, which compromise the function of proteins that control membrane excitability and subcellular calcium dynamics. Altered proteolytic cleavage of the β-amyloid precursor protein (APP) in response to the aging process in combination with genetic and environmental factors results in the production and accumulation of neurotoxic forms of amyloid β-peptide (Aβ). Aβ undergoes a self-aggregation process and concomitantly generates reactive oxygen species that can trigger membrane-associated oxidative stress which, in turn, impairs the functions of ion-motive ATPases and glutamate and glucose transporters thereby rendering neurons vulnerable to excitotoxicity and apoptosis. Mutations in presenilin-1 that cause early-onset AD increase Aβ production, but also result in an abnormal increase in the size of endoplasmic reticulum calcium stores. Some of the events in the neurodegenerative cascade can be counteracted in animal models by manipulations that stabilize neuronal calcium homeostasis including dietary energy restriction, agonists of glucagon-like peptide 1 receptors and drugs that activate mitochondrial potassium channels. Emerging knowledge of the actions of calcium upstream and downstream of Aβ provides opportunities to develop novel preventative and therapeutic interventions for AD. This article is part of a Special Issue entitled: 11th European Symposium on Calcium.     

Mitochondrial Enzymes and Endoplasmic Reticulum Calcium Stores as Targets of Oxidative Stress in Neurodegenerative Diseases

Considerable evidence indicates that oxidative stress accompanies age-related neurodegenerative diseases. Specific mechanisms by which oxidative stress leads to neurodegeneration are unknown. Two targets of oxidative stress that are known to change in neurodegenerative diseases are the mitochondrial enzyme alpha-ketoglutarate dehydrogenase complex (KGDHC) and endoplasmic reticulum calcium stores. KGDHC activities are diminished in all common neurodegenerative diseases and the changes are particularly well documented in Alzheimer's disease (AD). A second change that occurs in cells from AD patients is an exaggerated endoplasmic reticulum calcium store [i.e., bombesin-releasable calcium stores (BRCS)]. H(2)O(2), a general oxidant, changes both variables in the same direction as occurs in disease. Other oxidants selectively alter these variables. Various antioxidants were used to help define the critical oxidant species that modifies these responses. All of the antioxidants diminish the oxidant-induced carboxy-dichlorofluorescein (cDCF) detectable reactive oxygen species (ROS), but have diverse actions on these cellular processes. For example, alpha-keto-beta-methyl-n-valeric acid (KMV) diminishes the H(2)O(2) effects on BRCS, while trolox and DMSO exaggerate the response. Acute trolox treatment does not alter H(2)O(2)-induced changes in KGDHC, whereas chronic treatment with trolox increases KGDHC almost threefold. The results suggest that KGDHC and BRCS provide targets by which oxidative stress may induce neurodegeneration and a useful tool for selecting antioxidants for reversing age-related neurodegeneration.     

Aberrant subcellular neuronal calcium regulation in aging and Alzheimer's disease

In this mini-review/opinion article we describe evidence that multiple cellular and molecular alterations in Alzheimer's disease (AD) pathogenesis involve perturbed cellular calcium regulation, and that alterations in synaptic calcium handling may be early and pivotal events in the disease process. With advancing age neurons encounter increased oxidative stress and impaired energy metabolism, which compromise the function of proteins that control membrane excitability and subcellular calcium dynamics. Altered proteolytic cleavage of the β-amyloid precursor protein (APP) in response to the aging process in combination with genetic and environmental factors results in the production and accumulation of neurotoxic forms of amyloid β-peptide (Aβ). Aβ undergoes a self-aggregation process and concomitantly generates reactive oxygen species that can trigger membrane-associated oxidative stress which, in turn, impairs the functions of ion-motive ATPases and glutamate and glucose transporters thereby rendering neurons vulnerable to excitotoxicity and apoptosis. Mutations in presenilin-1 that cause early-onset AD increase Aβ production, but also result in an abnormal increase in the size of endoplasmic reticulum calcium stores. Some of the events in the neurodegenerative cascade can be counteracted in animal models by manipulations that stabilize neuronal calcium homeostasis including dietary energy restriction, agonists of glucagon-like peptide 1 receptors and drugs that activate mitochondrial potassium channels. Emerging knowledge of the actions of calcium upstream and downstream of Aβ provides opportunities to develop novel preventative and therapeutic interventions for AD. This article is part of a Special Issue entitled: 11th European Symposium on Calcium.     

