Peripheral levels of glutathione and protein oxidation as markers in the development of Alzheimer's disease from Mild Cognitive Impairment

There is a great interest in the relationship between Mild Cognitive Impairment (MCI) and the progression to Alzheimer's disease (AD). Several studies show the importance of oxidative stress in the pathogenesis of AD. The purpose of this study was the link between oxidative damage, MCI and AD. It analysed protein carbonyls and erythrocyte glutathione system plasma levels of 34 subjects with MCI, 45 subjects with AD and 28 age-matched control subjects. The results showed an increase in protein modification, a decrease in GSH levels and GSH/GSSG ratio in AD and MCI patients compared to age-matched control subjects (p<0.05). The present study shows that some peripheral markers of oxidative stress appear in MCI with a similar pattern to that observed in AD, which suggests that oxidative stress might represent a signal of the AD pathology. AD and MCI are biochemically equivalent. MCI does not necessarily need to progress to AD on a biochemical level.     

Protein Levels and Activity of Some Antioxidant Enzymes in Hippocampus of Subjects with Amnestic Mild Cognitive Impairment

Mild cognitive impairment (MCI) is generally referred to the transitional zone between normal cognitive aging and early dementia or clinically probable Alzheimer’s disease (AD). Most individuals with amnestic MCI eventually develop AD, which suggests that MCI may be the earliest phase of AD. Oxidative stress is observed in brain from subjects with both AD and MCI. Among others, two possibilities for elevated oxidataive stress are decreased activity or elevated expression of antioxidant enzymes, the latter as a response to the former. Accordingly, in the current study, the protein levels and activity of some antioxidant enzymes in the hippocampus of control and MCI brain were measured using Western blot analysis and spectrophotometric methods, respectively. Alterations in the levels and activity of a number of antioxidant enzymes in MCI brain compared to age-matched controls were found. These results are consistent with the hypothesis that oxidative stress may be an early event in the progression of amnestic MCI to AD. Special issue article in honor of Dr. Anna Maria Giuffrida-Stella.     

Analysis of lipophilic fluorescent products in blood of Alzheimer's disease patients

Alzheimer's disease (AD) is a severe neurodegenerative disorder characterized by cognitive decline. Prodromal stage of AD, also called mild cognitive impairment (MCI), especially its amnestic type (aMCI), precedes dementia stage of AD. There are currently no reliable diagnostic biomarkers of AD in the blood. Alzheimer's disease is accompanied by increased oxidative stress in brain, which leads to oxidative damage and accumulation of free radical reaction end‐products. In our study, specific products of lipid peroxidation in the blood of AD patients were studied. Lipophilic extracts of erythrocytes (AD dementia = 19, aMCI = 27, controls = 16) and plasma (AD dementia = 11, aMCI = 17, controls = 16) were analysed by fluorescence spectroscopy. The level of these products is significantly increased in erythrocytes and plasma of AD dementia and aMCI patients versus controls. We concluded that oxidative stress end‐products are promising new biomarkers of AD, but further detailed characterisation of these products is needed.     

Oxidative stress and APO E polymorphisms in Alzheimer's disease and in mild cognitive impairment

Abstract

A number of evidences indicates oxidative stress as a relevant pathogenic factor in Alzheimer's disease (AD) and mild cognitive impairment (MCI). Considering its recognized major genetic risk factors in AD, apolipoprotein (APO E) has been investigated in several experimental settings regarding its role in the process of reactive oxygen species (ROS) generation. The aim of this work has been to evaluate possible relationships between APO E genotype and plasma levels of selected oxidative stress markers in both AD and MCI patients.

APO E genotypes were determined using restriction enzyme analysis. Plasma levels of oxidative markers, advanced oxidation protein products, iron-reducing ability of plasma and, in MCI, activity of superoxide dismutases were evaluated using spectrophotometric analysis.

