Oxidized proteins in Alzheimer's plasma

The levels of oxidatively modified proteins were examined in blood from Alzheimer's disease (AD) patients, non-AD controls, and AD relatives. Oxidative modification was measured by reacting the protein carbonyls with 2,4-dinitrophenyl hydrazine (DNPH). The total oxidized proteins were determined by HPLC, while specific protein oxidation was assessed from Western blots of electrophoretic gels using antibody to the DNP derivatives. Statistically significant elevations (P < 0.05) of total oxidized proteins were observed in both AD subjects and AD relatives when compared with non-AD controls. Moreover, a protein band (e.g., MW = 78-kDa) was uniquely oxidized in the plasma of AD subjects. Furthermore, this protein from AD subjects was more susceptible to in vitro oxidation. These data suggest that such oxidized proteins may be useful as biomarkers for the detection and evaluation of AD.     

Aberrant profiles of native and oxidized glycoproteins in Alzheimer plasma

A proteomic approach was employed to elucidate possible differential expression of native and oxidized glycoproteins using pooled plasma samples derived from ten patients with sporadic Alzheimer's disease (AD) and pooled plasma samples from nine normal elderly control (NEC) subjects. The plasma samples were fractionated by sequential affinity chromatography on heparin-agarose (HepA) and concanavalin A-agarose (ConA) columns followed by separation on one-dimensional and two-dimensional polyacrylamide gels. Carbonylation (oxidation) of proteins was monitored by in-strip derivatization with 2,4-dinitrophenylhydrazine (DNP) and anti-DNP immunoblotting. Nine spots representing glycoproteins which showed enrichment or high specific oxidation indices in AD HepA-ConA 2-D gels relative to NEC samples were analyzed by matrix-assisted laser desorption-time of flight-mass spectrometry and identified with high probability (p < 0.001) as isoforms of human transferrin (Tf), hemopexin (Hpx) and alpha-1-antitrypsin (alpha-1-AT). These glycoproteins were concentrated, respectively, 5-, 6.5- and 107-fold in HepA-ConA eluates derived from AD plasma relative to the NEC samples. Specific oxidation indices of the identified Tf and Hpx isoforms in AD plasma were respectively, 7.4 and 2.8 relative to NEC. Our findings provide further evidence for systemic derangements in heme/iron/redox homeostasis and activation of the acute phase response in sporadic AD. Moreover, the data implicate isoforms of Tf, Hpx and alpha-1-AT as potential biological markers of this condition.     

Anti-apoptotic proteins are oxidized by Abeta25-35 in Alzheimer's fibroblasts

We have examined the effects of the beta-amyloid peptide (Abeta(25-35)) on fibroblasts derived from subjects with Alzheimer's disease (AD) and from age-matched controls. The peptide was significantly more cytotoxic to the AD-derived fibroblasts. The level of protein oxidation was also greater in the cells from AD subjects. Two-dimensional electrophoresis (2-DE) coupled with immunostaining for protein carbonylation revealed specific oxidation-sensitive proteins (OSPs) in both the control and AD-derived cells. Two specific OSPs were identified by mass spectrometry as heat shock protein 60 (HSP 60) and vimentin. Exposure of the cells to Abeta(25-35) resulted in a twofold increase in the level of oxidation of these two OSPs in the cells derived from controls, but a ninefold increase in their level of oxidation in the fibroblasts from AD subjects. These observations are of particular interest because of the proposed anti-apoptotic roles of both HSP 60 and vimentin and our recent observation that these same two proteins are particularly susceptible to oxidation in neuronally derived cells.     