Sporadic Alzheimer disease fibroblasts display an oxidative stress phenotype

Alzheimer disease (AD) is a major health problem in the United States, affecting one in eight Americans over the age of 65. The number of elderly suffering from AD is expected to continue to increase over the next decade, as the average age of the U.S. population increases. The risk factors for and etiology of AD are not well understood; however, recent studies suggest that exposure to oxidative stress may be a contributing factor. Here, microarray gene expression signatures were compared in AD-patient-derived fibroblasts and normal fibroblasts exposed to hydrogen peroxide or menadione (to simulate conditions of oxidative stress). Using the 23K Illumina cDNA microarray to screen expression of >14,000 human genes, we identified a total of 1017 genes that are chronically up- or downregulated in AD fibroblasts, 215 of which were also differentially expressed in normal human fibroblasts within 12h after exposure to hydrogen peroxide or menadione. Pathway analysis of these 215 genes and their associated pathways revealed cellular functions that may be critically dysregulated by oxidative stress and play a critical role in the etiology and/or pathology of AD. We then examined the AD fibroblasts for the presence of oxidative DNA damage and found increased accumulation of 8-oxo-guanine. These results indicate the possible role of oxidative stress in the gene expression profile seen in AD.     

alpha-keto-beta-methyl-n-valeric acid diminishes reactive oxygen species and alters endoplasmic reticulum Ca(2+) stores

Mitochondrial dysfunction and oxidative stress occur in neurodegenerative diseases. Other results show that bombesin-releasable calcium stores (BRCS) from the endoplasmic reticulum (ER) are exaggerated in fibroblasts from patients with Alzheimer's disease (AD) compared with controls and in fibroblasts from a young control treated with H(2)O(2). We hypothesize that alterations in oxidative stress underlie the exaggeration in BRCS in AD, and that appropriate antioxidants may be useful in treating this abnormality. Two indicators of different oxidant species were used to determine the effects of select oxidants on cellular oxidation status: carboxydichlorofluorescein (c-DCF) to detect reactive oxygen species (ROS), and 4-amino-5-methylamino-2',7'-difluorofluorescein (DAF) to detect nitric oxide (NO(.-)). Various conditions that induce ROS, including H(2)O(2), oxygen/glucose deprivation, and 3-morpholinosyndnonimine (SIN-1), were used to test the ability of alpha-keto-ss-methyl-n-valeric acid (KMV) to scavenge ROS. KMV diminished c-DCF-detectable ROS that were induced by H(2)O(2), oxygen/glucose deprivation, or SIN-1 in PC12 cells, primary neuronal cultures, or fibroblasts. Furthermore, KMV reduced the H(2)O(2)-induced increase in BRCS and diminished the elevation in BRCS in cells from AD patients to control levels. On the other hand, DAF-detectable NO(.-) induced by SIN-1 was not scavenged by KMV and did not exaggerate BRCS. The results indicate that KMV is an effective antioxidant of c-DCF-detectable ROS. The effects of KMV are not cell type specific, but are ROS specific. The same H(2)O(2)-induced ROS that reacts with KMV may also underlie the changes in BRCS related to AD. Thus, KMV ameliorates the effects of ROS on calcium homeostasis related to oxidative stress and to AD.     

Insulin resistance and amyloidogenesis as common molecular foundation for type 2 diabetes and Alzheimer's disease

Characterized as a peripheral metabolic disorder and a degenerative disease of the central nervous system respectively, it is now widely recognized that type 2 diabetes mellitus (T2DM) and Alzheimer's disease (AD) share several common abnormalities including impaired glucose metabolism, increased oxidative stress, insulin resistance and amyloidogenesis. Several recent studies suggest that this is not an epiphenomenon, but rather these two diseases disrupt common molecular pathways and each disease compounds the progression of the other. For instance, in AD the accumulation of the amyloid-beta peptide (Aβ), which characterizes the disease and is thought to participate in the neurodegenerative process, may also induce neuronal insulin resistance. Conversely, disrupting normal glucose metabolism in transgenic animal models of AD that over-express the human amyloid precursor protein (hAPP) promotes amyloid-peptide aggregation and accelerates the disease progression. Studying these processes at a cellular level suggests that insulin resistance and Aβ aggregation may not only be the consequence of excitotoxicity, aberrant Ca²⁺ signals, and proinflammatory cytokines such as TNF-α, but may also promote these pathological effectors. At the molecular level, insulin resistance and Aβ disrupt common signal transduction cascades including the insulin receptor family/PI3 kinase/Akt/GSK3 pathway. Thus both disease processes contribute to overlapping pathology, thereby compounding disease symptoms and progression.     

Transcriptional changes in OXPHOS complex I deficiency are related to anti-oxidant pathways and could explain the disturbed calcium homeostasis

Defective complex I (CI) is the most common type of oxidative phosphorylation disease, with an incidence of 1 in 5000 live births. Here, whole genome expression profiling of fibroblasts from CI deficient patients was performed to gain insight into the cell pathological mechanism. Our results suggest that patient fibroblasts responded to oxidative stress by Nrf2-mediated induction of the glutathione antioxidant system and Gadd45-mediated activation of the DNA damage response pathway. Furthermore, the observed reduced expression of selenoproteins, might explain the disturbed calcium homeostasis previously described for the patient fibroblasts and might be linked to endoplasmic reticulum stress. These results suggest that both glutathione and selenium metabolism are potentially therapeutic targets in CI deficiency.     