We found, compared to controls, increased levels of oxidized proteins and decreased values of plasma-reducing capacity in both AD patients (p < 0.0001) and MCI patients (p < 0.001); the difference between AD and MCI patients was significant only for plasma-reducing capacity (p < 0.0001), the former showing the lowest values. Superoxide dismutase activity was reduced, although not at statistical level, in MCI compared with that in controls. E4 allele was statistically associated (p < 0.05) with AD patients. When comparing different APO E genotype subgroups, no difference was present, as far as advanced oxidation protein products and iron-reducing ability of plasma levels were concerned, between E4 and non-E4 carriers, in both AD and MCI; on the contrary, E4 carriers MCI patients showed significantly decreased (p < 0.05) superoxide dismutase activity with respect to non-E4 carriers.

This study, in confirming the occurrence of oxidative stress in AD and MCI patients, shows how it can be related, at least for superoxide dismutase activity in MCI, to APO E4 allele risk factor.     

Involvements of the lipid peroxidation product,HNE, in the pathogenesis and progression of Alzheimer's disease

Alzheimer's disease (AD) is an age-related neurodegenerative disorder. A number of hypotheses have been proposed to explain AD pathogenesis. One such hypothesis proposed to explain AD pathogenesis is the oxidative stress hypothesis. Increased levels of oxidative stress markers including the markers of lipid peroxidation such as acrolein, 4-hydroxy-2-trans-nonenal (HNE), malondialdehyde, etc. are found in brains of AD subjects. In this review, we focus principally on research conducted in the area of HNE in the central nervous system (CNS) of AD and mild cognitive impairment (MCI), and further, we discuss likely consequences of lipid peroxidation with respect to AD pathogenesis and progression. Based on the research conducted so far in the area of lipid peroxidation, it is suggested that lipid accessible antioxidant molecules could be a promising therapeutic approach to treat or slow progression of MCI and AD.     

Effects of oxidative and nitrosative stress in brain on p53 proapoptotic protein in amnestic mild cognitive impairment and Alzheimer disease

Many studies reported that oxidative and nitrosative stress might be important for the pathogenesis of Alzheimer's disease (AD) beginning with arguably the earliest stage of AD, i.e., as mild cognitive impairment (MCI). p53 is a proapoptotic protein that plays an important role in neuronal death, a process involved in many neurodegenerative disorders. Moreover, p53 plays a key role in the oxidative stress-dependent apoptosis. We demonstrated previously that p53 levels in brain were significantly higher in MCI and AD IPL (inferior parietal lobule) compared to control brains. In addition, we showed that in AD IPL, but not in MCI, HNE, a lipid peroxidation product, was significantly bound to p53 protein. In this report, we studied by means of immunoprecipitation analysis, the levels of markers of protein oxidation, 3-nitrotyrosine (3-NT) and protein carbonyls, in p53 in a specific region of the cerebral cortex, namely the inferior parietal lobule, in MCI and AD compared to control brains. The focus of these studies was to measure the oxidation and nitration status of this important proapoptotic protein, consistent with the hypothesis that oxidative modification of p53 could be involved in the neuronal loss observed in neurodegenerative conditions.     

Distinct transthyretin oxidation isoform profile in spinal fluid from patients with Alzheimer’s disease and mild cognitive impairment

Background

Transthyretin (TTR), an abundant protein in cerebrospinal fluid (CSF), contains a free, oxidation-prone cysteine residue that gives rise to TTR isoforms. These isoforms may reflect conditions in vivo. Since increased oxidative stress has been linked to neurodegenerative disorders such as Alzheimer’s disease (AD) it is of interest to characterize CSF-TTR isoform distribution in AD patients and controls. Here, TTR isoforms are profiled directly from CSF by an optimized immunoaffinity-mass spectrometry method in 76 samples from patients with AD (n = 37), mild cognitive impairment (MCI, n = 17)), and normal pressure hydrocephalus (NPH, n = 15), as well as healthy controls (HC, n = 7). Fractions of three specific oxidative modifications (S-cysteinylation, S-cysteinylglycinylation, and S-glutathionylation) were quantitated relative to the total TTR protein. Results were correlated with diagnostic information and with levels of CSF AD biomarkers tau, phosphorylated tau, and amyloid β_1-42 peptide.