Oxidized plasma high-density lipoprotein is decreased in Alzheimer's disease

Oxidative stress is implicated in the pathogenesis of Alzheimer's disease (AD), and the enzyme myeloperoxidase (MPO) has been identified as one source of reactive oxidants. MPO-mediated oxidation of high-density lipoprotein (HDL) plays an important role in the pathogenesis of atherosclerosis and although several links between cardiovascular disease and AD have been reported, surprisingly little is known about the role of HDL oxidation in AD. We show that MPO binding to isolated HDL depends on the lipidation state of apolipoprotein A-I (apo A-I), the major protein constituent of HDL. When quantifying apo A-I and oxidized HDL in plasma of AD patients and cognitive healthy, age- and gender matched controls, we observed similar apo A-I levels in AD patients (263 +/- 70 mg/dl) and controls (268 +/- 70 mg/dl, p = 0.83). In striking contrast, oxidized HDL was significantly reduced in AD patients (4.72 +/- 1.91 U/dl) compared to controls (6.98 +/- 3.32 U/dl, p = 0.012). The marked decrease of oxidized HDL in AD patients is surprising considering the current oxidation hypothesis. We suggest that additional mechanisms, including increased antioxidant production and/or altered lipoprotein metabolism, might be involved in AD pathology.     

Proteomic identification of oxidatively modified proteins in Alzheimer's disease brain. Part I: creatine kinase BB,glutamine synthase,and ubiquitin carboxy-terminal hydrolase L-1

Oxidative alterations of proteins by reactive oxygen species (ROS) have been implicated in the progression of aging and age-related neurodegenerative disorders such as Alzheimer's disease (AD). Protein carbonyls, a marker of protein oxidation, are increased in AD brain, indicating that oxidative modification of proteins is relevant in AD. Oxidative damage can lead to several events such as loss in specific protein function, abnormal protein clearance, depletion of the cellular redox-balance and interference with the cell cycle, and, ultimately, to neuronal death. Identification of specific targets of protein oxidation represents a crucial step in establishing a relationship between oxidative modification and neuronal death in AD, and was partially achieved previously in our laboratory through immunochemical detection of creatine kinase BB and beta-actin as specifically oxidized proteins in AD brain versus control brain. However, this process is laborious, requires the availability of specific antibodies, and, most importantly, requires a reasonable guess as to the identity of the protein in the first place. In this study, we present the first proteomics approach to identify specifically oxidized proteins in AD, by coupling 2D fingerprinting with immunological detection of carbonyls and identification of proteins by mass spectrometry. The powerful techniques, emerging from application of proteomics to neurodegenerative disease, reveal the presence of specific targets of protein oxidation in Alzheimer's disease (AD) brain: creatine kinase BB, glutamine synthase, and ubiquitin carboxy-terminal hydrolase L-1. These results are discussed with reference to potential involvement of these oxidatively modified proteins in neurodegeneration in AD brain. Proteomics offers a rapid means of identifying oxidatively modified proteins in aging and age-related neurodegenerative disorders without the limitations of the immunochemical detection method.     

Redox proteomics analysis of HNE-modified proteins in Down syndrome brain: clues for understanding the development of Alzheimer disease

Down syndrome (DS) is the most common genetic cause of intellectual disability, due to partial or complete triplication of chromosome 21. DS subjects are characterized by a number of abnormalities including premature aging and development of Alzheimer disease (AD) neuropathology after approximately 40 years of age. Several studies show that oxidative stress plays a crucial role in the development of neurodegeneration in the DS population. Increased lipid peroxidation is one of the main events causing redox imbalance within cells through the formation of toxic aldehydes that easily react with DNA, lipids, and proteins. In this study we used a redox proteomics approach to identify specific targets of 4-hydroxynonenal modifications in the frontal cortex from DS cases with and without AD pathology. We suggest that a group of identified proteins followed a specific pattern of oxidation in DS vs young controls, probably indicating characteristic features of the DS phenotype; a second group of identified proteins showed increased oxidation in DS/AD vs DS, thus possibly playing a role in the development of AD. The third group of comparison, DS/AD vs old controls, identified proteins that may be considered specific markers of AD pathology. All the identified proteins are involved in important biological functions including intracellular quality control systems, cytoskeleton network, energy metabolism, and antioxidant response. Our results demonstrate that oxidative damage is an early event in DS, as well as dysfunctions of protein-degradation systems and cellular protective pathways, suggesting that DS subjects are more vulnerable to oxidative damage accumulation that might contribute to AD development. Further, considering that the majority of proteins have been already demonstrated to be oxidized in AD brain, our results strongly support similarities with AD in DS.     

