Decreased serum and red blood cell kynurenic acid levels in Alzheimer's disease

Kynurenine aminotransferases (KAT I and KAT II) are responsible for the transamination of kynurenine (KYN) to form kynurenic acid (KYNA), an excitatory amino acid receptor antagonist. Since these members of the kynurenine pathway (KP) are proposed to be involved in the pathogenesis of Alzheimer's dementia (AD), the activities of these enzymes and the levels of these metabolites were measured in the plasma and red blood cells (RBCs) of AD and control subjects together with the inheritance of the apolipoprotein (APOE) epsilon4 allele. KYNA levels were significantly decreased both in the plasma and in the RBCs in AD, but the levels of KYN and the activities of KAT I and KAT II remained unchanged. No association has been found with the possession of the epsilon4 allele. These findings indicate an altered peripheral KP in AD regardless of the APOE status of the probands.     

Serum clusterin levels are not increased in presymptomatic Alzheimer's disease

Increased plasma levels of clusterin have recently been found to be associated with severity and progression in Alzheimer's disease (AD). We have investigated clusterin levels in serum of elderly people with presymptomatic AD from a population-based prospective cohort study. During 10 years follow-up, 43 participants were diagnosed with AD after on average 4.2 years (±2.6 years SD) after the initial blood sampling. At the time of blood sampling, these participants showed normal cognitive function. For each presymptomatic AD case, a control was matched on gender and age. Furthermore, the selected controls had to remain dementia-free and still be alive at the end of follow-up. Quantitative serum clusterin levels were measured with a newly developed multiple reaction monitoring (MRM) assay. Results of the assay showed no significant difference in clusterin levels between presymptomatic AD and controls (p-value 0.54). In conclusion, serum clusterin is not an early, presymptomatic biomarker for AD.     

Decreased DNA repair in familial Alzheimer's disease

Alterations in the capacity of a cell to repair DNA lesions play an important role in a number of human diseases. We and others have demonstrated defective DNA repair of alkylation damage in cells from patients with Alzheimer's disease. It has been hypothesized that this defect is related to the cause of Alzheimer's disease and results in the accumulation of lesions in the central nervous system neurons. One prediction of this hypothesis is that in dominantly inherited Alzheimer's disease, the repair defect will be present in half of the offspring of affected patients long before they develop symptoms of the disease. In order to test the hypothesis that decreased DNA repair is responsible for familial Alzheimer's disease and their at-risk offspring we have studied DNA repair in these individuals after exposure of lymphoblasts to alkylating agents. Our results indicate that cell lines from affected patients repair significantly less damage in 3 h than cell lines from healthy controls. A small number of at-risk individuals were also studied and some of these had lower levels of repair, although more cell lines from individuals in this group must be studied. These findings provide further support for defective DNA repair playing a role in the pathogenesis of Alzheimer's disease.     

Selenium regulation of thioredoxin reductase activity and mRNA levels in rat liver

Mammalian thioredoxin reductase (TRR; NADPH₂:oxidized thioredoxin oxidoreductase, E.C. 1.6.4.5) is a new member of the family of selenocysteine-containing proteins. TRR activity in Se-deficient rat liver is reported to decrease to 4.5 to 15% of the activity in Se-adequate rat liver, similar to the fall in Se-dependent glutathione peroxidase-1 activity. Both glutathione peroxidase-1 enzyme activity and mRNA levels decrease dramatically in Se deficiency, whereas glutathione peroxidase-4 activity only decreases to 40% of Se-adequate levels and mRNA level is little affected by Se deficiency. The purpose of these experiments is to study the effect of Se status on TRR mRNA levels and enzyme activity in our well-characterized rat model, and to compare this regulation directly to the regulation of other Se-dependent proteins in male weanling rats fed Se-deficient diets or supplemented with dietary Se for 28 days. In two experiments, TRR activity in Se-deficient liver decreased to 15% of Se-adequate activity as compared to 2% and 40% of Se-adequate levels for GPX1 and GPX4, respectively. Using ribonuclease protection analysis, we found that TRR mRNA levels in Se-deficient rat liver decreased to 70% of Se-adequate levels. This decrease in TRR mRNA was similar to the GPX4 mRNA decrease in Se-deficient liver in these experiments, whereas GPX1 mRNA levels decreased to 23% of Se-adequate levels. This study clearly shows that TRR represents a third pattern of Se regulation with dramatic down-regulation of enzyme activity in Se deficiency but with only a modest decrease in mRNA level. The conservation of TRR mRNA in Se deficiency suggests that this is a valued enzyme; the loss of TRR activity in Se deficiency may be the cause of some signs of Se deficiency.     