Results

Preliminary data highlighted the high risk of artifactual TTR modification due to ex vivo oxidation and thus the samples for this study were all collected using strict and uniform guidelines. The results show that TTR is significantly more modified on Cys(10) in the AD and MCI groups than in controls (NPH and HC) (p ≤ 0.0012). Furthermore, the NPH group, while having normal TTR isoform distribution, had significantly decreased amyloid β peptide but normal tau values. No obvious correlations between levels of routine CSF biomarkers for AD and the degree of TTR modification were found.

Conclusions

AD and MCI patients display a significantly higher fraction of oxidatively modified TTR in CSF than the control groups of NPH patients and HC. Quantitation of CSF-TTR isoforms thus may provide diagnostic information in patients with dementia symptoms but this should be explored in larger studies including prospective studies of MCI patients. The development of methods for simple, robust, and reproducible inhibition of in vitro oxidation during CSF sampling and sample handling is highly warranted. In addition to the diagnostic information the possibility of using TTR as a CSF oxymeter is of potential value in studies monitoring disease activity and developing new drugs for neurodegenerative diseases.     

Oxidative stress in brain aging, neurodegenerative and vascular diseases: an overview

According to the free radical theory, aging can be considered as a progressive, inevitable process partially related to the accumulation of oxidative damage into biomolecules -- nucleic acids, lipids, proteins or carbohydrates -- due to an imbalance between prooxidants and antioxidants in favor of the former. More recently also the pathogenesis of several diseases has been linked to a condition of oxidative stress. In this review we focus our attention on the evidence of oxidative stress in aging brain, some of the most important neurodegenerative diseases -- Alzheimer's disease (AD), mild cognitive impairment (MCI), Parkinson's disease (PD), amyotrophic lateral sclerosis (ALS) and Huntington's disease (HD) -- and in two common and highly disabling vascular pathologies--stroke and cardiac failure. Particular attention will be given to the current knowledge about the biomarkers of oxidative stress that can be possibly used to monitor their severity and outcome.     

Metalloproteinase’s Activity and Oxidative Stress in Mild Cognitive Impairment and Alzheimer’s Disease

Matrix metalloproteinases (MMPs) and oxidative stress have been implicated in neurological diseases such as Alzheimer’s disease (AD). Plasma MMP-2 and MMP-9 activities were assessed in Mild Cognitive Impairment (MCI) and AD subjects compared with aged-matched controls, and subsequently analysed in relation to oxidative stress markers. Both MMP-2 and MMP-9 showed no significant changes versus control subjects. Plasma glutathione peroxidase Se-dependent (GPx-Se) activity and malondialdehyde (MDA) levels were higher in AD than in controls (P < 0.05), suggesting a role for GPx-Se in controlling oxidative stress in AD. Negative correlations were observed between MMPs and MDA in AD and MCI patients (P < 0.05). In conclusion, oxidative stress events did not include activation of MMPs and this similar pattern in AD and MCI suggests that both are biochemically equivalent.     

Elevation of glutathione as a therapeutic strategy in Alzheimer disease

Oxidative stress has been associated with the onset and progression of mild cognitive impairment (MCI) and Alzheimer disease (AD). AD and MCI brain and plasma display extensive oxidative stress as indexed by protein oxidation, lipid peroxidation, free radical formation, DNA oxidation, and decreased antioxidants. The most abundant endogenous antioxidant, glutathione, plays a significant role in combating oxidative stress. The ratio of oxidized to reduced glutathione is utilized as a measure of intensity of oxidative stress. Antioxidants have long been considered as an approach to slow down AD progression. In this review, we focus on the elevation on glutathione through N-acetyl-cysteine (NAC) and γ-glutamylcysteine ethyl ester (GCEE) as a potential therapeutic approach for Alzheimer disease. This article is part of a Special Issue entitled: Antioxidants and Antioxidant Treatment in Disease.     