Oxidative stress and muscular dystrophy

Oxidative stress may be the fundamental basis of many of the structural, functional and biochemical changes characteristic of the inherited muscular dystrophies in animals and humans. The presence of by-products of oxidative damage, and the compensatory increases in cellular antioxidants, both indicate oxidative stress may be occurring in dystrophic muscle. Changes in the proportions and metabolism of cellular lipids, abnormal functions of cellular membranes, altered activity of membrane-bound enzymes such as the SR Ca2+-ATPase, disturbances in cellular protein turnover and energy production and a variety of other changes all indicate that these inherited muscular dystrophies appear more like the results of oxidative stress to muscle than any other type of underlying muscle disturbance. Particular details of these altered characteristics of dystrophic muscle, in combination with current knowledge on the processes of oxidative damage to cells, may provide some insight into the underlying biochemical defect responsible for the disease, as well as direct research towards the ultimate goal of an effective treatment.     

The α-ketoglutarate-dehydrogenase complex

Damage from oxidative stress and mitochondrial dysfunction occur together in many common neurodegenerative diseases. The enzymes that form the mitochondrial alpha-ketoglutarate- dehydrogenase complex (KGDHC), a key and arguably rate-limiting enzyme system of the tricarboxylic acid cycle, might mediate the interaction of these processes. KGDHC activity is reduced in numerous age-related neurodegenerative diseases and is diminished by oxidative stress. In Alzheimer's disease (AD), the reduction correlates highly to diminished mental performance. Thus, research has focused on the mechanisms by which select oxidants reduce KGDHC and the consequences of such a reduction. Diminished KGDHC in cells is associated with apoptosis without changes in the mitochondrial membrane potential. Studies of isolated mitochondria and of animal models suggest that a reduction in KGDHC can predispose to damage by other toxins that promote neurodegeneration. Diminished oxidative metabolism can be plausibly linked to pathological features of neurodegenerative diseases (e.g., reduced mental function, the plaques and tangles in AD). Thus, reductions in KGDHC might be central to the pathophysiology of these diseases. Studies of proteins, cells, animal models, and humans suggest that treatments to diminish, or bypass, the reduction in KGDHC might be beneficial in age-related neurodegenerative disorders.     

Protective effect of new S-acylglutathione derivatives against amyloid-induced oxidative stress

Recent data support the role of oxidative stress in the pathogenesis of Alzheimer disease (AD). In particular, glutathione (GSH) metabolism is altered and its levels are decreased in affected brain regions and peripheral cells from AD patients and in experimental models of AD. In the past decade, interest in the protective effects of various antioxidants aimed at increasing intracellular GSH content has been growing. Because much experimental evidence suggests a possible protective role of unsaturated fatty acids in age-related diseases, we designed the synthesis of new S-acylglutathione (acyl-SG) thioesters. S-Lauroylglutathione (lauroyl-SG) and S-palmitoleoylglutathione (palmitoleoyl-SG) were easily internalized into the cells and they significantly reduced Abeta42-induced oxidative stress in human neurotypic SH-SY5Y cells. In particular, acyl-SG thioesters can prevent the impairment of intracellular ROS scavengers, intracellular ROS accumulation, lipid peroxidation, and apoptotic pathway activation. Palmitoleoyl-SG seemed more effective in cellular protection against Abeta-induced oxidative damage than lauroyl-SG, suggesting a valuable role for the monounsaturated fatty acid. In this study, we demonstrate that acyl-SG derivatives completely avoid the sharp lipoperoxidation in primary fibroblasts from familial AD patients occurring after exposure to Abeta42 aggregates. Hence, we put forward these derivatives as new antioxidant compounds which could be excellent candidates for therapeutic treatment of AD and other oxidative stress-related diseases.     

The effects of oxidative stress and altered intracellular calcium levels on vesicular transport of apoE-EGFP

Apolipoprotein E (apoE) plays a role in the distribution of lipid within many organs and cell types in the human body, including the central nervous system (CNS). The apoE4 isoform is also an established risk factor for late-onset Alzheimer's disease (AD), however its role in the aetiology of the disease remains largely unknown. Therefore, as AD is a late-onset disease, we sort to investigate how conditions hypothesised to model ageing affect apoE metabolism, such as the transport of apoE along the secretory pathway. Two of these models include oxidative stress and calcium deregulation. Using apoE-EGFP-expressing astrocytoma cell lines we established that vesicle number and velocity are up-regulated under oxidative stress conditions, and slowed under KCl induced calcium deregulation. Although these findings apply to cells in general under these two stress conditions, the up-regulation of apoE in particular may be a response to cell injury with implications for neurodegeneration such as that found with late-onset AD.