Deep proteomic network analysis of Alzheimer’s disease brain reveals alterations in RNA binding proteins and RNA splicing associated with disease

Background

The complicated cellular and biochemical changes that occur in brain during Alzheimer’s disease are poorly understood. In a previous study we used an unbiased label-free quantitative mass spectrometry-based proteomic approach to analyze these changes at a systems level in post-mortem cortical tissue from patients with Alzheimer’s disease (AD), asymptomatic Alzheimer’s disease (AsymAD), and controls. We found modules of co-expressed proteins that correlated with AD phenotypes, some of which were enriched in proteins identified as risk factors for AD by genetic studies.

Methods

The amount of information that can be obtained from such systems-level proteomic analyses is critically dependent upon the number of proteins that can be quantified across a cohort. We report here a new proteomic systems-level analysis of AD brain based on 6,533 proteins measured across AD, AsymAD, and controls using an analysis pipeline consisting of isobaric tandem mass tag (TMT) mass spectrometry and offline prefractionation.

Results

Our new TMT pipeline allowed us to more than double the depth of brain proteome coverage. This increased depth of coverage greatly expanded the brain protein network to reveal new protein modules that correlated with disease and were unrelated to those identified in our previous network. Differential protein abundance analysis identified 350 proteins that had altered levels between AsymAD and AD not caused by changes in specific cell type abundance, potentially reflecting biochemical changes that are associated with cognitive decline in AD. RNA binding proteins emerged as a class of proteins altered between AsymAD and AD, and were enriched in network modules that correlated with AD pathology. We developed a proteogenomic approach to investigate RNA splicing events that may be altered by RNA binding protein changes in AD. The increased proteome depth afforded by our TMT pipeline allowed us to identify and quantify a large number of alternatively spliced protein isoforms in brain, including AD risk factors such as BIN1, PICALM, PTK2B, and FERMT2. Many of the new AD protein network modules were enriched in alternatively spliced proteins and correlated with molecular markers of AD pathology and cognition.

Conclusions

Further analysis of the AD brain proteome will continue to yield new insights into the biological basis of AD.

     

Oxidative damage of DJ-1 is linked to sporadic Parkinson and Alzheimer diseases

Mutations in DJ-1 cause an autosomal recessive, early onset familial form of Parkinson disease (PD). However, little is presently known about the role of DJ-1 in the more common sporadic form of PD and in other age-related neurodegenerative diseases, such as Alzheimer disease (AD). Here we report that DJ-1 is oxidatively damaged in the brains of patients with idiopathic PD and AD. By using a combination of two-dimensional gel electrophoresis and mass spectrometry, we have identified 10 different DJ-1 isoforms, of which the acidic isoforms (pI 5.5 and 5.7) of DJ-1 monomer and the basic isoforms (pI 8.0 and 8.4) of SDS-resistant DJ-1 dimer are selectively accumulated in PD and AD frontal cortex tissues compared with age-matched controls. Quantitative Western blot analysis shows that the total level of DJ-1 protein is significantly increased in PD and AD brains. Mass spectrometry analyses reveal that DJ-1 is not only susceptible to cysteine oxidation but also to previously unsuspected methionine oxidation. Furthermore, we show that DJ-1 protein is irreversibly oxidized by carbonylation as well as by methionine oxidation to methionine sulfone in PD and AD. Our study provides new insights into the oxidative modifications of DJ-1 and indicates association of oxidative damage to DJ-1 with sporadic PD and 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.     