Processing of the Platelet Amyloid Precursor Protein in the Mild Cognitive Impairment (MCI)

It has been suggested that mild cognitive impairment (MCI) patients deteriorate faster than the healthy elderly population and have an increased risk of developing dementia. Certain blood molecular biomarkers have been identified as prognostic markers in Alzheimer’s disease (AD). The present study was aimed to assess the status of the platelet amyloid precursor protein (APP) metabolism in MCI and AD subjects and establish to what extent any variation could have a prognostic value suggestive of predictive AD in MCI patients. Thirty-four subjects diagnosed with MCI and 45 subjects with AD were compared to 28 healthy elderly individuals for assessing for protein levels of APP, β-APP cleaving enzyme 1 (BACE1), presenilin 1 (PS1) and a disintegrin and metalloproteinase-10 (ADAM-10) by western blot, and for the enzyme activities of BACE1 and γ-secretase by using specific fluorogenic substrates, in samples of platelets. A similar pattern in the healthy elderly and MCI patients was found for BACE1 and PS1 levels. A reduction of APP levels in MCI and AD patients compared with healthy elderly individuals was found. Augmented levels of ADAM-10 in both MCI and AD were displayed in comparison with age-matched control subjects. The ratio ADAM-10/BACE1 was higher for the MCI group versus AD group. Whereas BACE1 and PS1 levels were only increased in AD regarding to controls, BACE1 and γ-secretase activities augmented significantly in both MCI and AD groups. Finally, differences and similarities between MCI and AD patients were observed in several markers of platelet APP processing. Larger sample sets from diverse populations need to be analyzed to define a signature for the presence of MCI or AD pathology and to early detect AD at the MCI stage.     

Copper subtype of Alzheimer's disease (AD): Meta-analyses,genetic studies and predictive value of non-ceruloplasmim copper in mild cognitive impairment conversion to full AD

Alzheimer's disease (AD) is the most common form of dementia. A myriad of complex factors contribute to AD, promoting the deposition in plaques of amyloid-beta (Aβ), which is the main constituent of this pathognomonic sign of AD at autopsy brain inspection. Aβ toxicity is related to oxidative stress, which results in synaptic loss in specific brain areas, eventually leading to cognitive decline. Metal, and especially copper, dyshomeostasis is a key factor in these processes. Recent studies have demonstrated that the serum fraction of copper that is not bound to ceruloplasmin (Non-Cp copper, also known as ‘free’ or labile copper) increases in a percentage of AD patients and mild cognitive impairment (MCI) subjects; this is considered a precursor of AD. Non-Cp copper is the exchangeable fraction of low molecular weight copper in serum. It is distinguished from the copper structurally bound to the ceruloplasmin protein, a master protein of iron metabolism. Non-Cp copper levels are higher than normal reference values (range 0–1.6 μmol/L) in about 50% of amnestic MCI subjects and 60% of AD patients, typifying them in a subset of AD. Meta-analyses, genetic studies and a prognostic study evaluating the predictive value of Non-Cp copper in MCI conversion to full AD demonstrate the existence of this copper phenotype of AD.     

Oxidatively modified,mitochondria-relevant brain proteins in subjects with Alzheimer disease and mild cognitive impairment

Alzheimer disease (AD) is an age-related neurodegenerative disorder, characterized histopathologically by the presence of senile plaques (SP), neurofibrillary tangles and synapse loss in selected brain regions. Positron emission tomography (PET) studies of glucose metabolism revealed decreased energetics in brain of subjects with AD and arguably its earliest form, mild cognitive impairment (MCI), and this decrease correlated with brain structural studies using MRI. The main component of senile plaques is amyloid beta-peptide (Aβ), a 40–42 amino acid peptide that as oligomers is capable of inducing oxidative stress under both in vitro and in vivo conditions and is neurotoxic. In the mitochondria isolated from AD brain, Aβ oligomers that correlated with the reported increased oxidative stress markers in AD have been reported. The markers of oxidative stress have been localized in the brain regions of AD and MCI that show pathological hallmarks of this disease, suggesting the possible role of Aβ in the initiation of the free-radical mediated process and consequently to the build up oxidative stress and AD pathogenesis. Using redox proteomics our laboratory found a number of oxidatively modified brain proteins that are directly in or are associated with the mitochondrial proteome, consistent with a possible involvement of the mitochondrial targeted oxidatively modified proteins in AD progression or pathogenesis. The precise mechanistic link between mitochondrial oxidative damage and role of oligomeric Aβ has not been explicated. In this review, we discuss the role of the oxidation of mitochondria-relevant brain proteins to the pathogenesis and progression of AD.     