ZnT3 mRNA levels are reduced in Alzheimer's disease post-mortem brain

Extensive genetic, biochemical, and histological evidence has implicated the amyloid-β peptide (Aβ) in Alzheimer's disease pathogenesis, and several mechanisms have been suggested, such as metal binding, reactive oxygen species production, and membrane pore formation. However, recent evidence argues for an additional role for signaling mediated by the amyloid precursor protein, APP, in part via the caspase cleavage of APP at aspartate 664. Here we review the effects and implications of this cleavage event, and propose a model of Alzheimer's disease that focuses on the critical nature of this cleavage and its downstream effects.     

Ultrastructural demonstration of thioredoxin and thioredoxin reductase in rat hepatocytes

Electron microscopic immunocytochemistry, in conjunction with the immunogold technique, was used to demonstrate the ultrastructural localization of thioredoxin and thioredoxin reductase in rat liver hepatocytes. Gold particles representing thioredoxin and thioredoxin reductase antigenic sites were found throughout the cell, but particularly densely associated with the granular endoplasmic reticulum and the cisternae of the Golgi complex. Label was also distributed over the cytosol and in the chromatin of the nucleus. We conclude that thioredoxin and thioredoxin reductase are present in several different cellular compartments including the nucleus. In particular, the enrichment of thioredoxin and thioredoxin reductase to the endoplasmic reticulum is consistent with functions in protein processing, secretion and the formation of nascent protein disulfides.     

Immunohistochemical localization of thioredoxin and thioredoxin reductase in adult rats

Rabbit antisera against homogeneous rat liver thioredoxin and thioredoxin reductase (NADPH-oxidized thioredoxin oxidoreductase, E.C. 1.6.4.5) were prepared and used for immunohistochemical analysis in adult rats. Immunoreactive thioredoxin and thioredoxin reductase were widely distributed in tissues and organs, but varied a lot between cell types. Generally, epithelial cells, neuronal cells and secretory cells, both exocrine and endocrine, showed high immunoreactivity whereas mesenchymal cells with exceptions showed low activity. Surface lining epithelial and keratinizing cells showed high activity. The immunofluorescence was localized in the cytoplasm of cells with enrichments at secretory granules, at the plasma membrane or in the subplasma membrane zone. Variations in secretory cells were seen related to feeding and starvation and to metabolic activity. The distribution of thioredoxin and thioredoxin reductase is compatible with function in thiol-disulfide interchange reaction related to protein synthesis, intracellular transport and different forms of secretion.     

APP-BP1 mediates APP-induced apoptosis and DNA synthesis and is increased in Alzheimer's disease brain

APP-BP1, first identified as an amyloid precursor protein (APP) binding protein, is the regulatory subunit of the activating enzyme for the small ubiquitin-like protein NEDD8. We have shown that APP-BP1 drives the S- to M-phase transition in dividing cells, and causes apoptosis in neurons. We now demonstrate that APP-BP1 binds to the COOH-terminal 31 amino acids of APP (C31) and colocalizes with APP in a lipid-enriched fraction called lipid rafts. We show that coexpression of a peptide representing the domain of APP-BP1 that binds to APP, abolishes the ability of overexpressed APP or the V642I mutant of APP to cause neuronal apoptosis and DNA synthesis. A dominant negative mutant of the NEDD8 conjugating enzyme hUbc12, which participates in the ubiquitin-like pathway initiated by APP-BP1, blocks neuronal apoptosis caused by APP, APP(V642I), C31, or overexpression of APP-BP1. Neurons overexpressing APP or APP(V642I) show increased APP-BP1 protein levels in lipid rafts. A similar increase in APP-BP1 in lipid rafts is observed in the Alzheimer's disease brain hippocampus, but not in less-affected areas of Alzheimer's disease brain. This translocation of APP-BP1 to lipid rafts is accompanied by a change in the subcellular localization of the ubiquitin-like protein NEDD8, which is activated by APP-BP1.     