Proteomic identification of oxidatively modified proteins in Alzheimer's disease brain. Part II: dihydropyrimidinase-related protein 2, alpha-enolase and heat shock cognate 71

Alzheimer's disease (AD) is a neurodegenerative disorder in which oxidative stress has been implicated as an important event in the progression of the pathology. In particular, it has been shown that protein modification by reactive oxygen species (ROS) occurs to a greater extent in AD than in control brain, suggesting a possible role for oxidation-related decrease in protein function in the process of neurodegeneration. Oxidative damage to proteins, assessed by measuring the protein carbonyl content, is involved in several events such as loss in specific protein function, abnormal protein clearance, depletion of the cellular redox-balance and interference with the cell cycle, and, ultimately, neuronal death. The present investigation represents a further step in understanding the relationship between oxidative modification of protein and neuronal death in AD. Previously, we used our proteomics approach, which successfully substitutes for labor-intensive immunochemical analysis, to detect proteins and identified creatine kinase, glutamine synthase and ubiquitin carboxy-terminal hydrolase L-1 as specifically oxidized proteins in AD brain. In this report we again applied our proteomics approach to identify new targets of protein oxidation in AD inferior parietal lobe (IPL). The dihydropyrimidinase related protein 2 (DRP-2), which is involved in the axonal growth and guidance, showed significantly increased level in protein carbonyls in AD brain, suggesting a role for impaired mechanism of neural network formation in AD. Additionally, the cytosolic enzyme alpha-enolase was identified as a target of protein oxidation and is involved the glycolytic pathway in the pathological events of AD. Finally, the heat shock cognate 71 (HSC-71) revealed increased, but not significant, oxidation in AD brain. These results are discussed with reference to potential involvement of these oxidatively modified proteins in neurodegeneration in AD brain.     

Increased lipoprotein oxidation in Alzheimer's disease

Oxidation has been proposed to be an important factor in the pathogenesis of Alzheimer's disease (AD) and amyloid beta is considered to induce oxidation. In biological fluids, including cerebrospinal fluid (CSF), amyloid beta is found complexed to lipoproteins. On the basis of these observations, we investigated the potential role of lipoprotein oxidation in the pathology of AD. Lipoprotein oxidizability was measured in vitro in CSF and plasma from 29 AD patients and found to be significantly increased in comparison to 29 nondemented controls. The levels of the hydrophilic antioxidant ascorbate were significantly lower in CSF and plasma from AD patients. In plasma, alpha-carotene was significantly lower in AD patients compared to controls while alpha-tocopherol levels were indistinguishable between patients and controls. In CSF, a nonsignificant trend to lower alpha-tocopherol levels among AD patients was found. Polyunsaturated fatty acids, the lipid substrate for oxidation, were significantly lower in the CSF of AD patients. Our findings suggest that (i) lipoprotein oxidation may be important in the development of AD and (ii) the in vitro measurement of lipid peroxidation in CSF might become a useful additional marker for diagnosis of AD.     

Plasma biomarkers of brain atrophy in Alzheimer's disease

Peripheral biomarkers of Alzheimer''s disease (AD) reflecting early neuropathological change are critical to the development of treatments for this condition. The most widely used indicator of AD pathology in life at present is neuroimaging evidence of brain atrophy. We therefore performed a proteomic analysis of plasma to derive biomarkers associated with brain atrophy in AD. Using gel based proteomics we previously identified seven plasma proteins that were significantly associated with hippocampal volume in a combined cohort of subjects with AD (N = 27) and MCI (N = 17). In the current report, we validated this finding in a large independent cohort of AD (N = 79), MCI (N = 88) and control (N = 95) subjects using alternative complementary methods—quantitative immunoassays for protein concentrations and estimation of pathology by whole brain volume. We confirmed that plasma concentrations of five proteins, together with age and sex, explained more than 35% of variance in whole brain volume in AD patients. These proteins are complement components C3 and C3a, complement factor-I, γ-fibrinogen and alpha-1-microglobulin. Our findings suggest that these plasma proteins are strong predictors of in vivo AD pathology. Moreover, these proteins are involved in complement activation and coagulation, providing further evidence for an intrinsic role of these pathways in AD pathogenesis.     