Plasma protein profiling of Mild Cognitive Impairment and Alzheimer’s disease using iTRAQ quantitative proteomics

Background

With the promise of disease modifying treatments, there is a need for more specific diagnosis and prognosis of Alzheimer’s disease (AD) and mild cognitive impairment (MCI). Plasma biomarkers are likely to be utilised to increase diagnostic accuracy and specificity of AD and cognitive decline.

Methods

Isobaric tags (iTRAQ) and proteomic methods were used to identify potential plasma biomarkers of MCI and AD. Relative protein expression level changes were quantified in plasma of 411 cognitively normal subjects, 19 AD patients and 261 MCI patients. Plasma was pooled into 4 groups including normal control, AD, amnestic single and multiple domain MCI (aMCI), and nonamnestic single and multiple domain MCI (nMCI). Western-blotting was used to validate iTRAQ data. Integrated function and protein interactions were explored using WEB based bioinformatics tools (DAVID v6.7 and STRING v9.0).

Results

In at least two iTRAQ replicate experiments, 30 proteins were significantly dysregulated in MCI and AD plasma, relative to controls. These proteins included ApoA1, ApoB100, complement C3, C4b-binding protein, afamin, vitamin D-binding protein precursor, isoform 1 of Gelsolin actin regulator, Ig mμ chain C region (IGHM), histidine-rich glycoprotein and fibrinogen β and γ chains. Western-blotting confirmed that afamin was decreased and IGHM was increased in MCI and AD groups. Bioinformatics results indicated that these dysregulated proteins represented a diversity of biological processes, including acute inflammatory response, cholesterol transport and blood coagulation.

Conclusion

These findings demonstrate that expression level changes in multiple proteins are observed in MCI and AD plasma. Some of these, such as afamin and IGHM, may be candidate biomarkers for AD and the predementia condition of MCI.     

Heme oxygenase-1 posttranslational modifications in the brain of subjects with Alzheimer disease and mild cognitive impairment

Alzheimer disease (AD) is a neurodegenerative disorder characterized by progressive cognitive impairment and neuropathology. Oxidative and nitrosative stress plays a principal role in the pathogenesis of AD. The induction of the heme oxygenase-1/biliverdin reductase-A (HO-1/BVR-A) system in the brain represents one of the earliest mechanisms activated by cells to counteract the noxious effects of increased reactive oxygen species and reactive nitrogen species. Although initially proposed as a neuroprotective system in AD brain, the HO-1/BVR-A pathophysiological features are under debate. We previously reported alterations in BVR activity along with decreased phosphorylation and increased oxidative/nitrosative posttranslational modifications in the brain of subjects with AD and those with mild cognitive impairment (MCI). Furthermore, other groups proposed the observed increase in HO-1 in AD brain as a possible neurotoxic mechanism. Here we provide new insights about HO-1 in the brain of subjects with AD and MCI, the latter condition being the transitional phase between normal aging and early AD. HO-1 protein levels were significantly increased in the hippocampus of AD subjects, whereas HO-2 protein levels were significantly decreased in both AD and MCI hippocampi. In addition, significant increases in Ser-residue phosphorylation together with increased oxidative posttranslational modifications were found in the hippocampus of AD subjects. Interestingly, despite the lack of oxidative stress-induced AD neuropathology in cerebellum, HO-1 demonstrated increased Ser-residue phosphorylation and oxidative posttranslational modifications in this brain area, suggesting HO-1 as a target of oxidative damage even in the cerebellum. The significance of these findings is profound and opens new avenues into the comprehension of the role of HO-1 in the pathogenesis of AD.     