Quinone reductase (NQO1), a sensitive redox indicator, is increased in Alzheimer's disease

NAD(P)H:quinone oxidoreductase 1 (NQO1), a redox-regulated flavoenzyme, plays a central role in monitoring cellular redox state. NQO1 acts to protect against oxidative stress induced by a variety of metabolic situations, including metabolism of quinones and other xenobiotics, by: (i) functioning as a two electron donor to provide a shunt that competes with the formation of reactive oxygen species; (ii) maintaining reduced coenzyme Q; and (iii) regulating the stress activated kinase pathway. In Alzheimer's disease, while there is abundant evidence for the involvement of oxidative stress, the cause or the consequences are largely unresolved. We suspected that increased NQO1 could signal a major shift in redox balance in Alzheimer's disease and, in this study, found that NQO1 is localized not only to neurofibrillary tangles but also the cytoplasm of hippocampal neurons. By marked contrast, there is very little NQO1 in the same neuronal populations in young and age-matched controls. This novel association of NQO1 further buttresses the nexus of oxidative stress, via free radicals, with selective neuronal vulnerability and also supports a fundamental abnormality in redox balance in Alzheimer's disease.     

Quinone reductase (NQO1), a sensitive redox indicator,is increased in Alzheimer's disease

Abstract

NAD(P)H:quinone oxidoreductase 1 (NQO1), a redox-regulated flavoenzyme, plays a central rolein monitoring cellular redox state. NQO1 acts to protect against oxidative stress induced by a variety of metabolic situations, including metabolism of quinones and other xenobiotics, by: (i)functioning as a two electron donor to provide a shunt that competes with the formation of reactive oxygen species; (ii) maintaining reduced coenzyme Q; and (iii) regulating the stress activated kinase pathway. In Alzheimer's disease, while there is abundant evidence for the involvement of oxidative stress, the cause or the consequences are largely unresolved. We suspected that increased NQO1 could signal a major shift in redox balance in Alzheimer's disease and, in this study, found that NQO1 is localized not only to neurofibrillary tangles but also the cytoplasm of hippocampal neurons. By marked contrast, there is very little NQO1 in the same neuronal populations in young and age-matched controls. This novel association of NQO1 further buttresses the nexus of oxidative stress, via free radicals, with selective neuronal vulnerability and also supports a fundamental abnormality in redox balance in Alzheimer's disease.     

Glutathione reductase and thioredoxin reductase at the crossroad: the structure of Schistosoma mansoni thioredoxin glutathione reductase

Thioredoxin glutathione reductase (TGR) is a key flavoenzyme expressed by schistosomes that bridges two detoxification pathways crucial for the parasite survival in the host's organism. In this article we report the crystal structure (at 2.2 A resolution) of TGR from Schistosoma mansoni (SmTGR), deleted in the last two residues. The structure reveals the peculiar architecture of this chimeric enzyme: the small Glutaredoxin (Grx) domain at the N-terminus is joined to the large thioredoxin reductase (TR) one via an extended complementary surface, involving residues not conserved in the Grx superfamily; the TR domain interacts with an identical partner via its C-terminal domain, forming a dimer with a twisted "W" shape. Although lacking the penultimate Selenocysteine residue (Sec), the enzyme is still able to reduce oxidized glutathione. These data update the interpretation of the interdomain communication in TGR enzymes. The possible function of this enzyme in pathogenic parasites is discussed.     

Purification of thioredoxin, thioredoxin reductase, and glutathione reductase by affinity chromatography.

A scheme is described for the large scale purification of thioredoxin, thioredoxin reductase, and glutathione reductase. The scheme is based on an initial separation of thioredoxin from the two reductases by affinity chromatography on agarose-bound N6-(6-aminohexyl)-adenosine 2',5'-bisphosphate (agarose-2',5'-ADP). The two reductases were then separated by hydrophobic chromatography and purified separately to homogeneity. Thioredoxin was purified to homogeneity by immunoadsorption to agarose containing immobilized goat anti-thioredoxin. Overall yields for thioredoxin, thioredoxin reductase, and glutathione reductase exceeded 80% in each case. Both reductases exhibit an absorption band at approximately 320 nm which appears due to a residual amount of tightly bound NADP. Presence of this absorption band has no apparent effect on the specific activity of either enzyme.     