BAG-1M is up-regulated in hippocampus of Alzheimer's disease patients and associates with tau and APP proteins

The accumulation of tau and amyloid beta proteins is the major molecular pathology of Alzheimer's disease (AD). The mechanisms leading to the accumulation of these proteins are not completely clear. Hsc-70/Hsp-70, a chaperone protein, has been shown to bind both these proteins and regulate their degradation. We have previously shown that the co-chaperone protein BAG-1 can inhibit the degradation of tau by forming a complex with Hsc-70 and tau. In this current work, we show that there is an increase in the BAG-1M isoform in the hippocampus of AD patients. In addition, BAG-1 binds to both tau and amyloid precursor protein physically, and is found highly expressed in the same neurons that contain intracellular tau or amyloid in hippocampal sections from AD patients. Over-expression of BAG-1M in cell culture also induced an increase in both tau and amyloid precursor protein levels. In conclusion, we report a specific increase of BAG-1M in human AD patients, which is both physically and functionally associated to the two major molecular markers of AD.     

Upregulation of Glycolytic Enzymes,Mitochondrial Dysfunction and Increased Cytotoxicity in Glial Cells Treated with Alzheimer’s Disease Plasma

Alzheimer’s disease (AD) is a neurodegenerative disorder associated with increased oxidative stress and neuroinflammation. Markers of increased protein, lipid and nucleic acid oxidation and reduced activities of antioxidant enzymes have been reported in AD plasma. Amyloid plaques in the AD brain elicit a range of reactive inflammatory responses including complement activation and acute phase reactions, which may also be reflected in plasma. Previous studies have shown that human AD plasma may be cytotoxic to cultured cells. We investigated the effect of pooled plasma (n = 20 each) from healthy controls, individuals with amnestic mild cognitive impairment (aMCI) and Alzheimer’s disease (AD) on cultured microglial cells. AD plasma and was found to significantly decrease cell viability and increase glycolytic flux in microglia compared to plasma from healthy controls. This effect was prevented by the heat inactivation of complement. Proteomic methods and isobaric tags (iTRAQ) found the expression level of complement and other acute phase proteins to be altered in MCI and AD plasma and an upregulation of key enzymes involved in the glycolysis pathway in cells exposed to AD plasma. Altered expression levels of acute phase reactants in AD plasma may alter the energy metabolism of glia.     

Differential oxidation of apolipoprotein E isoforms and interaction with phospholipids

Accumulation of oxidized proteins has been demonstrated in the brain of patients suffering from Alzheimer's disease (AD). Among the proteins found in cerebral amyloid deposits, apolipoprotein (apo) E is a polymorphic protein which one specific isoform, apo E4, has been widely associated with AD. Apo E may be linked with AD by its isoform-specific interaction with lipids or other proteins in amyloid plaques. Using the myeloperoxidase oxidative system, we report that oxidation of the three recombinant apo E isoforms is differential (as estimated using immunoblot and high-performance liquid chromatography analysis), with apo E4 being more susceptible than apo E3, which in turn is much more susceptible than apo E2. In addition, susceptibility to thrombin proteolysis is reduced when apo E is oxidized, and oxidation of apo E decreases its incorporation into phospholipid discs by approximately 50%. Oxidation of apo E may contribute to inefficient lipid recycling in the brain, particularly regarding apo E4 and E3. Our results link and strengthen both the E4 allele linkage with AD and the role of protein oxidation in AD. The cerebral mechanisms underlying apo E oxidation and/or myeloperoxidase functions in vivo remain to be assessed.     

Proteomic analysis of oxidatively modified proteins in Alzheimer's disease brain: insights into neurodegeneration.