Phosphoproteomics of Alzheimer disease brain: Insights into altered brain protein regulation of critical neuronal functions and their contributions to subsequent cognitive loss

Alzheimer disease (AD) is the major locus of dementia worldwide. In the USA there are nearly 6 million persons with this disorder, and estimates of 13–20 million AD cases in the next three decades. The molecular bases for AD remain unknown, though processes involving amyloid beta-peptide as small oligomeric forms are gaining attention as known agents to both lead to oxidative stress and synaptic dysfunction associated with cognitive dysfunction in AD and its earlier forms, including amnestic mild cognitive impairment (MCI) and possibly preclinical Alzheimer disease (PCAD).Altered brain protein phosphorylation is a hallmark of AD, and phosphoproteomics offers an opportunity to identify these altered phosphoproteins in order to gain more insights into molecular mechanisms of neuronal dysfunction and death that lead to cognitive loss. This paper reviews what, to this author, are believed to be the known phosphoproteomics studies related to in vitro and in vivo models of AD as well as phosphoproteomics studies of brains from subjects with AD, and in at least one case in MCI and PCAD as well. The results of this review are discussed with relevance to new insights into AD brain protein dysregulation in critical neuronal functions and to potential therapeutic targets to slow, or in favorable cases, halt progression of this dementing disorder that affects millions of persons and their families worldwide.     

Peripheral Mitochondrial Dysfunction in Alzheimer’s Disease: Focus on Lymphocytes

Alzheimer's disease (AD) is the most common progressive neurodegenerative disease. Today, AD affects millions of people worldwide and the number of AD cases will increase with increased life expectancy. The AD brain is marked by severe neurodegeneration like the loss of synapses and neurons, atrophy and depletion of neurotransmitter systems in the hippocampus and cerebral cortex. Recent findings suggest that these pathological changes are causally induced by mitochondrial dysfunction and increased oxidative stress. These changes are not only observed in the brain of AD patients but also in the periphery. In this review, we discuss the potential role of elevated apoptosis, increased oxidative stress and especially mitochondrial dysfunction as peripheral markers for the detection of AD in blood cells especially in lymphocytes. We discuss recent not otherwise published findings on the level of complex activities of the respiratory chain comprising mitochondrial respiration and the mitochondrial membrane potential (MMP). We obtained decreased basal MMP levels in lymphocytes from AD patients as well as enhanced sensitivity to different complex inhibitors of the respiratory chain. These changes are in line with mitochondrial defects obtained in AD cell and animal models, and in post-mortem AD tissue. Importantly, these mitochondrial alterations where not only found in AD patients but also in patients with mild cognitive impairment (MCI). These new findings point to a relevance of mitochondrial function as an early peripheral marker for the detection of AD and MCI.     

A Subset of Cerebrospinal Fluid Proteins from a Multi-Analyte Panel Associated with Brain Atrophy,Disease Classification and Prediction in Alzheimer’s Disease

In this exploratory neuroimaging-proteomic study, we aimed to identify CSF proteins associated with AD and test their prognostic ability for disease classification and MCI to AD conversion prediction. Our study sample consisted of 295 subjects with CSF multi-analyte panel data and MRI at baseline downloaded from ADNI. Firstly, we tested the statistical effects of CSF proteins (n = 83) to measures of brain atrophy, CSF biomarkers, ApoE genotype and cognitive decline. We found that several proteins (primarily CgA and FABP) were related to either brain atrophy or CSF biomarkers. In relation to ApoE genotype, a unique biochemical profile characterised by low CSF levels of Apo E was evident in ε4 carriers compared to ε3 carriers. In an exploratory analysis, 3/83 proteins (SGOT, MCP-1, IL6r) were also found to be mildly associated with cognitive decline in MCI subjects over a 4-year period. Future studies are warranted to establish the validity of these proteins as prognostic factors for cognitive decline. For disease classification, a subset of proteins (n = 24) combined with MRI measurements and CSF biomarkers achieved an accuracy of 95.1% (Sensitivity 87.7%; Specificity 94.3%; AUC 0.95) and accurately detected 94.1% of MCI subjects progressing to AD at 12 months. The subset of proteins included FABP, CgA, MMP-2, and PPP as strong predictors in the model. Our findings suggest that the marker of panel of proteins identified here may be important candidates for improving the earlier detection of AD. Further targeted proteomic and longitudinal studies would be required to validate these findings with more generalisability.