Unique gene organization of thioredoxin and thioredoxin reductase in Mycobacterium leprae

The thioredoxin system comprising thioredoxin (Trx), thioredoxin reductase (TR) and NADPH operates via redox-active disulphides and provides electrons for a wide variety of different metabolic processes in prokaryotic and eukaryotic cells. Thioredoxin is also a general protein disulphide reductase involved in redox regulation. In bacteria, the Trx and TR proteins previously identified were encoded by separate genes (trxA and trxB). In this study, we report a novel genomic organization of TR and Trx in mycobacteria and show that at least three modes of organization of TR and Trx genes can exist within a single bacterial genus: (i) in the majority of mycobacterial strains the genes coding for TR and Trx are located on separate sites of the genome; (ii) interestingly, in all pathogenic Mycobacterium tuberculosis complex mycobacteria both genes are found on the same locus, overlapping in one nucleotide; (iii) in the pathogen Mycobacterium leprae, TR and Trx are encoded by a single gene. Sequence analysis of the M. leprae gene demonstrated that the N-terminal part of the protein corresponds to TR and the C-terminal part to Trx. A corresponding single protein product of approximately 49 kDa was detected in cell extracts of M. leprae. These findings demonstrate the very unusual phenomenon of a single gene coding for both the substrate (thioredoxin) and the enzyme (thioredoxin reductase), which seems to be unique to M. leprae.     

The involvement of thioredoxin and thioredoxin reductase in the dimethyl sulphoxide reductase system of Saccharomyces cerevisiae

Abstract Dimethyl sulphoxide (DMSO) reductase activity in crude extracts of Saccharomyces cerevisiae NCYC240 was stimulated by addition of thioredoxin, but not by addition of thioredoxin reductase. The activity was partially purified. DEAE-cellulose could be used to separate thioredoxin and its reductase (which bound to the column) from the terminal DMSO-reductase protein (which failed to bind). The highly unstable purified terminal reductase so obtained required both thioredoxin and thioredoxin reductase to reconstitute activity with either dithiothreitol (DTT) or NADPH as electron donor. Partially purified terminal reductase had an M _r of about 15000.     

Serum thioredoxin reductase levels increase in response to chemically induced acute liver injury

Background

Mammalian thioredoxin reductases (TrxR) are selenoproteins with important roles in antioxidant defense and redox regulation, principally linked to functions of their main substrates thioredoxins (Trx). All major forms of TrxR are intracellular while levels in serum are typically very low.

Methods

Serum TrxR levels were determined with immunoblotting using antibodies against mouse TrxR1 and total enzyme activity measurements were performed, with serum and tissue samples from mouse models of liver injury, as triggered by either thioacetamide (TAA) or carbon tetrachloride (CCl₄).

Results

TrxR levels in serum increased upon treatment and correlated closely with those of alanine aminotransferase (ALT), an often used serum biomarker for liver damage. In contrast, Trx1, glutathione reductase, superoxide dismutase or selenium-containing glutathione peroxidase levels in serum displayed much lower increases than TrxR or ALT.

Conclusions

Serum TrxR levels are robustly elevated in mouse models of chemically induced liver injury.

General significance

The exaggerated TrxR release to serum upon liver injury may reflect more complex events than a mere passive release of hepatic enzymes to the extracellular milieu. It can also not be disregarded that enzymatically active TrxR in serum could have yet unidentified physiological functions.     

Decreased protein levels of nicotinic receptor subunits in the hippocampus and temporal cortex of patients with Alzheimer's disease

Deficits of cortical nicotinic acetylcholine receptors (nAChRs) have been observed in Alzheimer's disease (AD) by receptor binding assays. Little is known about the receptor subunit specificity influenced by AD, and it might be of importance for therapeutic strategies. In the present study, the protein levels of nAChR alpha3, alpha4, alpha7, and beta2 subunits were investigated using western blot analysis on postmortem brains of patients with AD and age-matched controls. The results showed that in human postmortem brain samples, bands with molecular masses of 52, 42, and 50 kDa were detected by anti-alpha4, anti-alpha7, and anti-beta2 antibodies, respectively. When anti-alpha3 antibody was used, one major band of 49 kDa and two minor bands of 70 and 38 kDa were detected. In AD patients, as compared with age-matched controls, the alpha4 subunit was reduced significantly by approximately 35 and 47% in the hippocampus and temporal cortex, respectively. A significant reduction of 25% in the alpha3 subunit was also observed in the hippocampus and a 29% reduction in the temporal cortex. For the alpha7 subunit, the protein level was reduced significantly by 36% in the hippocampus of AD patients, but no significant change was detected in the temporal cortex. In neither the hippocampus nor the temporal cortex was a significant difference observed in the beta2 subunit between AD patients and controls. These results reveal brain region-specific changes in the protein levels of the nAChR alpha3, alpha4, and alpha7 subunits in AD.