Identification of oxidized proteins in Alzheimer's disease (AD) brain is hypothesized to lead to new insights into mechanisms of neurodegeneration and synapse loss in this dementing disorder that are associated with oxidative stress. Previous studies had shown increased oxidation of proteins in AD brain, but identifying those particular proteins that were specifically oxidized using standard immunochemical methods is a daunting task when one considers how many proteins there are in brain. To address this issue, proteomics has been used to identify specifically modified proteins in AD brain. This review outlines the nature of proteomics, the proteins identified in AD brain that are specifically oxidatively modified, and provides rational consequences related to neurodegeneration and synapse loss as sequelae to loss of function, due to oxidation and consistent with the known pathological and biochemical alteration in AD brain. The use of proteomics to learn about disease mechanisms is still embryonic, but the emerging techniques of proteomics represent a promising means to elucidate mechanisms of disease at the protein level. There are limitations to proteomics, and these, too, are discussed.     

Increased protein hydrophobicity in response to aging and Alzheimer disease

Increased levels of misfolded and damaged proteins occur in response to brain aging and Alzheimer disease (AD), which presumably increase the amount of aggregation-prone proteins via elevations in hydrophobicity. The proteasome is an intracellular protease that degrades oxidized and ubiquitinated proteins, and its function is known to be impaired in response to both aging and AD. In this study we sought to determine the potential for increased levels of protein hydrophobicity occurring in response to aging and AD, to identify the contribution of proteasome inhibition to increased protein hydrophobicity, and last to identify the contribution of ubiquitinated and oxidized proteins to the pool of hydrophobic proteins. In our studies we identified that aging and AD brain exhibited increases in protein hydrophobicity as detected using Bis ANS, with dietary restriction (DR) significantly decreasing age-related increases in protein hydrophobicity. Affinity chromatography purification of hydrophobic proteins from aging and AD brains identified increased levels of oxidized and ubiquitinated proteins in the pool of hydrophobic proteins. Pharmacological inhibition of the proteasome in neurons, but not astrocytes, resulted in an increase in protein hydrophobicity. Taken together, these data indicate that there is a relationship between increased protein oxidation and protein ubiquitination and elevations in protein hydrophobicity within the aging and the AD brain, which may be mediated in part by impaired proteasome activity in neurons. Our studies also suggest a potential role for decreased oxidized and hydrophobic proteins in mediating the beneficial effects of DR.     

In-vivo glutathione elevation protects against hydroxyl free radical-induced protein oxidation in rat brain

Glutathione deficiency has been associated with a number of neurodegenerative diseases including Lou Gehrig's disease, Parkinson's disease, and HIV. A crucial role for glutathione is as a free radical scavenger. Alzheimer's disease (AD) brain is characterized by oxidative stress, manifested by protein oxidation, lipid oxidation, oxidized glutathione, and decreased activity of glutathione S-transferase, among others. Reasoning that elevated levels of endogenous glutathione would offer protection against free radical-induced oxidative stress, rodents were given in vivo injections of N-acetylcysteine (NAC), a known precursor of glutathione, to study the vulnerability of isolated synaptosomal membranes treated with Fe2+/H2O2, a known hydroxyl free radical producer. Protein carbonyls, a marker of protein oxidation, were measured. NAC significantly increased endogenous glutathione levels in cortical synaptosome cytosol (P < 0.01). As reported previously, protein carbonyl levels of the Fe2+/H2O2-treated synaptosomes were significantly higher compared to that of non-treated controls (P < 0.01), consistent with increased oxidative stress. In contrast, protein carbonyl levels in Fe2+/H2O2-treated synaptosomes isolated from NAC-injected animals were not significantly different from saline-injected non-treated controls, demonstrating protection against hydroxyl radical induced oxidative stress. These results are consistent with the notion that methods to increase endogenous glutathione levels in neurodegenerative diseases associated with oxidative stress, including AD, may be promising.     

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.